CHR Program & Abstract Book OMA
TIN Chromatin STR
UCTURE & FUNCTION 2006 Structure&Function Punta Cana, Dominican Republic 5 - 8 December 2006 Pr o g r am & Abstr act Book
Organized By: Tony Kouzarides and Abcam
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Program & Abstract Book
The third Chromatin Structure & Function Punta Cana, Dominican Republic 5 - 8 December 2006
Organizers: Tony Kouzarides (University of Cambridge) and Abcam
Table of contents
Conference Program ...... Page 2
Poster Index ...... Page 6
Abstracts - Oral ...... Page 18
Abstracts - Poster ...... Page 51
Resort Information ...... Page 179
Disclaimer: Material contained within this booklet should be citied only with permission from the author(s). No live recording or photography is permitted during the oral or poster sessions.
Copyright © 2006 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.
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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 Conference Program Tuesday 5th December Meeting room - Allegro Plaza
Keynote Speaker - introduced by Tony Kouzarides 18:00 - 19:00 Steve Henikoff ...... Page 18 Epigenetic patterns generated by histone replacement
Welcome reception and buffet at poolside.
Allegro Live resort show
Wednesday 6th December Chair: Jerry Workman
09:00 - 09:30 Yang Shi ...... Page 19 The identification of histone demethylases established the dynamic and reversible nature of histone methylation regulation
09:30 - 09:45 Jesper Christensen ...... Page 20 The retinoblastoma tumor suppressor binding protein RBP2 is a transcriptional repressor demethylating tri- and dimethylated lysine 4 on Histone H3
09:45 - 10:00 Mischa Machius ...... Page 21 Structural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase
10:00 - 10:30 Ramin Shiekhattar ...... Page 22 Functional and biochemical characterization of histone demethylase complexes
Drinks break in hotel lobby
11:00 - 11:30 Yi Zhang ...... Page 23 Histone demethylation by the JmjC domain-containing proteins
11:30 - 11:45 Francis Stewart ...... Page 24 Epigenetic aspects of lineage commitment
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Conference Program
11:45 - 12:00 Henk Stunnenberg ...... Page 25 Title and abstract unavailable
12:00 - 12:30 Ali Shilatifard ...... Page 26 H2B monoubiquitination and H3K4 methylation via COMPASS
Lunch at the Beach Buffet Restaurant and free time Chair: Yang Shi
16:00 - 16:30 Paulo Sassone-Corsi ...... Page 27 A chromatin remodeling clock
16:30 - 16:45 Sung Hee Baek ...... Page 28 A Novel Link between SUMO Modification of a Chromatin Remodeling Complex and Cancer Metastasis
16:45 - 17:00 Laszlo Tora ...... Page 29 The simultaneously dimethylated Lys-9 and phosphorylated Ser-10 tails of histone H3 adopt different conformations during mitosis
17:00 - 17:30 Tony Kouzarides ...... Page 30 Characterisation of novel histone modifications
18:00 - 21:00 Posters and buffet by the pool
Allegro Live resort show
Thursday 7th December Chair: Genevieve Almouzni
09:00 - 09:30 Thomas Jenuwein ...... Page 31 Epigenetic control by histone methylation
09:30 - 09:45 Roberta Benetti ...... Page 32 The role of Dicer in the regulation of chromatin at telomeres
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09:45 - 10:00 Mareike Puschendorf ...... Page 33 Ezh2 independent targeting of PRC1 proteins to paternal constitutive heterochromatin in mouse pre-implantation embryos
10:00 - 10:30 Edith Heard ...... Page 34 The nuclear and epigenetic dynamics of X-chromosome inactivation in the mouse
Drinks break in hotel lobby
11:00 - 11:30 Adrian Bird ...... Page 35 MeCP2: molecular interactions and phenotypic stability in a mouse model of Rett Syndrome
11:30 - 11:45 Jon Penterman ...... Page 36 DNA demethylation in Arabidopsis thaliana
11:45 - 12:00 Francois Fuks ...... Page 37 The Polycomb Group protein EZH2 is recruited to promoters by MECP2
12:00 - 12:30 Shelley Berger ...... Page 38 Factor and histone covalent modifications in genome regulation
Lunch at the Beach Buffet Restaurant and free time Chair: Ramin Shiekhattar
16:00 - 16:30 Danny Reinberg ...... Page 39 A molecular understanding of epigenetics
16:30 - 16:45 Jessica Tyler ...... Page 40 The mechanistic basis for the requirement of promoter chromatin disassembly for transcriptional activation
16:45 - 17:00 Gratien Prefontaine ...... Page 41 Epigenetic mechanisms influencing pituitary gene expression
17:00 - 17:30 Bob Kingston ...... Page 42 Possible roles in silencing for piRNAs
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Conference Program
18:00 - 19:30 Posters and drinks by the pool
Beach barbeque and live band on the sand
Friday 8th December Chair: Edith Heard
09:00 - 09:30 Michael Grunstein ...... Page 43 Deacetylation of histone H4 K16 regulates gene activity in yeast
09:30 - 09:45 Ann Ehrenhofer-Murray ...... Page 44 A role for the HDAC Rpd3 in establishing eurchromatin- heterochromatin boundaries at yeast telomeres
09:45 - 10:00 Wyatt Yue ...... Page 45 CARM1 and histone methylation - a structural study
10:00 - 10:30 Sharon Dent ...... Page 46 Common and unique factors regulate Set1-mediated methylation of the Dam1 kinetochore protein and histone H3
Drinks break in hotel lobby
11:00 - 11:30 Genevieve Almouzni ...... Page 47 Chromatin assembly factors, histone H3 variants and cell cycle
11:30 - 11:45 Dmitry Fyodorov ...... Page 48 ATP-dependant deposition of Histone H3.3 by Drosophila CHD1 in vivo
11:45 - 12:00 Roberto Mantovani ...... Page 49 The histone fold trimer NF-Y is required to define positive histone marks in CCAAT-promotors: a genome wide analysis
12:00 - 12:30 Jerry Workman ...... Page 50 Histone modification and chromatin remodeling in transcription
Lunch at the Beach Buffet Restaurant
Conference ends
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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 Poster Index Abstract P1 Karl Agger ...... Page 51 The role of the polycomb group protein RYBP in oncogene induced senescence
Abstract P2 Helena Ahlfors ...... Page 52 A novel player in T helper cell differentiation
Abstract P3 Barbara Alberter ...... Page 53 Histone modification pattern of the T lymphotropic Herpesvirus saimiri genome in latency
Abstract P4 Marco Alvarez ...... Page 54 Histone variant macroH2A is an epigenetic factor involved in the modulation of ribosomal gene expression during seasonal adaptation of carp fish
Abstract P5 Terra Arnason ...... Page 55 Rsp5 is required for nuclear shuttling of the Snf1 kinase complex in yeast
Abstract P6 Stuart Atkinson ...... Page 56 Epigenetic mechanisms of pluripotency and differentiation
Abstract P7 Joanne Attema ...... Page 57 Epigenetic features of hematopoietic stem cells using small numbers of highly purified primary cells
Abstract P8 Kristin Baetz ...... Page 58 NuA4 is a cellular “Hub”: an integrative map of physical and genetic interactions mediated by the NuA4 histone acetyltransferase
Abstract P9 Slobodan Barbaric ...... Page 59 Chromatin remodeling activities at the yeast PHO84 promoter
Abstract P10 Vivian Bardwell ...... Page 60 Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets
Abstract P11 Amrita Basu ...... Page 61 Computational prediction of histone and non-histone proteins
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Poster Index
Abstract P12 Mark Bedford ...... Page 62 Screening for the methylated proteome
Abstract P13 Sukesh R. Bhaumik ...... Page 63 Regulation of transcriptional activation by mRNA cap-binding complex in vivo
Abstract P14 Marjorie Brand ...... Page 64 The Ash2L/MLL2 methyltransferase complex is important for ß- globin transcription during erythroid differentiation
Abstract P15 Lauren Buro ...... Page 65 Histone methylation patterns at interferon-gamma inducible gene loci
Abstract P16 Jill Butler ...... Page 66 CXXC-finger protein 1 regulates Dnmt1 protein expression
Abstract P17 Jim Cakouros ...... Page 67 Identification of a novel enzyme which regulates the kinetics of histone arginine methylation in Drosophila melanogaster
Abstract P18 Raymond Camahort ...... Page 68 Genome-wide analysis of the budding yeast histone variant Cse4 reveals occupancy at a single centromeric nucleosome as well as additional non-centromeric locations
Abstract P19 Dylan Carney ...... Page 69 The RAG2 PHD Finger links the histone code to V(D)J recombination
Abstract P20 Beverly Chilton ...... Page 70 Analysis of RUSH/SMARCA3 isoforms and their interactions with Egr-1 and c-Rel in the regulation of transcription
Abstract P21 Alexandra Chittka ...... Page 71 Signalling by a novel p75 neurotrophin receptor interacting protein, SC1/PRDM4
Abstract P22 Leslie Chu ...... Page 72 Inheritance of epigenetic chromatin states
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Abstract P23 Mair Churchill ...... Page 73 Structural basis for the histone chaperone activity of Asf1
Abstract P24 Jeffrey Craig ...... Page 74 What makes centromeres localise and cluster in interphase nuclei?
Abstract P25 Valerie Crusselle-Davis ...... Page 75 Regulation of beta-globin expression through the recruitment of chromatin modifying enzymes by TFII-I and USF Abstract P26 Eullia de Nadal ...... Page 76 Control of gene expression by the yeast Hog1 MAPK.
Abstract P27 Foteini Davrazou ...... Page 77 Molecular mechanism of histone H3K4me3 recognition by the PHD finger of ING2
Abstract P28 Roger Deal ...... Page 78 Repression of flowering in Arabidopsis requires histone H2A.Z deposition by a putative SWR1 complex
Abstract P29 Laurent Delva ...... Page 79 The Transcription Intermediary Factor 2 is required for zebrafish development
Abstract P30 Luisa Di Stefano ...... Page 80 Lsd1 mutation in Drosophila disrupt normal level of H3K4 methylation and affects viability and fertility
Abstract P31 Stephan Diekmann ...... Page 81 In vivo dynamic (FRAP, FCS) and neighbourhood relation (AB- FRET, FLIM) studies of human inner kinetochore proteins
Abstract P32 Jeffrey Dilworth ...... Page 82 MEF2 helps establish muscle specific pattern of gene expression by recruiting Trithorax Group proteins to specific promoters
Abstract P33 Ivana Djuretic ...... Page 83 T-bet and Runx3 cooperate to activate Interferon gamma and silence Interleukin-4 in T helper-1 cells
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Poster Index
Abstract P34 Tom Donndelinger ...... Page 84 Seeing cells in a new light: Improving resolution with a scientific approach to tissue processing
Abstract P35 Bojan Drobic ...... Page 85 Characterization of Histone H3 kinases, MSK1 and MSK2
Abstract P36 Danielle Ellis ...... Page 86 Histone acetylation of SRC and p21 promoters in response to histone deacetylase inhibitor treatment; implications of HDAC activity and SRC expression
Abstract P37 Alexander Erkine ...... Page 87 Differential mechanisms of nucleosome displacement at yeast heat shock gene promoters
Abstract P38 Ragnhild Eskeland ...... Page 88 HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors
Abstract P39 George Feehery ...... Page 89 CpG methylated DNA standards and control primers for use in methyl sensitive PCR and bisulphite sequencing
Abstract P40 Barna Fodor ...... Page 90 Identification of novel pericentric proteins by their localization
Abstract P41 Maria Fousteri ...... Page 91 Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo
Abstract P42 Robert Gillespie ...... Page 92 Retinoid regulated association of transcriptional coregulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARß2, and Cyp26A1 in F9 embryonal carcinoma cells
Abstract P43 Clara Goday ...... Page 93 Chromatin modifications in germline chromosomes of sciarid flies
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Abstract P44 Aaron Goldberg ...... Page 94 HIRA-dependent incorporation of histone H3.3 marks active genes in mouse embryonic stem cells
Abstract P45 Elizabeth Goneska ...... Page 95 Phosphorylation of the SQ H2A.X motif is required for proper meiosis and mitosis in Tetrahymena thermophila
Abstract P46 Susana Gonzalo ...... Page 96 Telomere epigenetic modifications: a control of telomere length and a stop on recombination
Abstract P47 Tanya Gustafson ...... Page 97 Epigenetic silencing of Singleminded-2 in breast cancer
Abstract P48 Soon-Ki Han ...... Page 98 Role of plant CBP/p300-like genes in the regulation of flowering time
Abstract P49 Christin Hanigan ...... Page 99 Identification of an HDAC2 mutation in colorectal cancer and its consequences
Abstract P50 Troy Harkness ...... Page100 Rsp5 is required for nuclear shuttling of the Snf1 kinase complex in yeast
Abstract P51 Tiffany Hung ...... Page 101 ING4 recognition of histone H3 trimethylated at lysine 4
Abstract P52 David Johnson ...... Page 102 E2F1 and GCN5 facilitate the recruitment of nucleotide excision repair factors to sites of UV-induced DNA damage
Abstract P53 Paul Kalitsis ...... Page 103 Nucleosome spacing analysis of repeat DNA regions in the mouse genome
Abstract P54 Min-Jeong Kang ...... Page 104 Role of a RPD3/HDA1 family histone deacetylase in the regulation of phytochrome-mediated light respases in Arabidopsis
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Poster Index
Abstract P55 Panagiota Karagianni ...... Page 105 ICBP90, a putative link between histone ubiquitination and cell cycle progression
Abstract P56 Emmanuel Kas ...... Page 106 Altering the structure and functional properties of heterochromatin with satellite-specific minor-groove binders
Abstract P57 Chul Geun Kim ...... Page 107 PIAS1 confers erythroid cell specific α-globin gene regulation by the CP2 transcription factor family
Abstract P58 Keun Il Kim ...... Page 108 A novel link between SUMO modification of a chromatin remodeling complex and cancer metastasis
Abstract P59 Sarah Kimmins ...... Page 109 Methylation of Histone H3 at lysine 4 is dynamic and tightly regulated during male germ cell development
Abstract P60 Robert Klose ...... Page 110 JmjC-domain-containing proteins and histone demethylation
Abstract P61 Christoph Koch ...... Page 111 The landscape of activating histone modifications across 1% of the human genome
Abstract P62 Ryoki Kujiki ...... Page 112 1alpha,25(OH)2D3-induced transrepression on 1alpha- hydroxylase gene promoter mediates chromatin remodeling through WINAC
Abstract P63 Sharmistha Kundu ...... Page 113 SWI/SNF establishes transcriptional memory at the Saccharomyces cerevisiae GAL1 gene
Abstract P64 Georg Kustatscher ...... Page 114 Metabolite-sensitive and metabolite-insensitive chromatin surfaces through the human histone macroH2A
Abstract P65 Hyockman Kwon ...... Page 115 BAF53-dependent higher-order chromatin structure as the compartment of replication and repair foci
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Abstract P66 Monika Lachner ...... Page 116 Studying lysine methylation in non-histone proteins
Abstract P67 Brian Larsen ...... Page 117 Caspase 3 mediated DNA strand breaks contribute to genomic reorganization during skeletal muscle terminal differentiation
Abstract P68 Richard Lawrence ...... Page 118 Mechanisms controlling dynamic Swi6/HP1 binding in S. pombe facilitate de novo heterochromatin formation
Abstract P69 Frederic Leduc ...... Page 119 Presence of gamma-H2AX in elongating spermatids: involvement of NHEJ?
Abstract P70 Min Gyu Lee ...... Page 120 Functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein
Abstract P71 Niraj Lodhi ...... Page 121 Histone acetylation (H3K9) and methylation (H3K4) of the nucleosome over core promoter are associated with the induction of tobacco PR-1a gene
Abstract P72 Mattias Mannervik ...... Page 122 An HDAC3/SMRTER/Ebi complex required for Snail repressor function in Drosophila development
Abstract P73 Robert Martin ...... Page 123 Chromatin labeling and distribution in living cells
Abstract P74 Peter McKeown ...... Page 124 Chromatin components of the Arabidopsis thaliana nucleolus
Abstract P75 Rosalind Meldrum ...... Page 125 Visualisation of DNA repair and chromatin dynamics
Abstract P76 Brendon Monahan ...... Page 126 Purification and characterization of the fission yeast Swi/Snf and RSC chromatin remodeling complexes
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Poster Index
Abstract P77 Antonin Morillon ...... Page 127 Transcriptional co-suppression in S. cerevisiae
Abstract P78 Ashby Morrison ...... Page 128 Mec1/Tel1-dependent phosphorylation of a chromatin remodeling complex influences the DNA damage checkpoint pathway
Abstract P79 Raul Mostoslavsky ...... Page 129 Genomic instability and aging-like phenotype in the absence of mammalian SIRT6
Abstract P80 Takahiro Nakayama ...... Page 130 Drosophila GAGA factor promotes histone H3.3 replacement that prevents the heterochromatin spreading
Abstract P81 Zuyao Ni ...... Page 131 The tumor suppressor BRG1 silences the distal silencers at interferon-responsive genes
Abstract P82 Olivia Osborn ...... Page 132 Transcriptional targets of Af4
Abstract P83 Julia Pagan ...... Page 133 A novel corepressor, BCOR-L1, functions through CTBP and class 2 HDACs
Abstract P84 Maria Panchenko ...... Page 134 Role of Jade-1 in the HAT HBO1 complex
Abstract P85 Tej Pandita ...... Page 135 Mammalian ortholog of Drosophila MOF is critical for embryogenesis and DNA repair
Abstract P86 Maëlle Pannetier ...... Page 136 Imprinting perturbation in mouse hepatocarcinoma: link between DNA methylation and histone methylation
Abstract P87 Janet Partridge ...... Page 137 Establishment and maintenance of centromeric heterochromatin in fission yeast are functionally separable
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Abstract P88 Kelly Perkins ...... Page 138 Activated HIV-1 provirus forms a gene loop, connecting viral transcriptional initiation with termination
Abstract P89 David Picketts ...... Page 139 SNF2L-mediated control of cell number in the developing brain
Abstract P90 Romina Ponzielli ...... Page 140 Optimization of experimental design parameters of ChIP-on- chip studies
Abstract P91 Ryan Raisner ...... Page 141 Single nucleosome resolution mapping of the histone variant H2A.Z in a developing organism
Abstract P92 Rama Natarajan ...... Page 142 Genome-wide analysis of histone lysine methylation variations caused by diabetic conditions in human monocytes
Abstract P93 Edward Ramos ...... Page 143 Global characterization and function of Gypsy-like endogenous insulators in Drosophila melanogaster
Abstract P94 William Renthal ...... Page 144 Class II histone deacetylases regulate the behavioral adaptations to chronic cocaine and stress
Abstract P95 Karsten Rippe ...... Page 145 Activities of histone chaperone NAP1: Association states and interactions with histones, nucleosome assembly and effect on the chromatin fiber conformation
Abstract P96 Charles Roberts ...... Page 146 The Swi/Snf chromatin remodeling complex regulates lineage specific transcription programs during development and impairment of this activity causes cancer
Abstract P97 Paul Sadowski ...... Page 147 Post-translational modification of the insulator protein, CTCF
Abstract P98 Teresa Sanchez Alcaraz ...... Page 148 Role of USP7 and GMP synthetase in deubiquitination of human histone H2B
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Poster Index
Abstract P99 Annette Scharf ...... Page 149 Dynamics of histone modifications during chromatin assembly
Abstract P100 Stefan Schoeftner ...... Page 150 Screening for miRNAs regulating mammalian telomeres
Abstract P101 Gunnar Schotta ...... Page 151 A genome-wide transition to H4K20 mono-methylation impairs stress-induced and programd DNA damage response in the mouse
Abstract P102 David Schrump ...... Page 152 Brother of the Regulator of Imprinted Sites (BORIS) recruits Sp1 to modulate NY-ESO-1 expression in lung cancer cells
Abstract P103 Bonnie Scott ...... Page 153 Evolution of centromere-binding proteins and their interactions with centromere DNA in Arabidopsis
Abstract P104 David Shechter ...... Page 154 Histone H2A arginine3 is mono- and symmetrically-di methylated by a complex of PRMT5 and the WD-repeat protein MEP50 in Xenopus laevis eggs
Abstract P105 Yoichi Shinkai ...... Page 155 H3K9 methylation and germ cell development
Abstract P106 Krishna Sinha ...... Page 156 Inhibition of the transcriptional activity of osterix by interactions with NO66, a jumonji family chromatin protein
Abstract P107 Karen Smith ...... Page 157 Identification and characterization of novel HDAC-associated proteins that regulate cancer cell growth
Abstract P108 Matthew Smith ...... Page 158 Chromatin- mediated silencing of immune response genes
Abstract P109 Hae-Ryong Song ...... Page 159 Coordination of transcriptional regulation and chromatin modification of Arabidopsis circadian clock genes
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Abstract P110 Stacey Southall ...... Page 160 Structural studies of histone methyltransferases
Abstract P111 Maike Stam ...... Page 161 Molecular analysis of chromatin changes involved in b1 paramutation, an allele-dependent transfer of epigenetic information
Abstract P112 Sean Taverna ...... Page 162 Connecting H3 methylation and acetylation: The role of Yng1 in transcription
Abstract P113 Tage Thorstensen ...... Page 163 The Arabidopsis SUVR proteins define a novel subgroup of SET domain proteins associated with the nucleolus
Abstract P114 Christopher Topp ...... Page 164 Unusually-sized centromeric RNAs associate with maize centromeric chromatin
Abstract P115 Martin Tribus ...... Page 165 Molecular mechanisms of histone variant H3.3 assembly by the motor protein CHD1
Abstract P116 Christopher Vakoc ...... Page 166 A profile of histone lysine methylation generated by mammalian gene transcription
Abstract P117 Claudius Vincenz ...... Page 167 Visualizing polycomb group protein interactions with histones in vivo
Abstract P118 Vikki Weake ...... Page 168 The SAGA histone acetyltransferase complex functions in the development of neuronal connectivity in the Drosophila compound eye
Abstract P119 Stephanie Williams ...... Page 169 Mechanistic insights into promoter chromatin disassembly
Abstract P120 Jon Wilson ...... Page 170 Structural studies of SET domain methyltransferases
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Poster Index
Abstract P121 Zhaodong Xu ...... Page 171 Remote elements critical for cytokine induced gene expression
Abstract P122 Xiaofang Yang ...... Page 172 Dissecting SWI/SNF ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae
Abstract P123 Juan I. Young ...... Page 173 Post-transcriptional functions of MeCP2
Abstract P124 Veronica Yu ...... Page 174 Over-expression of Cks proteins causes gene derepression in Saccharomyces cerevisiae
Abstract P125 Rebekah Zinn ...... Page 175 hTERT is expressed in cancer despite promoter DNA methylation by preservation of unmethylated DNA and active chromatin around the transcription start site
Additional poster submissions
Abstract P126 Yoshimitsu Takahashi ...... Page 176 Degree of SUMO modification as a differential tag for targeting to specific chromosomal domains
Abstract P127 Marna S. Costanzo ...... Page 177 The evolutionary conservation of chromatin modifying proteins in malaria
Abstract P128 Philippe Prochasson ...... Page 178 Functional characterization of the HIR corepressor complex
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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 Abstracts – Oral Steve Henikoff Abstract 1 Epigenetic patterns generated by histone replacement
Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109- 1024, U.S.A.
Histone H3 is deposited at replication, but it is replaced at active genes by the constitutive histone variant, H3.3. We have used chromatin affinity purification of biotin-tagged H3.3 to map histone replacement throughout the Drosophila genome. Replacement is especially prominent at active genes, corresponding to sites of abundant RNA polymerase II and methylated H3 lysine-4 throughout the genome. Active genes are depleted of histones at promoters and are enriched in H3.3 from upstream to downstream of transcription units. Histone replacement patterns differ between the dosage compensated X-chromosome and autosomes downstream of gene promoters, suggesting that dosage compensation is achieved by modulating transcriptional elongation. Histone replacement is low overall at the Bithorax Complex, but surprisingly, Polycomb Response Elements are sites of conspicuously high histone turnover, whose peaks precisely correspond to nuclease hypersensitive sites. We also observe high levels of histone turnover at the “poised” promoters of heat shock genes. We propose that the remodeling process responsible for histone replacement patterns at cis-regulatory elements maintains continuous accessibility of DNA to trans-acting factors, providing a simple general mechanism for cellular memory.
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Abstracts - Oral
Yang Shi Abstract 2 The identification of histone demethylases established the dynamic and reversible nature of histone methylation regulation
In this presentation, I will discuss our continued efforts to catalog histone demethylases and to understand their roles in development and human diseases.
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Jesper Christensen Abstract 3 The retinoblastoma tumor suppressor binding protein RBP2 is a transcriptional repressor demethylating tri- and dimethylated lysine 4 on Histone H3
Jesper Christensen1, Karl Agger1, Paul A. C. Cloos1, Diego Passini1, Klaus H. Hansen1 and Kristian Helin1, 2
1Biotech Research & Innovation Centre, Fruebjergvej 3,2100 Copenhagen, Denmark; 2Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
The Retinoblastoma tumor suppressor protein, pRB, is a key regulator of cell-cycle progression, differentiation and senescence, and is often found deregulated in cancer. To repress transcription upon cell-cycle exit induced by differentiation or oncogene-induced senescence, pRB binds directly to members of the E2F transcription factor family and cellular factors, thereby bridging chromatin modifiers to E2F-regulated genes causing chromatin condensation. Here we show that the Jumonji domain containing protein, Retinoblastoma Binding Protein 2 (RBP2), is a transcriptional co-repressor, which modifies chromatin by demethylating tri- and dimethylated lysine 4 on histone 3 (H3K4), a chromatin mark present on active genes. Ectopic expression of RBP2 in human TIG3 fibroblasts induced a senescent-like phenotype with reduced H3K4 methylation. Similarly, ectopic expresion of RBP2 in U2OS cells strongly reduced H3K4 methylation when analyzed by immunofluorescence. Mutation of the Jumonji domain of RBP2 abolished the demethylation activity. Furthermore, purified recombinant RBP2 efficiently demethylated tri- or dimethylated H3K4 in vitro using purified calf thymus histones or HeLa cell nucleosomes as substrate for the enzyme reactions, while other histone methylation marks at H3K9, H3K27, H3K36 and H4K20 were unaffected. Finally, the enzymatic specificity of RBP2 was confirmed by testing tri-, di-, and monomethylated H3K4 peptides as substrate and subsequent mass spectrometry analysis of the reaction products. The biological function of RBP2 is currently not fully elucidated. However, considering the pRB binding and the demethylation activity of RBP2, a role for RBP2 in chromatin demethylation and repression of pRB regulated genes is a possibility and is currently being explored.
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Abstracts - Oral
Mischa Machius Abstract 4 Structural Basis for CoREST-Dependent Demethylation of Nucleosomes by the Human LSD1 Histone Demethylase
Mischa Machius, Maojun Yang, Christian B. Gocke, Xuelian Luo, Dominika Borek, Diana R. Tomchick, Zbyszek Otwinowski and Hongtao Yu
University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, U.S.A.
Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain lysine-specific demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and di- methylated H3-K4 of nucleosomes. We report the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 likely recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoRESTdependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome-binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosome-binding modules.
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Ramin Shiekhattar Abstract 5 Functional and biochemical characterization of histone demethylase complexes
Ramin Shiekhattar
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104
Schizosaccharomyces pombe contains two proteins, SWIRM1 and SWIRM2, with close homology to human histone H3 lysine 4 demethylase. Both proteins contain the amino oxidase catalytic domain and a recently described DNA interaction SWIRM domain. Our results indicate that while SWIRM2 is an essential gene, cells lacking SWIRM1 are viable. We found that SWIRM1 and SWIRM2 are stably associated in a multiprotein complex, but intriguingly, unlike their human counterpart, S. pombe SWIRM complex contains neither a histone deacetylase (HDAC) nor any detectable demethylase activity. Genome-wide chromatin immunoprecipitation unexpectedly showed the absence of both SWIRM proteins from heterochromatic domains. Instead, consistent with biochemical analyses, SWIRM1 and SWIRM2 co-localize to a common set of target gene promoters whose functions are implicated in diverse processes including mitochondrial metabolism and transcriptional regulation. Importantly, we show that SWIRM1 is not only required for optimum transcription of its target genes but also display a global role in regulation of antisense transcription.
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Abstracts - Oral
Yi Zhang Abstract 6 Histone demethylation by the JmjC domain-containing proteins
Yi Zhang
Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A.
Posttranslational histone modifications play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues and participates in diverse range of biological processes including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. While acetylation, phosphorylation, and ubiquitylation are dynamically regulated by enzymes that catalyze the addition and removal of a particular modification, enzymes that are capable of removing methyl groups were not known until recently. Using a novel demethylase assay, we have identified a family of JmjC domain-containing histone demethylases. The mechanism of demethylation and biological significance of these demethylases will be discussed.
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Francis Stewart Abstract 7 Epigenetic aspects of lineage commitment
Glaser, S., Lubitz, S., Anastassiadis, K., Siebler, J., Schwenk, F. and Stewart, A.F.
BioInnovationsZentrum, Technische Universitaet Dresden, Germany; Artemis Pharmaceuticals, Cologne, Germany
In higher eukaryotes, somatic cells differ epigenetically from the pluripotent cells of early development. Consequently it is possible that epigenetic mechanisms play important roles in lineage commitment and cellular differentiation. In mammals, the simplest model suggests that the epiblast is pluripotent because its chromatin is epigenetically naive and lineage commitment restricts pluripotency via the imposition of epigenetic marks. A potential corollary to this model suggests that cellular identity in the adult is maintained, in part, by epigenetic mechanisms. Recent progress has highlighted the importance of three histone lysine methylations in epigenetics. Whereas methylation of histone 3 lysine 9 (H3 K9) and H3 K27 direct inheritable states of gene silencing, methylation of H3 K4 is associated with gene expression. Mammals have multiple enzymes for each of these methylations, including at least six for H3 K4. It is therefore possible that different gene expression programs are regulated by different methyltransferases. To explore these issues, we are studying two of the H3 K4 methyltransferases, Mll and Mll2, in mouse development. These two sister genes have arisen by a gene duplication and are closely related in many ways. However they regulate different genes. Notably Mll regulates the Hoxa complex whereas Mll2 the Hoxb complex. Based on experiments with conditional mutagenesis and studies in utero and in ES cells, we conclude that epigenetic mechanisms are not essential for lineage commitment decisions, rather they contribute to securing and co-ordinating decisions with notable effects on timing and the regulation of apoptosis.
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Abstracts - Oral
Henk Stunnenberg Abstract 8 Title and abstract unavailable
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Ali Shilatifard Abstract 9 H2B monoubiquitination and H3K4 methylation via COMPASS
Ali Shilatifard
Saint Louis University Cancer Center, Saint Louis University School of Medicine Saint Louis, MO 63104
Chromosomal rearrangements and translocations play a major role in the pathogenesis of hematological malignancies. The trithorax related mixed lineage leukemia (MLL) gene located on chromosome 11q23 is rearranged in a variety of aggressive human B and T lymphoid tumors as well as acute myeloid leukemia (AML) in both children and adults. In order to better define the role of MLL in pathogenesis of leukemia, we have been studying the biochemical properties of MLL and MLL-related proteins from several different organisms. We have demonstrated that the MLL homologue in yeast, the Set1 protein, exist in a macromolecular complex we call COMPASS. COMPASS is a histone methyltransferases capable of mono- di and trimethylating the fourth lysine of histone H3. Previously, we demonstrated that the ubiquitin-conjugating enzyme Rad6 and its E3 ligase Bre1 and several other factors are required for COMPASS mediated methylation of H3K4 through regulation of monoubiquitination of H2B at K123. Here, I will discuss our recent findings regarding the molecular mechanism and the role of H2B monoubiquitination in the regulation of H3K4 methylation by COMPASS.
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Paulo Sassone-Corsi Abstract 10 A chromatin remodeling clock
Jun Hirayama, Masao Doi, Saurabh Sahar, Benedetto Grimaldi, David Gauthier, Yasukazu Nakahata and Paolo Sassone-Corsi
Department of Pharmacology, School of Medicine, University of California, Irvine.
Circadian rhythms are the overt consequences of biological clocks, endogenous timers acting within cells. At the molecular level, circadian clocks are constituted by ‘clock genes’, some of which encode proteins able to feedback and inhibit their own transcription Circadian rhythms are regulated by clocks located in specific structures of the central nervous system – such as the suprachiasmatic nucleus (SCN) in mammals – but also by peripheral oscillators present in various other tissues. It is now established that an intrinsic circadian pacemaker functions in virtually each cell. Importantly, about 15% of all genes are expressed in a circadian manner. It is thereby conceivable to invoke large-scale events of chromatin remodeling in order to accommodate these global changes in gene expression. The molecular machinery that governs circadian rhythmicity comprises proteins whose interplay generates time-specific transcription of clock genes. The role of chromatin remodeling in a physiological setting such as the circadian clock has been unclear. We have shown that the protein CLOCK, a central component of the circadian pacemaker, has histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and activation of clock genes in Clock-mutant cells. Identification of CLOCK as a novel type of DNA-binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism and identifies unforeseen links between histone acetylation and cellular physiology.
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Sung Hee Baek Abstract 11 A Novel Link between SUMO Modification of a Chromatin Remodeling Complex and Cancer Metastasis
Jung Hwa Kim1,3, Hyejin Nam1,3, Hee June Choi1, Bogyou Kim1, Ji Min Lee1, Ik Soo Kim1, Keun Il Kim2, and Sung Hee Baek1
1Department of Biological Sciences, Seoul National University, Seoul 151-746, South Korea, 2Department of Biological Sciences, Sookmyung Women's University, Seoul 140-742, South Korea
3These authors contributed equally
Defining the functional modules with transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. We have reported the dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate a metastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumor metastasis, and further which signaling factors confer repressive function on reptin and hence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., Nature Cell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealed the presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptin that underlies its repressor function. DeSUMOylation of reptin alters the repressive function of reptin and its association with HDAC1. Further, SUMOylation status of reptin modulates the invasive activity in cancer cells with metastatic potential. This provides a clear definition of the functional model and a novel insight for linking SUMO modification to cancer metastasis. As a follow-up study, we will address novel findings on the function of newly identified histone methyltransferase as a component of reptin, linking chromatin remodeling process and cancer metastasis.
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Laszlo Tora Abstract 12 The simultaneously dimethylated Lys-9 and phosphorylated Ser-10 tails of histone H3 adopt different conformations during mitosis
Tora L., Eberlin A., Oulad-Abdelghani M., Robert F., Grauffel C., Spehner D., Wurtz J-M., Schultz P. and Dejaegere A.
Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGMBC), UMR 7104 CNRS, ULP, INSERM, INSERM U.596, Parc d’Innovation,1, rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France
Eukaryotic cells possess mechanisms for condensing and decondensing chromatin. Chromatin condensation is particularly evident during mitosis and cell death induced by apoptosis, whereas chromatin decondensation is necessary for replication, repair, recombination and transcription. Histones are among the numerous DNA binding proteins that control the level of DNA condensation, and post-translational modification of histone tails plays a critical role in the dynamic condensation/decondensation that occur at numerous cellular processes. Post-translational modifications, alone or in combination, can direct distinct downstream events. The association of lysine (K) 9 dimethylation (di-Me), a hallmark of the heterochromatin, with serine (S) 10 phosphorylation (P), a marker of mitosis, on the same histone H3 tail, as well as the idea of a structured histone-tail, has long been controversial. Interestingly, by using a specific antibody, we detect a histone H3 tail conformation, which contains simultaneously K9(di-Me) and S10(P) that appears only between the late prophase and the early anaphase steps, being the strongest during metaphase. This H3 tail conformation is different from another state, where the K9(di-Me) S10(P) modifications are also simultaneously recognized, but more widely during mitosis. Furthermore, results obtained by confocal and electron microscopy suggest that the conformation of K9(di-Me) and S10(P) histone H3 tails changes during mitosis and can adopt at least two different conformations. This observation has also been confirmed by biostructural docking and molecular dynamics modelling as well as by competition tests, using various modified peptides. The localisation and the role of these different conformations in gene regulation and mitotic chromosome condensation will be discussed.
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Tony Kouzarides Abstract 13 Characterisation of novel histone modifications
Claire Pike, Chris Nelson, Paul Hurd, Andy Bannister and Tony Kouzarides
The Gurdon Institute, Cambridge University, Tennis Court Road, Cambridge, U.K.
We are investigating the mechanism of action of several novel modifications on histone H3. We have previously identified an enzyme FPR4 in yeast that can isomerise prolines in the tail of H3. A mammalian enzyme that can accomplish similar functions has been identified and is under characterisation. In addition, we have identified a new phosphorylation site on human H3 by mass spectrometry. Specific antibodies raised against this site are now being used to establish the kinase pathways that mediate this phosphorylation event.
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Abstracts - Oral
Thomas Jenuwein Abstract 14 Epigenetic control by histone methylation
Thomas Jenuwein
IMP Vienna, Dr. Bohrgasse 7, Austria
Epigenetic mechanisms, such as histone modifications, control eukaryotic development beyond DNA-stored information. We are analyzing histone lysine methylation in mammalian chromatin to further dissect its role(s) in chromosome organization, gene regulation, genome stability and overall epigenetic control. While there is under-representation of repressive histone marks in quiescent (resting), stem and regenerating cells, there is a selective accumulation of aberrant histone lysine methylation profiles in aging, ‘stressed’ and tumor cells. We have generated mutant mice that lack crucial HMTases, such as the Suv4-20h enzymes. In these Suv4-20h double-null mice, there is a genome-wide transition from H4K20 tri- to H4K20 mono-methylation, which appears to impair stress-induced and programd DNA damage response. In addition, we have screened chemical libraries (in collaboration with Boehringer Ingelheim, Ridgefield U.S.A.) and identified a small molecule inhibitor for the G9a HMTase. This novel compound, BIX-01294, is the first HMTase inhibitor that can be used to transiently modulate H3K9me2 levels in mammalian chromatin. Finally, we have been characterizing jumonjiC-containing proteins that represent hydroxylases with the potential to remove repressive H3K9me3 marks. Together, these approaches promise to yield new insights into the plasticity of cell fate decisions and may offer novel strategies to revert aberrant development.
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Roberta Benetti Abstract 15 The role of Dicer in the regulation of chromatin at telomeres
Roberta Benetti1*, Susana Gonzalo1,4*, Stefan Schoeftner1, Isabel Jaco1, Purificacion Muntildedoz1, Elizabeth Murchison2, Thomas Andl3, Peter Klatt1, Sarah Millar3, Gregory Hannon2 and Maria A. Blasco1
1Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid E-28029, SPAIN; 2Cold Spring Harbor Laboratory, NY 11724, U.S.A.; 3Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104-6100, U.S.A.; 4Radiation and Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, U.S.A.
Dicer has been proposed to have a role in the maintenance of silencing at centromeres in several organisms, including mammals. Here we describe a role for Dicer in regulating mammalian telomeric chromatin. In particular, mouse ES cells and skin keratinocytes conditionally deleted for Dicer show aberrantly elongated telomeres compared to wild-type controls, concomitant with increased telomeric recombination. This occurs in the absence of changes in TRF1 and TRF2 expression and with decreased telomerase activity in Dicer-null cells. The long-telomere phenotype of Dicer-null cells is accompanied by an increased density of histone heterochromatic marks at telomeric chromatin, such as histone 3 lysine 9 (H3K9) and histone 4 lysine 20 (H4K20) tri-methylation, and by decreased histone acetylation, supporting the idea of a silencing of telomeric chromatin in the absence of Dicer. In support of this, we observed a decreased abundance of telomeric RNA transcripts in Dicer-null cells. All together, these results demonstrate an unprecedented role for Dicer in the regulation of mammalian telomeric chromatin. Dicer has been proposed to have a role in the maintenance of silencing at centromeres in several organisms, including mammals. Here we describe a role for Dicer in regulating mammalian telomeric chromatin. In particular, mouse ES cells and skin keratinocytes conditionally deleted for Dicer show aberrantly elongated telomeres compared to wild-type controls, concomitant with increased telomeric recombination. This occurs in the absence of changes in TRF1 and TRF2 expression and with decreased telomerase activity in Dicer-null cells. The long-telomere phenotype of Dicer-null cells is accompanied by an increased density of histone heterochromatic marks at telomeric chromatin, such as histone 3 lysine 9 (H3K9) and histone 4 lysine 20 (H4K20) tri-methylation, and by decreased histone acetylation, supporting the idea of a silencing of telomeric chromatin in the absence of Dicer. In support of this, we observed a decreased abundance of telomeric RNA transcripts in Dicer-null cells. All together, these results demonstrate an unprecedented role for Dicer in the regulation of mammalian telomeric chromatin
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Abstracts - Oral
Mareike Puschendorf Abstract 16 Ezh2 independent targeting of PRC1 proteins to paternal constitutive heterochromatin in mouse pre-implantation embryos
Mareike Puschendorf
Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4057 Basel Switzerland
In mammals, fertilization triggers a cascade of events leading to the formation of a totipotent embryo from two highly specialized gametes. During this process both parental genomes undergo major epigenetic reprogramming, suggesting a potential causal relationship between the two events. Several immunofluorescence studies indicate that chromatin states of maternal and paternal genomes are initially highly asymmetric. Whereas the maternal genome inherits many distinct types of histone lysine methylation, the paternal genome is de novo methylated at different lysine residues in a highly spatially and temporally coordinated manner after the protamine to histone exchange. At the maternal genome, constitutive heterochromatin is labeled by modifications characteristic of the Suv39h pathway (such as H3K9 and H4K20 tri-methylation and binding of HP1b). Importantly, in proliferating somatic cells Suv39h function is required to maintain mitotic genome stability. Surprisingly, paternal constitutive heterochromatin in early embryos is devoid of the canonical Suv39h-dependent chromatin marks. Instead, we observe that various proteins of the Polycomb Repressive Complex 1 (PRC1) are targeted to constitutive heterochromatin of only the paternal genome. By using embryos maternally and zygotically deficient for Ezh2, we demonstrate that the parental-origin-specific labeling is independent of Ezh2 function and H3K27 tri-methylation. PRC1 binding to paternal heterochromatin is stably transmitted over several mitotic divisions suggesting the existence of a memory of parental identity of constitutive heterochromatin in pre-implantation embryos.
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Edith Heard Abstract 17 The nuclear and epigenetic dynamics of X-chromosome inactivation in the mouse
Edith Heard
Mammalian Developmental Epigenetics Group, CNRS UMR 218 - Nuclear Dynamics and Genome Plasticity Curie Institute, 26 rue d’Ulm, 75248 Paris Cedex 05, France
In female mammals, one of the two X chromosomes is converted from the active euchromatic state into inactive heterochromatin during early embryonic development. This process, known as X-chromosome inactivation, results in the transcriptional silencing of over a thousand genes and ensures dosage compensation between the sexes. X inactivation is a dramatic example of mammalian epigenetics, involving differential regulation of two homologous chromosomes within the same nucleus, in a mitotically heritable but developmentally reversible manner. We are interested in the mechanisms and kinetics of this process in early mouse embryos and differentiating embryonic stem (ES) cells. X inactivation is a highly dynamic process during early development (Okamoto et al, 2004) and we are interested in defining the epigenetic marks that underlie its initiation and its maintenance. Given the mono- allelic character of X inactivation, we are also investigating the role of sub-nuclear compart- mentalization in this process, both at the level of the master control locus of X inactivation, the Xic, and the non-coding Xist transcript it produces, that is responsible for inducing transcriptional silencing in cis. In this context, we have recently discovered that the two Xics transiently co-localise just prior to random monoallelic up-regulation of Xist and the onset of X inactivation (Bacher et al, 2006). This co-localisation seems to be important for ensuring that X inactivation is triggered when more than one Xic is present. Recent evidence will be presented for a new region of the Xic that seems to be critical for bringing the two loci together in trans and that is characterized by specific histone modifications.
Refs: Okamoto,I., Otte,A., Allis,C.D., Reinberg,D. and Heard,E. (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science, 303, 644-649 Bacher,C., Guggiari,M., Brors,B., Augui,S., Avner,P., Eils,R. and Heard,E. (2006) Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nature Cell Biology, 8, 293-239.
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Abstracts - Oral
Adrian Bird Abstract 18 MeCP2: molecular interactions and phenotypic stability in a mouse model of Rett Syndrome
Jacky Guy1, Xinsheng Nan1, Jianghui Hou2, Skirmantas Kriaucionis1 and Adrian Bird1
1Wellcome Trust Centre for Cell Biology, The University of Edinburgh, The Kings Buildings, Edinburgh EH9 3JR, U.K., 2Molecular Medicine Centre, The University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, U.K.
Rett Syndrome (RTT) is a profound neurological disorder that almost exclusively affects girls. More than 80% of patients carry a new mutation in one copy of the X-linked MECP2 gene and this is now established as the primary cause of the condition. Overt symptoms show delayed onset in girls between 6 and 18 months of age and include developmental delay, loss of purposeful limb use and breathing abnormalities. As there is no obvious neurodegeneration in post-mortem brains of RTT patients, the question of reversibility arises and is of obvious relevance for therapeutic approaches to RTT. We earlier created a mouse model for RTT that lacks an intact Mecp2 gene and mimicks several features of the disorder including late inset. Using a mouse with an Mecp2 allele that can be conditionally activated, we are asking whether neuronal defects in the young adult can be rectified if MeCP2 is provided after abnormal neuronal morphology and symptoms have arisen. Can switching on MeCP2 in these animals reverse the phenotype, or is it too late? In addition to these physiological studies, we have identified the Swi/Snf motor protein ATRX as an MeCP2 binding partner. Mutations in both MECP2 and ATRX genes cause X-linked mental retardation and we have preliminary evidence for interdependence in the mouse brain.
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Jon Penterman Abstract 19 DNA demethylation in Arabidopsis thaliana
Jon Penterman1, Daniel Zilberman2, Jin Hoe Huh1, Tracy Ballinger2,3, Steven Henikoff2,3, Robert Fischer1
1Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, U.S.A. 2Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109, U.S.A. 3Howard Hughes Medical Institute
Cytosine DNA methylation is an epigenetic modification that functions in a number of processes, one of which is genome defense against transposons and repetitive elements. In Arabidopsis thaliana DNA is methylated by methyltransferases whose specificity is determined by parental methylation patterns, histone modifications, and/or small RNAs. Here we show that methylation at many loci throughout the genome is actively removed by a DNA demethylation pathway. The DEMETER-LIKE (DML) DNA glycosylases, which excise 5-methylcytosine and initiate the base excision DNA repair pathway, mediate this process. Using genome-tiling arrays, we detected nearly two hundred discrete loci that are demethylated in a DML-dependent manner. We find that DML demethylation primarily occurs at the 5’ and 3’ ends of genes, a pattern opposite to the overall distribution of wild- type DNA methylation. Our results show that DML-dependent DNA demethylation is a fundamental pathway that edits the Arabidopsis methylation profile. We believe that DML demethylation provides a protective buffer against the methylation pathway of Arabidopsis, which might indirectly enable Arabidopsis to have a robust defense pathway for repressing transposons and repetitive elements.
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Abstracts - Oral
Francois Fuks Abstract 20 The Polycomb Group protein EZH2 is recruited to promoters by MECP2
Emmanuelle Vire1, Helene Denis1, Esteban Ballestar2, Yvan de Launoit3, Manel Esteller2 and Francois Fuks1
1Free University of Brussels, Faculty of Medicine, Laboratory of Cancer Epigenetics, 808 route de Lennik, 1070 Brussels, Belgium; 2CNIO, Cancer Epigenetics Group, C/ Melchor Fernandez Almagro 3,28029-Madrid, Spain; 3Institut de Biologie de Lille, 1 rue Calmette, 59021 Lille, Cedex, France
Polycomb Group (PcG) proteins and DNA methylation are fundamental epigenetic systems involved in gene silencing. Recently we have uncovered a close connection between these two systems: the PcG protein EZH2 can control DNA methylation (1). Here we show that conversely, CpG methylation can influence EZH2 function through the methyl-CpG-binding protein MECP2. We demonstrate that EZH2 interacts physically with MECP2 in vivo. Chromatin immunoprecipitations indicate that the presence of MECP2 is required for binding of EZH2 to target promoters. Genome-wide location analysis with antibodies against these proteins are under way to explore whether there is a cross-talk between EZH2 and MECP2 across promoters within the genome. Our results suggest that MECP2 may act as a molecular scout for PcG recruitment to chromatin. They could shed light on the poorly understood mechanisms by which mammalian Polycomb Group proteins are targeted to promoters.
Ref. (1) Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F. Nature. 2006 Feb 16:871-4.
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Shelley Berger Abstract 21 Factor and histone covalent modifications in genome regulation
Shelley L. Berger
The Wistar Institute, Philadelphia, PA, 19104, U.S.A.
Genomic structure and function is regulated in part through covalent post-translational modifications (PTMs) of factors and histones, including acetylation (ac), methylation (me), phosphorylation (ph), ubiquitylation (ub), and sumoylation (su). There are an enormous number of factor and histone PTMs. To make sense of this bewildering complexity, we focuses on patterns, temporal sequences, and cross-talk between PTMs in the yeast S. cerevisiae and mammalian cells. In mammals we study PTMs of DNA-bound transcription factors, using the tumor suppressor and transcription factor p53 as a model. We currently focus on methylation by the SET domain methyltransferase Smyd2. Smyd2 methylates p53 at K370, adjacent to the previously identified methylation site at K372. K372me is associated with transcriptional activation by p53, and inhibits K370me, which in contrast, is associated with transcriptional repression by p53. Thus, there is regulatory cross-talk between activating and repressing lysine methylation. Our most recent results indicate that demethylation occurs within p53. A methylation/demethylation pathway will be discussed in detail.
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Abstracts - Oral
Danny Reinberg Abstract 22 A molecular understanding of epigenetics
Danny Reinberg
NYU-School of Medicine, NY, U.S.A.
Epigenetics encompasses changes in gene expression profiles that occur without alterations in the genomic DNA sequence of a cell. This arises from the dynamic processes that structure regions of chromosomal DNA through a range of compaction in eukaryotes. The altered pattern of gene expression is pivotal to cellular differentiation and development and is inherited by daughter cells thereby maintaining the integrity, specifications, and functions for a given cell type. Aberrancies in this epigenetic process give rise to perturbations that are also inherited and disruptive to normal cellular properties. The histone proteins that package DNA into chromatin are subject to post-translational modifications generating different chromatin structures. While euchromatin has a relaxed structure permissive to transcription, constitutive heterochromatin is densely packed and inaccessible to transcription factors. On the other hand, facultative heterochromatin is repressive, but can be altered in its properties to become active. Our goals are to identify the molecular mechanisms controlling the formation of facultative heterochromatin and the epigenetic parameters that ensure its propagation through cell divisions.
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Jessica Tyler Abstract 23 The mechanistic basis for the requirement of promoter chromatin disassembly for transcriptional activation
Jessica K Tyler1, Stephanie Williams1, Melissa Adkins1, Christine English1 and Mair Churchill2
1 Department of Biochemistry and Molecular Genetics, 2Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, Aurora CO 80045
Nucleosomes appear to be disassembled from the promoters of transcriptionally active genes in Eukaryotic species spanning from yeast to humans. We have previously shown that this promoter chromatin disassembly is essential for transcriptional activation of the budding yeast PHO5 and PHO8 genes. Promoter chromatin disassembly is mediated by the highly conserved histone H3/H4 chaperone Anti-silencing function 1 (Asf1) during activation of the PHO5 and PHO8 genes upon phosphate depletion and during activation of the ADY2 and ADH2 genes upon glucose removal. Using PHO5 as our model system to learn how promoter chromatin disassembly activates gene expression, we have discovered that Asf1-mediated promoter chromatin disassembly is required for recruitment of TBP and RNA polymerase II, but not for recruitment of the Pho4 and Pho2 activators. Furthermore, accumulation of SWI/SNF and SAGA at PHO5 required promoter chromatin disassembly. We have also uncovered a novel requirement for SWI/SNF and SAGA in chromatin disassembly to facilitate activator recruitment to the nucleosome-buried binding site in the PHO5 promoter that is distinct from the stable recruitment of SWI/SNF and SAGA after chromatin disassembly. Towards addressing the mechanism whereby Asf1 mediates chromatin disassembly, we have solved the crystal structure of Asf1 in complex with histones H3/H4 to 1.7 Angstrom resolution; this is the first time histone proteins have been “seen” outside of the nucleosome or octamer structures (see Mair Churchill’s abstract for more details). Using mutants designed from the Asf1-H3/H4 structure, we show that Asf1 needs to bind to the H3:H3 dimerization surface of the H3/H4 heterodimer in order to achieve chromatin disassembly from the PHO5 promoter. Furthermore, binding of Asf1 to the C-terminal beta strand of histone H4, which in itself induces an 180˚ flipping-out of this region of H4 as compared to the nucleosome, is also required for chromatin disassembly by Asf1. From these results we propose a “strand capture” model for chromatin disassembly.
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Gratien Prefontaine Abstract 24 Epigenetic mechanisms influencing pituitary gene expression.
Prefontaine G.G., Lunyak, V. and Rosenfeld M.G.
Department of Medicine, University of California, San Diego, 9500 Gilman Drive, CMM- West #345, La Jolla, CA, 92093-0648
During development, tissue-specific transcription factors direct covalent modifications of chromatin, laying down the foundation for regulated gene transcription. These factors act through cis-acting elements located in promoter proximal and distal elements. The pituitary gland provides an excellent model system for studying epigenetic changes in gene regulation. Common pluripotent primordial ectodermal cells differentiate to produce a pituitary gland composed of 5 major cell types characterized by types of hormone they produce and secrete, including: lactotropes, somatotropes, thyrotropes, corticotropes and gonadotropes. I have performed a comprehensive analysis of the CpG DNA methylation status of the growth hormone (GH) promoter in mouse pituitary. Early in development the embyonic murine GH promoter was largely CpG methylated. Later postnatally, the GH promoter was demethylated in a subset of cell types. I have linked the DNA demethylation of the promoter, genetically and spatially with the occupancy of a cell-type specific factor. Furthermore, using a combination of techniques, I show the CpG methylation status of the promoter associated with specific histone modifications. These results have allowed us to propose a model where the GH gene is differentially repressed by polycomb (long term silencing) and HP1 (short term repression) proteins in distinct pituitary cell types. Using BAC recombination to substitute a fluorescent gene into the GH gene locus and to delete upstream distally located regulatory elements, I have created multiple transgenic mice that demonstrate the importance of a GH enhancer or locus control region and a repetitive element that are critical for proper regulation of the GH locus.
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Bob Kingston Abstract 25 Possible roles in silencing for piRNAs
Bob Kingston, Anita Seto, Nelson Lau, Jinkuk Kim, David Bartel, Jonathan Dennis and Caroline Woo
Massachusetts General Hospital, 185 Cambridge Street, CPZN7250, Boston MA 02114, U.S.A.
Maintaining a silent state requires the targeting of epigenetic regulatory complexes to specific genes. Small noncoding RNAs have been proposed to play a role in this targeting, based on studies in numerous model organisms. Because these RNAs appeared to be enriched meiotic cells in model organsims, we made extracts from Rat testes to try to identify RNAs that might be involved in silencing in mammals. From these extracts, we purified a mammalian complex that might function in transcriptional gene silencing (TGS). This complex, called piRC, contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29–30 nt in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 small (<100 kb) genomic loci. Within these loci, the piRNAs distribute across only one genomic strand, or distribute on two genomic strands but in a divergent, non-overlapping manner. Preparations of piRC contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Recombinant RecQ1 and Piwi family proteins appear to interact directly. Piwi has been genetically linked to TGS in flies, and the purified complex has piRNA-directed slicer activity. These results are consistent with a gene silencing role for piRC in mammals.
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Abstracts - Oral
Michael Grunstein Abstract 26 Deacetylation of histone H4 K16 regulates gene activity in yeast
Wei Xie, Amy Wang and Michael Grunstein
Department of Biological Chemistry and the Molecular Biology Institute, Boyer Hall, UCLA, Los Angeles, CA. 90095, U.S.A.
We have found that of all the H4 sites of acetylation, K16 when deacetylated is uniquely involved in the silencing of heterochromatin through its interaction with the silencing protein Sir3. This site is also uniquely involved in gene activation in euchromatin. Surprisingly, while histone hyperacetylation is generally correlated with gene activity, it is the hypoacetylation of K16 that is associated with gene activity genome wide. This occurs in part through its recruitment of the bromodomain containing transcription factor, Bdf1. We describe here the enzymes which deacetylate and acetylate K16 and their recruitment during gene activity to dynamically regulate transcription.
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Ann Ehrenhofer-Murray Abstract 27 A role for the HDAC Rpd3 in establishing eurchromatin- heterochromatin boundaries at yeast telomeres
Stefan Ehrentraut and Ann E. Ehrenhofer-Murray
Universität Duisburg-Essen, 45117 Essen, Germany
Eukaryotic genomes are organized into euchromatic and heterochromatic regions. Cells need to ensure that the respective chromatin states are restricted to their location in order to prevent inappropriate gene expression. In Saccharomyces cerevisiae, spreading of the telomeric Sir2/ Sir3/ Sir4 heterochromatin complex is prevented by the activity of the HAT complex SAS-I, which acetylates lysine 16 of histone H4, a residue that is required in the deacetylated state for the SIR complex to bind to chromatin. In sas2-delete(D) cells, SIR spreads to more centromere-proximal positions and causes repression of subtelomeric genes. However, the SAS2 deletion is not lethal in yeast. Here, we performed a genetic screen to identify factors that become lethal in the absence of Sas2. Surprisingly, we found that the absence of the HDAC Rpd3 was synthetically lethal in combination with sas2D. The lethality was specific for rpd3D and sas2D in that no other HDAC deletion was lethal with sas2D, and no other HAT deletion was lethal with rpd3D. Furthermore, the lethality depended on the components of the Rpd3(L) complex and on all SAS-I components. Our observations suggest parallel functions of the two protein complexes despite their opposing enzymatic activities. Significantly, we found that the lethality of sas2D rpd3D cells was caused by inappropriate spreading of SIR complexes, because the lethality was suppressed by sir2/3/4D. In line with this, we found by ChIP analysis that Sir2 was more abundant at telomeres and in subtelomeric regions in rpd3D cells than in wild-type. Furthermore, subtelomeric genes were more repressed in rpd3D cells than in wild-type as measured by qRT-PCR, and the repression was abrogated in the absence of SIRs. Altogether, our data show a novel and unexpected role for Rpd3 in preventing spreading of SIR complexes into euchromatic regions, indicating that Rpd3 exerted a boundary function at yeast telomeres.
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Wyatt Yue Abstract 28 CARM1 and Histone Methylation - a Structural Study
W. W. Yu e 1, V. Thompson Vale1, M. Hassler1, S. Kisakye-Nambozo1, M. Roe1, T. Kouzarides2 and L.H. Pearl1
1Section of Structural Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK; 2Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, U.K.
Covalent histone modifications have important roles in transcriptional regulation by effecting changes in the chromatin structure. Coactivator-associated arginine methyltransferase 1 (CARM1) methylates histone H3 at Arg17 and Arg26 upon hormonal activation of nuclear receptors. It enhances transcriptional activation through synergistic interactions with coactivators such as GRIP-1 and the histone acetyltransferase CBP. CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic domain flanked by unique N- and C-terminal extensions. We have determined the 2.8 Å structures of the CARM1 catalytic domain alone, and in complex with the cofactor product AdoHcy. The catalytic domain consists of a cofactor binding region and a nine-stranded ß-barrel. The binding of AdoHcy allows the ordering of the N-terminal helix α1, which forms extensive contacts with AdoHcy. This almost completely buries the cofactor and limits the accessibility of the arginine substrate to a narrow channel. Helix α1 also forms the upper ridge of a proposed substrate binding groove lining the entrance to the active site. The bottom ridge of this groove is formed by the first 10 residues of the CARM1 C-terminal extension, which is not present in other PRMT structures. Using in vitro methylation and pull-down assays, we have demonstrated that the N- and C- terminal extensions are important in contributing to the enzyme activity of CARM1. In addition, CARM1 activity towards histone H3 Arg17 is potentiated by pre-acetylation at Lys18, suggesting a possible cross-talk mechanism between histone acetylation and methylation. We are in the process of determining the structure of a CARM1-cofactor- substrate complex, in order to establish the molecular basis for the substrate specificity of CARM1 towards histone H3, which is unique among PRMTs.
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Sharon Dent Abstract 29 Common and unique factors regulate Set1-mediated methylation of the Dam1 kinetochore protein and histone H3
Sharon Y.R. Dent, John A. Latham and Ke Zhang
Univ. Texas M.D. Anderson Cancer Center, Dept. Biochemistry and Molecular Biol., Unit 1000, 1515 Holcombe Blvd, Houston, Texas 77030, U.S.A.
We reported last year that deletion of the SET1 methyltransferase gene suppresses defects in chromosome segregation caused by mutations in the IPL1 aurora kinase in yeast (Zhang et al. Cell 122, 2005). Mutations in other components of the COMPASS complex also suppress the ipl1-2 mutation, but mutations in PAF1 or H2B K123 do not. These results indicate that Set1 and the COMPASS complex normally oppose functions of Ipl1, but that these effects are independent of the functions of Set1 in transcription initiation and elongation that are mediated by Paf1 and H2B ubiquitylation. Moreover, we determined ipl1-2 suppression is not related to loss of H3 K4 methylation in set1 mutant cells. Rather, Set1 is required for methylation of a kinetochore protein, Dam1, and Dam1 methylation at K233 limits phosphorylation of neighboring serines. We have now defined the effects of mutations in individual COMPASS components on Dam1 methylation in vivo. We have also begun to define upstream factors that are required for methylation of both H3 K4 and Dam1, and as well as unique factors required for methylation of each of these substrates. Our findings demonstrate that Set1 has important functions in mitosis, and they suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function.
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Abstracts - Oral
Geneviève Almouzni Abstract 30 Chromatin assembly factors, histone H3 variants and cell cycle
Dominique Ray-Gallet, Sophie Polo, Jean-Pierre Quivy, Anja Groth, Danièle Roche and Geneviève Almouzni
UMR 218 CNRS, Institut Curie – Recherche, 26 rue d’Ulm, F-75248 Paris cedex 05, France
The ordered assembly of chromatin produces a nucleoprotein template capable of regulating the expression and maintenance of the genome functions. Factors have been isolated from cell extracts that stimulate early steps in chromatin assembly in vitro. One such factor, chromatin assembly factor-1 (CAF-1), facilitates nucleosome formation coupled to DNA synthesis. It is thought to participate in a marking system at the crossroads of DNA replication and repair to monitor genome integrity and to define particular epigenetic states. We have begun to approach its critical importance during early development in Xenopus laevis and using mammalian cell systems. In addition, we have now identified a chromatin assembly pathway independent of DNA synthesis. The HIRA protein appears critical for this pathway in Xenopus egg extracts. Notably, CAF-1 was part of the the histone H3 complex, H3.1 complex (replicative form) and HIRA of the H3.3 complex (replacement form) (Tagami et al, 2004, Nakatani et al, 2004). A major goal in our laboratory is now to better integrate the function of these factors in vivo during development and also in connection with replication, repair and control of histone pools. We will discuss our recent findings on this topic and the interrelationships with other assembly factors.
Refs. Groth A., Ray-Gallet D., Quivy J.P., Lukas J., Bartek J. & Almouzni G. (2005) Human Asf1 regulates the flow of S-phase histones during replicational stress. Mol. Cell, 17, 301-311. Polo S. & Almouzni G. (2006) Chromatin assembly : a basic recipe with various flavors. Current Opinion in Genetics and Development, 16, 104-111. Gérard A., Koundrioukoff S., Ramillon V., Sergère J.C., Mailand N., Quivy J.P. & Almouzni G. (2006) The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of Chromatin Assembly Factor 1 with proliferating cell nuclear antigen. EMBO Reports , 7, 817-823. Polo S., Roche D. & Almouzni G. (2006) Evidence for new histone incorporation marking sites of UV-repair in human cells. Cell (in press). Loyola A., Bonaldi T., Roche D., Imhof A. & Almouzni G. (2006) Modifications on histone H3 variants before chromatin assembly potentiate their final epigenetic state. Mol. Cell (in press).
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Dmitry Fyodorov Abstract 31 ATP-dependent deposition of Histone H3.3 by Drosophila CHD1 in vivo
Alexander Konev1, Martin Tribus2, Alexandra Lusser2 and Dmitry Fyodorov1
1Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A.; 2Innsbruck Medical University, Innsbruck, A-6020, Austria.
The assembly of nucleosomes in vitro is mediated by the concerted action of two groups of factors – histone chaperones, such as CAF-1, NAP-1, Asf1 and HIRA, and ATP-utilizing enzymes, such as ACF/CHRAC, RSF and CHD1. However, the respective roles of these factors in the chromatin assembly process in vivo remain controversial. For instance, it is possible that ATP-dependent factors are dispensable for the histone deposition and participate only in the spacing of the nascent nucleosomes. Drosophila CHD1, in conjunction with the histone chaperone NAP-1, can mediate the assembly of periodic arrays of nucleosomes in vitro. To elucidate the biological function of CHD1 we generated mutant alleles of Chd1. Homozygous null Chd1 female flies survive to adulthood and lay fertilized eggs, but the embryos die before hatching. We have analyzed the chromosome structure in developing Chd1 embryos and found that the absence of CHD1 impedes decondensation of paternal sperm chromatin and consequently results in the development of haploid embryos. Sperm decondensation is the earliest developmental instance of genome-scale chromatin assembly. The male pronucleus undergoes profound chromatin reorganization, as paternal protamines are replaced with maternal histones. This process occurs in a replication- independent manner and involves H3.3 variant but not the canonical H3. We found that in the absence of maternal CHD1, H3.3 fails to become incorporated into male chromatin. Thus, we demonstrate that CHD1 directly mediates the loading of H3.3 onto DNA in vivo. The function of CHD1 in histone H3.3 deposition, combined with the recent finding that the delivery of H3.3 to the male pronucleus is dependent on the chaperone HIRA, support a molecular model in which CHD1 utilizes HIRA-delivered histones to assemble H3.3- containing nucleosomes. The combined action of CHD1 and HIRA defines a novel pathway for the replication-independent deposition of variant histones into chromatin. The nucleosome assembly in the male pronucleus takes place in the transcriptionally silent phase of Drosophila embryonic development. However, both H3.3 and CHD1 are expected to function in transcription-coupled nucleosome assembly. Therefore, we analyzed H3.3 incorporation into chromatin during later developmental stages, after the onset of zygotic transcription. We discovered that elimination of maternal CHD1 blocks the assembly of H3.3-containing nucleosomes in transcriptionally active chromatin. Our work provides the first conclusive evidence that ATP-dependent mechanisms are utilized for histone deposition during chromatin assembly in vivo. Hence, molecular motor proteins, such as CHD1, function not only in remodeling of existing nucleosomes but also in de novo nucleosome assembly from DNA and histones.
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Abstracts - Oral
Roberto Mantovani Abstract 32 The histone-fold trimer NF-Y is required to define positive histone marks in CCAAT-promoters: a genome-wide analysis.
Michele Ceribelli, Giacomo Donati, Diletta Dolfini, Giulio Pavesi, Alesaandra Viganò and Roberto Mantovani
Dipartimento di Scienze Biomolecolari, U. di Milano, Via Celoria 26 20133 Milano, Italy
The CCAAT box is a promoter element, bound by the NF-Y trimer, composed of an H2A- H2B-like dimer and a sequence-specific subunit, NF-YA. To gain an unbiased view of NF-Y binding in vivo, we performed ChIP on chips with anti-NF-YB antibodies on 3 platforms. (i) CpG islands arrays identified 300 genes targeted by NF-Y. Surprisingly, 41% of NF-Y sites are not in promoters, but in introns or at distant 3’ or 5’ loci (1). (ii) A more sensitive oligo- based chip containing 179 human promoters indicated that NF-Y binds to 40/54% of promoters, essentially all containing one -or more- CCAAT boxes (2). (iii) We performed location analysis on the Nimblegen tiling arrays of chromosomes 20/21/22. This method proved to be highly specific, as none of 24 negative locations scored positive, but missed some 30% of sites. Consistent with the CpG array, only a minority of the sites -15/20%- are in promoters. A positive correlation with active histone –H3-Acetylation and H3-K4- trimethylation- was established in parallel ChIP on Chip experiments on the same platform. ChIP analysis of cells infected with a dominant negative NF-YA expressing Adenovirus showed a remarkable decrease in NF-YB local promoter binding and in the above mentioned histone modifications, as well as H3-K79-dimethylation. Similar results were obtained transfecting cells with siRNA for NF-YB. Consequently, elimination of NF-Y binding leads to transcriptional impairment. These data establish NF-Y as a crucial factor in the formation of a positive epigenetic environment around the transcription start sites.
Refs 1) Testa et al. J. Biol Chem. 280, 13606-13615 (2005). 2) Cerebelli et al. Cell Cycle, In press (2006).
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Jerry Workman Abstract 33 Histone modification and chromatin remodeling in transcription
Bing Li, Kenneth Lee, Mark Chandy, Michael Carey and Jerry Workman
Stowers Institute for Medical Research 1000 East 50th Street Kansas City, MO 64110 U.S.A.
Nucleosome remodeling and histone modification play crucial roles in the process of gene transcription. Sequence-specific DNA binding transcription activators recruit the SAGA histone acetyltransferase complex to gene promoters during activation. Targeted acetylation of promoter nucleosomes by the SAGA complex marks them for subsequent displacement by the Swi/Snf nucleoosome remodeling complex. This generates nucleosome free regions where the general transcription factors and RNA polymerase II can form a preinitiation complex. Following initiation the elongating RNA polymerase associates with acetyltransferases that co-transcriptionally acetylate nucleosomes in the coding region. This acetylation is recognize by the bromo-domain containing RSC complex which remodels nucleosomes to assist polymerase passage. The Set2 histone methyltransferase also travels with the polymerase and co-transcriptionally methylates histone H3. This methylation is subsequently recognized by the chromodomain containing Rpd3S histone deacetylase complex which removes the co-transcriptional histone acetylation marks returning the stability of nucleosomes within the coding region.
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Abstracts - Poster Abstracts – Poster Karl Agger Abstract P1 The role of the polycomb group protein RYBP in oncogene induced senescence
Karl Agger1, Paul Cloos1, Michael Lees1 and Kristian Helin1,2
1Biotech Research & Innovation Centre, Fruebjergvej 3 Copenhagen, Denmark; 2Faculaty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark
Several links between deregulated chromatin modifying activities and cancer have been described. In our laboratory special attention has been applied to the role of PcG genes in the regulation of cell cycle progression and cancer because of their well- established oncogenic potential. In an attempt to identify additional proteins involved in PcG related chromatin modification we performed multiple Yeast two hybrid (YTH) screens using PcG genes as baits. From the results of these screens we constructed a protein interaction map of the human Polycomb group proteins. We chose to focus on one of the identified interactions between the PcG gene RYBP and the HMT SUV4-20H1. This interaction is interesting because it links a new repressive enzymatic activity to the PcG protein family. To further characterise the interaction we confirmed the binding between the two proteins by co-immunoprecipitation. We found that RYBP and SUV4-20H1 over-expression induced heterochromatin foci in U2OS cells. These foci resemble Senescence associated heterochromatin foci (SAHF). SAHFs are dapi dense heterochromatin foci that appears in cells undergoing senescence, a proliferative arrest that provides a barrier to malignant transformation and contributes to the antitumor activity of certain chemotherapies. We found that both RYBP and SUV4-20H1 can induce senescence in diploid fibroblasts upon over- expression. Additionally we found that ectopic RYBP activity can induce SAHF-like structures and the protein RYBP co-localise with these. Hence, RYBP could be directly involved in the formation of SAHF at the chromatin level and additionally be involved in senescence induction.
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Helena Ahlfors Abstract P2 A novel player in T helper cell differentiation
Helena Ahlfors1, Soile Tuomela1,2, Tiina Henttinen1, Riikka Lund1 and Riitta Lahesmaa1
1Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland, 2Turku Graduate School for Biomedical Sciences, Turku, Finland
Naïve CD4+ T helper lymphocytes can differentiate into two distinct subsets, termed as Th1 and Th2, according their cytokine expression profiles. These subsets of T helper cells are responsible for specific immune functions; Th1 cells contribute to cell-mediated inflammatory immunity, while Th2 cells are responsible for humoral responses. Defects in the T helper cell differentiation may result in the pathogenesis of various immune mediated diseases such as asthma and allergies. We have carried out genome wide gene expression profiling during various stages of Th cell polarization to Th1 or Th2 cells to identify potential new players involved in the process. In this study we have focused on one Th2-specific transcription factor (acronym TF2). The differential expression of TF2 observed with microarray experiments was confirmed both at mRNA and protein level during the early Th1 and Th2 cell polarization. TF2 is clearly induced by interleukin-4 at early time points both on mRNA and protein level, and its expression remains at increased level throughout the early differentiation process. As STAT6 plays a key role in Th2 cell differentiation, we investigated whether downregulation of STAT6 would regulate the expression of TF2. We transfected primary human Th cells with plasmid-based constructs of STAT6 siRNAs contaning a marker for cell selection and cultured the cells in polarizing conditions. Here we show that downregulation of STAT6 by siRNA downregulates the expression of TF2. Characterization of putative STAT6 binding sites in the promoter region of TF2 is in progress. In addition, we designed and optimized vector-based siRNAs to downregulate the expression of TF2. These constructs were transfected to primary human Th cells to elucidate the dose- dependent effects of TF2 in T helper cell differentiation, growth, proliferation and survival. After 24 and 48 hours and 7 days of polarization the cytokine production of the transfected cells was analyzed. Interestingly, the knockdown of TF2 changed the cytokine production profiles of cells polarized to Th1 and Th2 directions. Samples from the early time points were hybridized into Illumina beadarrays for genome wide detection of the genes regulated by TF2. Studies in progress aim at further characterization of the role of TF2 in Th cell differentiation.
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Abstracts - Poster
Barbara Alberter Abstract P3 Histone modification pattern of the T lymphotropic Herpesvirus saimiri genome in latency
Barbara Alberter and Armin Ensser
Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen- Nürnberg, D-91054 Erlangen, Germany
Herpesvirus saimiri (HVS) is the prototypic γ2-herpesvirus. Its circular dsDNA genome (155 kb) consists of an AT-rich coding region harbouring at least 77 open reading frames (orf) and a non-coding region which is made up of tandem repetitive elements with high GC content. After infection HVS establishes latency in its natural host, the new world primate Saimiri sciureus. Experimentally infected and thereby growth transformed human T cells also retain latent HVS genomes. Here, only the viral orf1 (bicistronic transcript, StpC and Tip) and orf73 (LANA homolog) are transcribed. StpC and Tip are essential for the transformation of T cells to antigen-independent growth and the orf73/LANA supports the episomal maintenance of the viral genome in those cells. It is not known how gene expression of latent genes on the one hand and repression of the major group of lytic genes on the other hand are epigenetically regulated in γ2-herpesviruses. In this study, we performed chromatin immunoprecipitation with seven different acetylation or methylation specific antibodies followed by quantitative SYBR Green PCR to profile the histone modifications in the herpesviral genome. Four studied promoters of lytic genes carry repressive marks, whereas the orf73 promoter revealed a variable modification pattern. As expected, the promoter of orf1 was found to be wrapped up in permissive chromatin, but to our surprise the most permissive chromatin structure was revealed in the non-coding repetitive elements at the ends of the genome.
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Marco Alvarez Abstract P4 Histone variant macroH2A is an epigenetic factor involved in the modulation of ribosomal gene expression during seasonal adaptation of carp fish
Pinto R., Bouvet P. 1, Dimitrov S. 2, Molina A., Vera M.I. and Alvarez M.
Departamento de Ciencias Biologicas, Universidad Andres Bello and Millennium Institute for Fundamental and Applied Biology, Santiago, Chile, 1Ecole Normale Superieure, Lyon, France, 2Institut Albert Bonniot, Grenoble, France
Cyprinus carpio acclimatization is a process mediated through molecular mechanisms that coordinate a homeostatic state in response to cyclic environmental changes. Consequently, molecular and cellular functions are reprogramd during seasonal adaptation of the fish. This “phenotypic plasticity” is the result of fine and coordinated regulation of gene expression. MacroH2A is a histone variant associated with epigenetic mechanisms of gene silencing. Previously, we have reported that high levels of macroH2A correlate with hypermethylation of the carp rDNA gene promoter during winter, concomitant with a lower transcription level of ribosomal genes. Altogether these observations seem to suggest that histone variant macroH2A could be involved into the seasonal regulation mechanisms of the carp ribosomal biogenesis. In the present work, by means of chromatin immunoprecipitation (CHIP), we demonstrated that macroH2A is present in the rDNA cistron during the cold season. Furthermore, real- time PCR analyses of these experiments confirmed that macroH2A is mainly enriched in the promoter region of the rDNA compared to summer season. Moreover, we tested the nuclease accessibility of carp rDNA promoter and here we show that the enzyme accessibility decrease during winter suggesting a more compact state of the chromatin. In conclusion, we postulate that an epigenetic mechanism like histone replacement by its variants, particularly the replacement of H2A by macroH2A, plays a central role in the regulation of ribosomal genes expression in carp fish.
FONDECYT 1040197; DI-UNAB 37-04
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Abstracts - Poster
Terra G. Arnason Abstract P5 Rsp5 is required for nuclear shuttling of the Snf1 kinase complex in yeast
Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A. Harkness
Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada
Chromatin assembly in yeast is regulated by a complex molecular network governed in part by the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) and the SCF. We have shown that Rsp5p, localized exclusively to the plasma membrane and adjacent to vacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors, such as the SCF. The APC then initiates replication-independent, but CAF-I-dependent, chromatin assembly. Here, we demonstrate another mechanism leading to Rsp5p-dependent APC activity. Mutation to RSP5 leads to increased histone H3 phosphorylation and decreased histone H3 acetylation at elevated temperatures. We show that the histone H3 kinase, Snf1p, is required for the rsp5 phenotype. Interestingly, we previously demonstrated that the Snf1 kinase complex, which shuttles across the nuclear membrane, is required for APC activity. Thus, we propose that Rsp5p is required for the transit of Snf1p across the nuclear membrane. In support of this theory, we show that GFP-tagged Snf1p, Snf4p (activator subunit) and Gal83p (localizing subunit) all fail to localize to the nucleus upon carbon stress in rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p, failed to exit the nucleus in rsp5 mutants. We next asked whether Snf4p, which requires ubiquitination for stability and function, requires Rsp5p or any of the Rsp5p associated E2 enzymes. In ubiquitin coimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4p bound to ubiquitin, but not GST alone. We observed that carbon stress induced an increase in ubiquitinated GST- Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4p ubc5p cells, GST-Snf4p was again recovered, but we failed to observe induction of ubiquitinated GST-Snf4p upon carbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5p interacting protein, Rod1p, and iii) Ubc7p, an E2 that physically interacts with Rsp5p, on Snf4p ubiquitination will be discussed.
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Stuart P. Atkinson Abstract P6 Epigenetic mechanisms of pluripotency and differentiation
Stuart P. Atkinson and Anna Golebiewska
Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ. U.K.
A great deal of recent research has concentrated on the epigenetic basis of pluripotency of human and mouse embryonic stem cells (ESCs) and studies have also suggested that the differences between various cell types may be due to differences in global epigenetic profiles. Understanding the epigenetic differences between pluripotent cell, such as human ESCs, and differentiated cell lines may allow further understanding of the role epigenetics plays in development. Human embryonal carcinoma (EC) cells are first utilised as a model. The chromatin environment of promoters of genes involved in pluripotency, self renewal and early stages of differentiation were studied in great detail in undifferentiated EC cells as well as after ATRA-mediated differentiation. An active chromatin configuration, such as high levels of H3K4 methylation and histone acetylation, was observed in EC cells at the promoters of genes involved in maintenance of pluripotency and self-renewal (e.g. Sox2, Oct4, and Nanog). A similar pattern was observed at the promoters of genes involved in early stages of differentiation (e.g. Gata2, Pax6) with additional H3K27 methylation suggesting that such genes were primed for expression, but still kept in repressed state in pluripotent EC cells. During differentiation, such promoter configurations become more active linking this to expression of such genes in differentiated ECs, whereas more repressed pattern was observed at promoters of genes involved in pluripotency and self- renewal (e.g. presence of H3K9me2). These studies and the growing knowledge of the epigenetic state of hES cells could potentially be used to devise reprogramming strategies to induce ‘stem-like’ phenotypes in differentiated cells through the modulation of epigenetic mechanisms. In the future these studies may facilitate the production of isogenic tissues for regenerative therapy without the ethical and logistical problems associated with therapeutic cloning in humans.
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Abstracts - Poster
Joanne L. Attema Abstract P7 Epigenetic features of hematopoietic stem cells using small numbers of highly purified primary cells
Joanne L. Attema1, Peter Papathanasiou1, E. Camilla Forsberg1, Jian Xu2, Stephen T. Smale2 and Irving L. Weissman1
1Institute of Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, U.S.A; 2Howard Hughes Medical Institute, Molecular Biology Institute and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California, U.S.A.
Hematopoietic stem cells (HSC) are endowed with the ability to produce all blood cell lineages through their capacity to self-renew and differentiate to descendent progenitors with restricted potential. Recent studies have shown that HSC express many different lineage-affiliated genes at low levels, leading to the hypothesis that epigenetic marks may exist at these loci for their critical expression during differentiation. We investigated this by examining histone and DNA modifications at lineage-affiliated genes in prospectively purified hematopoietic stem and progenitor cells. Here, we describe a method that allows for the analysis of DNA and associated histones from as few as 50,000 primary cells. We found that histone modifications and unmethylated CpG dinucleotides co-localize across defined regulatory regions of key lineage-affiliated genes in HSC that were either maintained or lost in the committed progenitors consistent with their expression. These data support a model in which epigenetic histone modifications are present at lineage-affiliated genes in HSC and could serve as an epigenetic-based mechanism that underlies multipotency.
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Kristin Baetz Abstract P8 NuA4 is a cellular “Hub”: an integrative map of physical and genetic interactions mediated by the NuA4 histone acetyltransferase
Leslie Mitchell, Wendan Chen, Maria Gerdes, Jean-Philippe Lambert, Daniel Figeys and Kristin Baetz
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
NuA4 is the only essential histone acetyltransferase in the budding yeast Saccharomyces cerevisiae and has established roles in transcription, DNA repair and faithful chromosome segregation. NuA4 acetylates histone H4, H2A and the histone variant Htz1, however the exact molecular mechanisms by which NuA4 mediates its cellular processes are poorly understood. Nor is it known if NuA4 has additional cellular roles or acetylation targets. To better understand the cellular functions of NuA4 we have exploited the biochemical and genetic amenabilities of the seven non-essential subunits of NuA4 – Eaf1, Eaf3, Eaf5, Eaf6, Eaf7, Yaf9 and Yng2. Physical and genetic interactions centered on the non-essential subunits of NuA4 were mapped at high resolution using systematic proteomic and genomic methods. Physical interactions were identified using large scale affinity purification of NuA4 complex in all seven non-essential NuA4 mutant backgrounds to establish the contribution of each subunit to NuA4 complex integrity and their role in mediating interactions with non- NuA4 proteins. Genetic interactions for each of the non-essential NuA4 subunits were uncovered using genome-wide synthetic genetic array technology. An extended network, consisting of more then 300 genetic and physical interactions, was found to connect NuA4 to a wide range of cellular functions and has identified several novel cellular roles for NuA4, including golgi-vacuole protein transport. The NuA4 network is helping to define roles for each of the non-essential subunits of NuA4 and we determined that there is a direct correlation between a subunit’s contribution to NuA4 complex integrity and the extent of genetic interactions identified. For example unlike most of the non-essential subunits, Eaf1 is crucial to NuA4 complex stability and displays greater than100 genetic interactions suggesting that Eaf1 may be a scaffold protein for the NuA4 complex. Our extensive network indicates that NuA4 is a “hub” of fundamental importance in the cell.
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Abstracts - Poster
Slobodan Barbaric Abstract P9 Chromatin remodeling activities at the yeast PHO84 promoter
B. Silic1, T. Luckenbach2, S. Stuerzl2,P. Korber2 and S. Barbaric1
1Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia and 2Adolf-Butenandt-Institut, Universitaet Muenchen, Schillerstr. 44, 80336 Muenchen, Germany
The yeast PHO84 promoter, which is coregulated with the well studied PHO5 and PHO8 promoters in response to phosphate availability, is the strongest promoter of the PHO family, containing five binding sites for the specific activator Pho4. Under repressive conditions there is a short hypersensitive region in the promoter containing two closely positioned Pho4 binding sites, but upon induction the promoter chromatin structure is altered, so that at least one nucleosome upstream and one downstream from the hypersensitive region are remodeled. Remodeling of chromatin structure leads to histone depletion from the promoter region. The rate of histone eviction and consequently the rate of promoter activation are strongly delayed in mutants deleted for either Snf2 or Gcn5. Nonetheless, after prolonged induction full activation is achieved, but in the absence of Snf2 chromatin remodeling is only partial, resulting in displacement of the downstream but not of the upstream nucleosome. Therefore, eviction of these two nucleosomes requires different chromatin remodeling activities. In contrast to Snf2, Gcn5 is not required for efficient remodeling of both nucleosomes upon full induction, either in the presence or absence of Snf2. Similarly to Gcn5, the absence of Ino80 also causes delay in the promoter activation without affecting the final extent of chromatin remodeling and the same is true for the histone chaperone Asf1. Taken together, Gcn5, Ino80, or Asf1 affects only the rate of remodeling at the PHO84 promoter, similar as we previously found for the PHO5 promoter. However, with respect to the requirement for Snf2, the PHO84 promoter chromatin structure possesses hybrid characteristics compared to the two coregulated promoters: the PHO8 is essentially dependent on Snf2 and at the PHO5 only the rate of chromatin remodeling is reduced in the absence of Snf2. We have also examined a possible role of the histone variant H2A.Z in regulation of chromatin remodeling at PHO promoters. Interestingly, the absence of H2A.Z has only a slight effect on activation of the PHO5 promoter, but causes a strong delay in the activation kinetics of the PHO84 promoter. This provides additional evidence that chromatin remodeling process at the two coregulated promoters involves different mechanisms.
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Vivian Bardwell Abstract P10 Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets
Micah Gearhart, Connie Corcoran, Joseph Wamstad and Vivian Bardwell
Department of Genetics, Cell Biology and Development and the Cancer Center, University of Minnesota, Minneapolis, MN 55455, U.S.A.
The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocar- diodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmuno- precipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.
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Amrita Basu Abstract P11 Computational prediction of histone and non-histone proteins
A. Basu1, Y. Zhao2,S.Hake1, K. Rose3, B. Ueberheide3, D. Hunt3,C.D. Allis1 and E. Segal4
1Laboratory of Chromatin Biology, The Rockefeller University, New York NY 10021,U.S.A., 2Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390,U.S.A., 3Department of Chemistry, University of Virginia, Charlottesville, VA 22904, U.S.A., 4Department of Computer Science and Applied Mathematics, The Weizmann Institute of Science, Rehovoth 76100, Israel
Acetylation, a well-studied post-translational modification,.plays essential regulatory roles in a broad spectrum of biological processes, notably gene regulation. Although many studies have been contributed on the molecular mechanism of acetylation dynamics, the intrinsic features of substrate site specificity are still elusive and remain to be critically defined to a point where predictions of unknown acetylation sites can be made with reasonable accuracy. Since many enzymatic processes that modify histones have been identified and characterized, it is highly probable that these histone-modifying enzymes target non- histones in mammalian cells, as illustrated by the p300/CBP requirement for p53 and HIV1 acetylation in human cells (1,2). Whether or not these enzymes target the substrate in a specific manner and whether sequence composition surrounding the modified amino acids plays an important role remains to be elucidated. In this work, using a sequence based bioinformatics approach, we show that there is a bias towards specific amino acids surrounding an acetylated lysine that may result from HAT recognition in histones and nuclear non-histone proteins. We hope that our approaches can be used as a predictive method in identifying acetylated substrates in the human proteome.
Refs: (1) Gu W and Roeder R.G, Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997. 90: pp. 595–606. (2) Kiernan R.E. et al., HIV-1 tat transcriptional activity is regulated by acetylation. EMBO J, 1999, 18: pp. 6106–6118.
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Mark T. Bedford Abstract P12 Screening for the methylated proteome
Donghang Cheng and Mark T. Bedford
University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis PO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A.
The coactivator associated arginine methyltransferase, CARM1, is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening approach for this purpose and identified CA150, SAP49, SmB and U1C as splicing factors that are specifically methylated by CARM1. We further showed that CA150, a molecule that links transcription to splicing, interacts with the tudor domain of the spinal muscular atrophy protein SMN, in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicate this enzyme in the regulation of alternative splicing and points towards its involvement in spinal muscular atrophy pathogenesis.
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Sukesh R. Bhaumik Abstract P13 Regulation of transcriptional activation by mRNA cap-binding complex in vivo
Pratibha Bajwa, Abhijit Shukla, Nadia Stanojevic and Sukesh R. Bhaumik
Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL-62901, U.S.A.
Eukaryotic gene regulation is largely controlled at the level of transcriptional activation by gene-specific activators which function by stimulating the assembly of general transcription factors to form a preinitiation complex (PIC) at the promoters of the active genes. Interestingly, we show here that mRNA cap-binding complex (CBC) dramatically stimulates formation of the PIC assembly and subsequently transcription in vivo. However, CBC is recruited to the active gene following formation of the PIC assembly. These results reveal that PIC and CBC are intimately coupled via their reciprocal synergism, providing a novel regulatory pathway of transcriptional activation by CBC in a positive feedback mechanism.
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Marjorie Brand Abstract P14 The Ash2L/MLL2 methyltransferase complex is important for ß-globin transcription during erythroid differentiation
Patrick Lai1, Jeffrey A. Ranish2, Celina Demers1, Gaetan Juban3, Francois Morle3, Ruedi Aebersold2,4, F. Jeffrey Dilworth1, Mark Groudine5 and Marjorie Brand1
1Sprott Center for Stem Cell Research, Ottawa Health Research Institute, Ottawa, ON, Canada, 2Institute for Systems Biology, Seattle, WA, USA, 3Centre de Genetique Moleculaire et Cellulaire, Villeurbanne, France, 4Institute for Molecular Systems Biology, Zurich, Switzerland, 5Fred Hutchinson Cancer Research Center, Seattle, WA, U.S.A.
The mammalian ß-globin locus is comprised of five clustered ß-like globin genes, whose developmental-specific expression is regulated by the distal locus control region (LCR), located dozens of kilobases upstream of the genes. Methylation of histone H3 at lysine 4 (H3-K4) occurs at the ßMaj-globin gene during erythroid differentiation and is dependent upon the hematopoietic-specific transcription factor NF-E2-p45. However, the enzyme responsible for H3-K4 methylation at the ß-globin locus is unknown.
Here, we set out to identify methyltransferase(s) interacting with NF-E2-p45 in erythroid cells using immunoprecipitation and mass spectrometry. Strikingly we found that NF-E2- p45 associates with 2 distinct histone H3 methyltransferases: G9a which modifies lysine 9, and MLL2 which is specific for lysine 4. Interestingly, in vitro these 2 methyltransferases display competing activities, which appear to be regulated by the acetylation status of their target, suggesting a new element to the histone-code. Using chromatin immunoprecipitation, we then showed that, during erythroid differentiation, the MLL2 complex is recruited to the ß-globin LCR, 38kb upstream of the ßMaj-Globin gene. Interestingly, MLL2 (but not the Ash2L subunit of the MLL2 complex) is also recruited across the entire ß-globin locus from the LCR to the transcribed ß-Majglobin area, suggesting that the H3-K4 methyltransferase is transported across the ß-globin locus via a spreading mechanism. Finally, in contrast to the recent proposition that trimethylation of H3- K4 and H3-K9 co-exists within the active ßMaj-globin gene, we demonstrate that these 2 modifications are mutually exclusive, trimethylated H3-K4 being instead strongly correlated to acetylated H3-K9. In fact, we found that the ß-Majglobin transcribed area (methylated on H3-K4 and acetylated on H3-K9) is flanked by regions that are enriched in trimethyl-H3-K9, while being poorly trimethylated on H3-K9 itself. This is in agreement with earlier suggestions that one role of trimethyl-H3-K4 is to prevent spreading of the repressive H3- K9 trimethyl mark. Thus altogether our results provide new insights into the regulation of transcription via recruitment and spreading of the H3-K4 methyltransferase MLL2 to specific genes during development.
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Abstracts - Poster
Lauren Buro Abstract P15 Histone methylation patterns at interferon-gamma inducible gene loci
Lauren Buro and Melissa Henriksen
Department of Biological Sciences, Fordham University, Bronx, NY 10458, U.S.A.
Here we examine the lysine methylation profile of histone H3 (H3) at several interferon gamma (IFN-() induced, STAT1 dependent gene loci. STATs are a family of transcription factors that, in response to a variety of extracellular ligands, are rapidly and transiently recruited from their latent state in the cytoplasm to the nucleus where they drive transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. The accessibility of the activated STAT to its DNA binding site and the succeeding gene expression are intimately tied to chromatin structure. Post-translational modifications of the residues in the amino terminal tails of the lysine residues of histones define the functional state of the chromatin. Methylation of lysine residues of H3 is described as either activating or repressive of gene expression, with the activity correlated to the position of the lysine residue (K4 or K9), the level of methylation (mono, di or trimethylation) and the location of the nucleosome itself (promoter or transcribed regions, euchromatin or heterochromatin). Chromatin immunoprecipitation followed by real-time PCR revealed K4 dimethylation (K4M2) and K4 trimethylation (K4M3) primarily in the control regions of all the IFN-( inducible genes assayed, independent of transcriptional activity. That such methylation was not dynamic suggests a role for these modifications only in poising these genes for transcription. Further experiments to determine if K4M2 and/or K4M3 are permissive for gene induction are underway. H3 K9 trimethylation (K9M3), a modification reportedly associated with transcription elongation was not observed in the coding regions of the IFN-gamma inducible genes.
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Jill S. Butler Abstract P16 CXXC-finger Protein 1 regulates Dnmt1 protein expression
Jill S. Butler and David G. Skalnik
Indiana University School of Medicine, Herman B Wells Center for Pediatric Research 1044 W. Walnut Street, R4 Room 324, Indianapolis, IN 46202, U.S.A.
Cytosine methylation and histone tail modifications are two epigenetic modifications that influence gene expression. Elucidation of epigenetic regulation is becoming increasingly important as deregulation of epigenetic processes is observed in many diseases, including cancer. The CXXC1 gene encodes CXXC finger protein 1 (CFP1), a transcriptional activator that specifically binds unmethylated CpG dinucleotides. This DNA binding activity of CFP1 makes it unique in that most CpG binding proteins bind methylated CpG dinucleotides and facilitate heterochromatin formation. CFP1 has recently been identified as a component of the mammalian SET1 histone H3 lysine 4 methyltransferase complex (Lee and Skalnik, JBC (2005) 280:41725-31). Disruption of CXXC1 in mice results in an early embryonic lethal phenotype (Carlone and Skalnik, MCB (2001) 21:7601-6), and embryonic stem (ES) cells lacking CFP1 exhibit multiple epigenetic defects including altered histone modifications and reduced global cytosine methylation (Carlone, et al, MCB (2005) 21:4881-91). DNA methyltransferase 1 (Dnmt1) is the major source of maintenance DNA methyltransferase activity in mammalian cells and is primarily responsible for copying cytosine methylation patterns during DNA replication. Dnmt1 protein level and DNA methyltransferase activity are decreased by ~50% in CXXC1-/- ES cells and are rescued by stable expression of murine CFP1. Northern blot analysis along with real-time PCR experiments revealed Dnmt1 transcript level is elevated ~ 40% in CXXC1 -/- ES cells. Additionally, immunoprecipitation experiments revealed an interaction between CFP1 and Dnmt1 in vivo. Regulation of Dnmt1 protein level by CFP1 is the first example of reduced Dnmt1 protein without direct disruption of Dnmt1 gene function. The functional significance of this novel intersection of epigenetic regulatory proteins is currently under investigation.
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Abstracts - Poster
Jim Cakouros Abstract P17 Identification of a novel enzyme which regulates the kinetics of histone arginine methylation in Drosophila melanogaster
D. Cakouros, T. Daish and S. Kumar
Hanson Institue, Department of Haematology, Frome Road, Adelaide, Australia
The sequential modifications of histones form the basis of the histone code which translates into either gene activation or repression. The dynamic fluctuations of histone methylation occurs in response to specific signals, regulated by the interplay of histone methyltransferases and histone demethylases. Nuclear receptors recruit a cohort of histone modifying enzymes in response to ligand binding and regulate proliferation, differentiation and programd cell death (PCD). In Drosophila, coactivators for the nuclear ecdysone receptor (EcR/UsP) have not been extensively examined. We have identified a novel cofactor for the EcR which is normally involved in amino acid catabolism. This enzyme contains two enzymatic domains in its amino and carboxyl termini respectively, binds EcR/UsP directly and potentiates ecdysone mediated transcription. We found that it can inhibit H3-R17 dimethylation mediated by the drosophila methyltransferase CARMER (DART4) by directly binding the amino tail of histone H3 specifically and this inhibition is dependant on the cofactors ketoglutarate and NADH. In drosophila cells this protein regulates the kinetics of H3-R17 dimethylation and knockdown by RNAi can compromise ecdysone mediated transcription and PCD. The in vivo function of this enzyme is currently being examined in flies.
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Raymond Camahort Abstract P18 Genome-wide analysis of the budding yeast histone variant Cse4 reveals occupancy at a single centromeric nucleosome as well as additional non-centromeric locations
Raymond Camahort1,2, Bing Li1, Brian Fleharty1, Jerry L. Workman1, Chris Seidel1, Jennifer L. Gerton1,2
1Stowers Institute for Medical Research, Kansas City, MO 64110, U.S.A., 2Department of Biochemistry and Molecular Biology, 4011 Wahl Hall East, 3901 Rainbow Blvd., University of Kansas Medical Center, Kansas City, KS 66160, U.S.A.
Cse4 is the S.cerevisiae centromeric H3 histone variant, also known as CENP-A. This histone variant is incorporated into nucleosomes that are located at centromeres in budding yeast and are required for proper kinetochore assembly and chromosome segregation. Centromeres in budding yeasts are defined by a specific 125 bp sequence that contains the elements CDEI, CDEII, and CDEIII. We demonstrate the localization of Cse4 to the sixteen centromeres of budding yeast chromosomes, as expected, and additionally the surprising result that Cse4 nucleosomes are located at other regions in the yeast genome, mainly repetitive regions including telomeres, rDNA, and Ty elements. To verify the localization of Cse4 to these regions, we fused Cse4 to a transcriptional activation domain and demonstrate that this chimeric protein can activate transcription from the rDNA and Ty elements. Using high resolution quantitative PCR we demonstrate that there is a single Cse4-containing nucleosome at centromeres in vivo and this nucleosome is positioned over centromere sequences. Finally, using purified components, we assemble canonical and Cse4-containing nucleosomes in vitro and demonstrate that they can assemble with high efficiency on non-centromeric DNA. Our results demonstrate that Cse4 alone is not sufficient to nucleate a kinetochore. We suggest that Cse4, in addition to its critical role at the centromere, may participate in repressing DNA metabolic processes such as transcription and recombination.
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Dylan Carney Abstract P19 The RAG2 PHD finger links the histone code to V(D)J recombination
Dylan Carney, Alex Kuo, Tina Christakos and Or Gozani
Gilbert Biological Sciences, 371 Serra Mall, Stanford, CA 94306, U.S.A.
The PHD finger (Plant Homeodomain) module is a signature chromatin-associated domain that is found throughout eukaryotic proteomes and is mutated in several human diseases. One such PHD-containing protein, the Recombination Activating Gene 2 (RAG2), is necessary for the V(D)J recombination reaction that is responsible for generating immunoglobulin and T-Cell Receptor (TCR) diversity among lymphocytes. Mutations within the RAG2 PHD finger have been implicated in Omenns Syndrome, which is a result of an inability to effectively carry out V(D)J recombination. Preliminary work has shown that the RAG2 PHD domain binds specifically to histone H3 trimethylated at lysine 4 (H3K4me3). Furthermore, we have shown that the very same mutation (W453R) implicated in several cases of Omenns Syndrome abrogates this binding. Our work suggests that the recognition of H3K4me3 by the RAG2 PHD finger plays an important role in V(D)J recombination and thus proper immune system function.
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Beverly S. Chilton Abstract P20 Analysis of RUSH/SMARCA3 isoforms and their interactions with Egr-1 and c-Rel in the regulation of transcription
Aveline Hewetson and Beverly S. Chilton
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street – MS6540, Lubbock, TX 79430, U.S.A.
RUSH, a SWI/SNF-related regulator of chromatin, was cloned from rabbit endometrium. Steroid- dependent alternative splicing yields two distinct isoforms (alpha and beta). Alpha has seven highly conserved DNA-dependent ATPase domains, and beta has four. Although both isoforms are capable of DNA binding, no studies have addressed their individual involvement in any biological process. RUSH is highly conserved in eukaryotes and was independently identified as HIP116 (human) and P113 (mouse). These names were replaced by the gene name, SMARCA3. In addition to alternative splicing, progesterone-induced transcriptional activation is mediated by a bipartite progesterone receptor (PRE) half-site/overlapping Y-box combination (-38/-26) in the proximal promoter (-162/+90). Activation by progesterone is achieved via the PRE half-site, and attenuated by NF-Y binding to the Y-box. Repression is also achieved at two GC-rich sequences in the proximal promoter and a distal RUSH site (-616/-611). At each GC-rich site, specific binding of Sp3 was 15-17-fold more abundant than Sp1. The rate of complex disassociation (off rate) for the distal A site (-131/-126) with the consensus Sp binding sequence (GGGGCGGGG) was half that of the proximal B site (-62/-53) with a variant (GGGGCGGAG) sequence. MatInspector (Genomatix) analysis revealed the complexity of the stronger A site. A putative Egr-1/Sp/MAZ/MZF1 site on the positive strand overlaps a putative c-Rel site on the negative strand (matrix similarity values ( 0.91). Supershift assays with nuclear extracts from progesterone-treated animals confirmed the binding of each candidate protein to the composite A site. In contrast, only Sp3 binds the A site in nuclear extracts from estrous animals, and protein binding to the B site is negligible. At the RUSH site, isoform-specific binding was demonstrated with supershift assays. Exclusive binding of alpha was confirmed with isoform-specific antibodies and nuclear extract from progesterone-treated animals. Exclusive binding of beta to the same site was confirmed with isoform-specific antibodies and nuclear extract from estrous animals. TransSignal TF-TF Interaction Arrays showed strong physical associations between alpha and DNA-bound Egr-1 and c-Rel. No physical interactions occurred between alpha and DNA-bound Sp1, MZF1 or NF-Y. Supershift assays confirmed alpha interacts physically with Egr-1 and c-Rel bound to DNA at the A site, and conversely Egr-1 and c-Rel interact with alpha bound to DNA at the distal RUSH site. ChIP assays confirmed alpha interacts physically with Egr-1 and c-Rel at each site in the transcriptionally active promoter. RUSH/Egr-1 and RUSH/c-Rel interactions were visualized by confocal microscopy. The proposed model for this interaction includes the fact that the authentic RUSH site is separated from the composite A site by 500-bp of 5(-sequence with a series of alternating pyrimidine-purine (CA) elements, which are anisotropically flexible and promote DNA bending. Thus progesterone acts via the PRE to induce transactivation. Fine-tuning the magnitude of this response includes isoform-specific autorepression in which newly synthesized alpha binds DNA in a sequence selective manner, and interacts physically with ligand-bound Egr-1 and c-Rel in the proximal promoter. Conversely, when the gene is silent (estrous), beta replaces alpha, and the Egr-1 and c-Rel partners are unavailable for binding interactions. Alternative splicing is a powerful means of generating macromolecular complexity. Understanding the functional capabilities of alternatively spliced isoforms of specific genes that are transcription factors with intrinsic helicase activity will enhance our understanding of isoform-distinctive mechanisms of transcriptional regulation.
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Abstracts - Poster
Alexandra Chittka Abstract P21 Signalling by a novel p75 neurotrophin receptor interacting protein, SC1/PRDM4
Alexandra Chittka1*, Juan Carlos Arevalo2, Maria Rodriguez-Guzman1, Pilar Perez3, Moses V. Chao2 and Michael Sendtner1
1*MRC centre for developmental neurobiology, King’s College, Guy’s Campus, London Bridge, London SE1 1UL, 1Institute for Clinical Neurobiology, University of Wuerzburg, 97080 Wuerzburg, Germany, 2Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, 3Instituto de Microbiologia Bioquimica, CSIC/Departamento de Microbiologia y Genetica, Universidad de Salamanca, 37007 Salamanca, Spain
Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) domain-containing zinc finger protein family, and it has been shown to be regulated by serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine (BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and analyzed the function of its various domains. We show that SC1 acts as a transcriptional repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the presence of its zinc finger domains and the PR domain. Additionally, these two domains are involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are also necessary to direct SC1’s nuclear localization. Lastly, SC1 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1. Currently, we are searching for additional target genes of SC1/PRDM4 to address the mechanistic role it plays during the development of the nervous system.
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Leslie Chu Abstract P22 Inheritance of epigenetic chromatin states
Leslie Chu and Joachim Li
University of California San Francisco, 600 16th Street, Genetech Hall, Rm S376, San Francisco, CA 94158 U.S.A.
In eukaryotes, DNA is packaged into either euchromatin or heterochromatin. Euchromatin is classically defined as actively transcribed regions of DNA, while heterochromatin is defined as transcriptionally silenced regions of DNA. This regional silencing requires the formation of a highly compact chromatin structure. Once established, the silent chromatin structure is stably maintained throughout the cell cycle and inherited in each subsequent generation. The inheritance of silent chromatin is surprisingly stable, given the stresses placed on chromatin through out the cell cycle. Previous work has shown that in each cell cycle, DNA replication disrupts nucleosomes, the fundamental unit of chromatin. This suggests that DNA replication also disrupts the silent chromatin structure. During each cell cycle, however, transcriptional silencing remains intact. These studies imply that if DNA replication disrupts silent chromatin, then the restoration of silencing is tightly coupled to replication. Our studies focus on understanding if DNA replication disrupts silencing, what serves as the molecular memory for silent chromatin and identifying proteins required for the inheritance of transcriptional silencing.
Using Saccharomyces cerevisiae, we show that Sir1, a heterochromatin establishment protein, and Asf1, a nucleosome deposition factor, are required for the inheritance of HMLalpha transcriptional silencing. We also show that progression through S phase, in the absence of Sir1 and Asf1, disrupts transcriptional silencing. This finding demonstrates that an S phase event, possibly DNA replication, perturbs silencing. We used chromatin immunoprecipitation to analyze the chromatin structure of HMLalpha. Interestingly, when silencing is not inherited (lost with progression through one cell cycle), an intermediate chromatin structure is formed. This intermediate structure contains both euchromatin and heterochromatin features. These remaining heterochromatin features may serve as a molecular memory to restore the silent chromatin structure. These results suggest that S phase disrupts transcriptional silencing, resulting in an intermediate chromatin structure. This intermediate chromatin structure may provide the molecular memory that directs Sir1 and Asf1 restorative silencing activity, resulting in the faithful inheritance of silencing.
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Abstracts - Poster
Mair Churchill Abstract P23 Structural basis for the histone chaperone activity of Asf1
Mair E.A. Churchill, Christine M. English, Melissa W. Adkins, Joshua J. Carson and Jessica K. Tyler
Department of Pharmacology, Department of Biochemistry and Molecular Genetics, U. Colorado Health Sciences Center, U.S.A.
Asf1 is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 shows how Asf1 binds the H3/H4 heterodimer, enveloping the C-terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C-terminus of histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome, undergoes a major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta- sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a strand- capture mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly / assembly that may be used ubiquitously by histone chaperones.
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Jeffrey Craig Abstract P24 What makes centromeres localise and cluster in interphase nuclei?
Irina Solovei1, Claudia Weierich1, Tatyana Karamysheva1, Paul Canham2, K.H. Andy Choo3, Thomas Cremer1 and Jeffrey M. Craig2
1Biozentrum (LMU), Grosshaderner Str. 2, Planegg-Martinsried, 82152 Germany, 2Epigenetics Research Laboratory and 3Chromosome and Chromatin Research Laboratory, Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia
Centromeres lie at the heart of chromosomes where they choreograph the multiple events of cell division. Centromeres are also a prime example of intergenerational epigenetic inheritance – their chromatin structure is preserved from one generation to the next. They contain large regions of heterochromatin and this has been shown to play a major role in establishing structured nuclear domains which can control gene expression. In mammals, these domains are composed of clusters of centromeres and are often located close to the nuclear periphery. Furthermore, centromere are also localise to the periphery of their individual chromosome territories. The roles of centromeres per se and of heterochromatin in this nuclear localisation and clustering are unclear. To separate out these roles we have looked at the nuclear localisation of human centromeres without tandemly-repetitive DNA. These neocentromeres contain only small domains of pericentric heterochromatin. We have looked at the three dimensional position of neocentromeres in the nuclei of human cell lines using in situ hybridisation and immunolofluorescence using antibodies to centromere proteins. We found that consistently, neocentromeres exhibited the same localisation as repetitive centromeres within a territory and within the whole nucleus. However, ongoing findings are showing that neocentromeres may not cluster with other centromeres in the interphase nucleus. These results imply that large regions of heterochromatin exert more influence on centromere clustering and the creation of large heterochromatic domains in interphase and less influence on positioning with chromosomes and nuclei.
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Valerie J. Crusselle-Davis Abstract P25 Regulation of beta-globin expression through the recruitment of chromatin modifying enzymes by TFII-I and USF
Valerie J. Crusselle-Davis, Archana Anantharaman, Tihomir Dodev and Jurg Bungert
Department of Biochemistry and Molecular Biology, Powell Gene Therapy Center, Center for Mammalian Genetics, and Shands Cancer Center, University of Florida, Gainesville, FL 32610 U.S.A.
The human beta-globin locus contains five functional genes which are arranged in the order of their developmental expression. Gene proximal cis-regulatory DNA elements and interacting proteins restrict expression of the genes to the embryonic, fetal, or adult stages of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region also contributes to the temporal regulation of the genes. To more fully understand adult beta-globin gene regulation, we examined the downstream promoter and found that transcription factors TFII-I and USF interact with elements within this region in erythroid cells. TFII-I was found to act as a repressor of beta-globin expression while USF proteins were found to act as activators of beta-globin expression. It is becoming increasingly clear that one role DNA binding proteins play is to recruit co-activators or co- repressors that modify histones or mobilize nucleosomes at regulatory sites. Therefore, to understand the mechanism behind the regulation of beta-globin expression by TFII-I and USF we investigated the recruitment of chromatin modifying enzymes to the beta-globin gene locus by these proteins in both an embryonic and adult environment. TFII-I was found to interact with HDAC3 exclusively in embryonic environment. Suz12, a component of the Polycomb group complexes 2,3, and 4 which contains histone methylase activity, was also found to interact at the beta-globin promoter in an embryonic environment but not in an adult environment suggesting a role in repression. USF was found to interact with activators in an adult environment. The role of USF in beta-globin expression was also further investigated in transgenic mice which express a dominant-negative protein to USF exclusively in erythroid cells.
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Foteini Davrazou Abstract P26 Molecular mechanism of histone H3K4me3 recognition by the PHD finger of ING2
Pedro V. Peña1, Foteini Davrazou1, Xiaobing Shi2, Kay L. Walter2, Vladislav V. Verkhusha3, Or Gozani2, Rui Zhao4 and Tatiana G. Kutateladze1
1Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, U.S.A. 2Department of Biological Sciences, Stanford University, Stanford, California 94305, U.S.A. 3Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, U.S.A. 4Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Aurora, Colorado 80045, U.S.A.
The PHD (plant homeodomain) finger is found in many chromatin remodeling complexes however its function remains unknown. We found that a subset of PHD fingers targets tri- methylated H3 histone (H3K4me3) tail representing a novel family of protein-effectors that recognize this epigenetic mark1,2. We have determined the structure of the PHD finger of ING2 (inhibitor of growth) tumor suppressor in complex with a histone H3K4me3 peptide and characterized its specificity toward post-translationally modified histone tails. The H3K4me3 peptide is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among other PHD fingers. The trimethylammonium group of Lys 4 is recognized by aromatic residues of the domain, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving five peptide residues, account for the PHD finger’s high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis, suggesting a novel tumor suppressive mechanism. Strong binding of other human ING and yeast YNG PHD fingers indicates that the recognition of the H3K4me3 histone code is a general function of this protein family.
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Abstracts - Poster
Eullia de Nadal Abstract P27 Control of gene expression by the yeast Hog1 MAPK
Eullia de Nadal, Meritxell Zapater, Glria Mas, Nuria Noriega, lex Vendrell, Sergi Regot and Francesc Posas
Cell Signaling Unit, Departament de Cincies Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain
Mitogen-activated protein kinase (MAP) cascades are common signaling modules found in both higher and lower eukaryotic cells. Budding yeast has several MAP kinase cascades one of which contains a relative of the p38 family of stress activated MAP kinases. This kinase coordinates cellular responses to increases in external osmolarity by inducing diverse osmo-adaptative response. Recent genome-wide transcriptional studies revealed that a great number of genes are regulated by osmotic stress in a Hog1 dependent manner, suggesting a key role for the MAP kinase in stress-induced gene expression. However, there is not a uniform mechanism by which stress-activated MAP kinase modulates gene expression. It has been reported that MAPK can modify gene regulation by direct phosphorylation of transcription factors, activators and repressors, such Smp1 and Sko1 proteins. Apart from the role of Hog1 in the modification of transcription factors, this kinase is associated specifically to chromatin in stress responsive promoters. Binding of the MAPK is critical for RNA Pol II and chromatin remodeling factors recruitment to osmostress responsive promoters and for gene expression. These data suggest a new dimension to gene regulation by signaling kinases and it prompted us to further study alternative mechanisms by which the MAP kinase Hog1 could regulate osmostress gene expression.
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Roger Deal Abstract P28 Repression of flowering in Arabidopsis thaliana requires histone H2A.Z deposition by a putative SWR1 complex
Roger B. Deal, Christopher N. Topp, Elizabeth C. McKinney and Richard B. Meagher
Department of Genetics (Deal, McKinney, Meagher) and Department of Plant Biology (Topp), University of Georgia, Athens, GA 30602, U.S.A.
In addition to the bulk histones that package the nascent genome during S phase, eukaryotes also encode variant histones that are deposited independently of DNA replication and serve to functionally specialize particular chromatin regions. The histone variant H2A.Z is universally conserved and has been implicated in a wide variety of chromatin-mediated processes including transcriptional activation and euchromatin maintenance in yeast, and heterochromatin formation in metazoans. In budding yeast and humans H2A.Z is deposited into chromatin through the action of a conserved protein complex known as SWR1 or SRCAP, respectively. Here we show that the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN 6 (ARP6) and the Snf2 protein PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), are part of a single protein complex and are each required for deposition of H2A.Z into chromatin at multiple loci. One of these loci is the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition from vegetative to reproductive development. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that H2A.Z is required for transcriptional activation of FLC to levels that inhibit flowering. Collectively these results support the existence of a SWR1-like complex in Arabidopsis thaliana and show that, similar to its role in yeast, H2A.Z can serve to potentiate transcriptional activation in plants.
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Laurent Delva Abstract P29 The Transcription Intermediary Factor 2 is required for zebrafish development
Julia Zhuravleva1, Bernard Thisse2, Christine Thisse2, Jean-Noël Bastie1 and Laurent Delva1
1Inserm U517, Faculté de Médecine, Université de Bourgogne, 7, bd Jeanne d’Arc, 21000 Dijon, France, 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/Inserm/ULP, 1, rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France
The TIF2 (Transcription Intermediary Factor 2) gene, encoding a histone acetyl transferase, belongs to the p160 family. TIF2 interacts with liganded nuclear receptors, enhancing transcription activity of the receptors. Because of partial genetic compensation effect, knock out TIF2 mice did not inform completely about the role of TIF2 in development. Therefore, we decided to use zebrafish as an animal model. tif2 is ubiquitously expressed in zebrafish development. tif2-knock down zebrafish embryos present embryonic alterations such as a smaller tail size, abnormalities in the notochord and mesenchyme. In addition, tif2 morphants did not harbor posterior intermediate cell mass (ICM) indicating putative hematopoietic differentiation defects. To better understand the nature of the defects observed in tif2 morphants, we performed molecular analysis of various differentiation markers by using whole-mount RNA in situ hybridization. Our results suggest the involvement of tif2 in embryonic development, particularly in primitive hematopoiesis. Furthermore, we observed that loss-of-function of zebrafish tif2 results in massive apoptosis in the mesenchyme.
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Luisa Di Stefano Abstract P30 Lsd1 mutation in Drosophila disrupt normal level of H3K4 methylation and affects viability and fertility
Di Stefano L, Moon NS, Ji JY and Dyson N.
Massachusetts General Hospital Cancer Research Center and Harvard Medical School, Charlestown, Massachusetts, U.S.A.
Covalent modifications of histone tails have fundamental roles in determining the chromatin structure and in regulating gene expression. One such modification, histone lysine methylation was considered irreversible until the recent discovery of histone demethylases. Two distinct classes of histone lysine demethylases have been characterized so far, the Jumonji-domain containing proteins and Lsd1. In mammalian cells, Lsd1 was shown to specifically demethylate mono and di-methyl histone H3 lysine 4 and, when associated with the androgen receptor, to act on dimethyl-H3K9. Lsd1 is highly conserved between organisms from yeast to human but its role has yet to be studied in vivo. Here we describe the effects of Lsd1 mutation in Drosophila. We find that mutation of dLsd1 strongly affects global level of methylation of mono and dimethyl H3K4 revealing that specificity towards these residues is conserved throughout evolution. In contrast the global levels of dimethyl- H3K9 are not affected in dLsd1 mutant suggesting either a functional difference to the human counterpart or that this regulation is restricted to specific tissues. As a consequence of dLsd1 mutation, animal viability is strongly affected in a gender specific manner. dLsd1 mutant flies are sterile and ovary development is strongly impaired. dLsd1 mutation strongly suppresses positional effect variegation (PEV) and affects gene expression. Taken together our results support an important role for histone H3K4 lysine demethylation in the regulation of chromatin structure and gene transcription in Drosophila development.
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Stephan Diekmann Abstract P31 In vivo dynamic (FRAP, FCS) and neighbourhood relation (AB-FRET, FLIM) studies of human inner kinetochore proteins
S. Diekmann, P. Hemmerich, S. Orthaus, S. Weidtkamp-Peters and C. Hoischen
Molecular Biology, FLI, Beutenbergstr. 11, D-07745 Jena, Germany
The kinetochore specifies a DNA/protein assembly at the surface of chromosomes that plays an essential role in faithful segregation of the genetic material. To gain a precise dynamic understanding of this complex, the mobility of GFP-tagged inner kinetochore proteins CENP-A, CENP-B, CENP-C, CENP-H, CENP-I, and hMis12 were analyzed in living human cells using fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). In interphase cells, CENP-A, CENP-B, CENP-C, CENP-H and CENP-I are stable components of the kinetochore over hours while hMis12 rapidly and completely exchanges within seconds. FCS detected soluble pools of kinetochore proteins in interphase cells with protein-specific diffusion coefficients indicating the absence of pre- assembled kinetochore protein subcomplexes. During mitosis CENP-A, CENP-C, CENP-H, and CENP-I remain stably associated with the kinetochores, while CENP-B becomes mobile and, strikingly, hMis12 becomes completely immobilized at the kinetochores. Thus, unlike all other chromatin binding complexes analysed so far, the kinetochore is not maintained by a constant flux of rapidly exchanging components but rather by a static assembly mechanism. Alterations in the mobility of specific kinetochore proteins such as Mis12, however, appear to be associated with the changing functional properties of kinetochores during mitosis. Many of the proteins involved in kinetochore formation are known, however, little information is available on molecular structures and complex architecture although structural phenomena seem to play an important role for kinetochore function. In addition to their dynamic behaviour, we also determined the neighbourhood relation (in the < 10 nm range) of the inner kinetochore proteins CENP-A, CENP-B, CENP-C and CENP-I as well as histones in living human HEp-2 cells by energy transfer (FRET and FLIM). The data can be well explained by a centromeric chromatin 30 nm fiber model. Our results elucidate the architecture of the human inner kinetochore complex.
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Jeffrey Dilworth Abstract P32 MEF2 helps establish muscle specific pattern of gene expression by recruiting Trithorax Group proteins to specific promoters
Shravanti Rampalli, Marjorie Brand and F. Jeffrey Dilworth
Sprott Stem Cell Research Center, Ottawa Health Research Institute, Ottawa, Ontario, K1H 8L6., Canada
Pluripotency of stem cells is proposed to be due to the fact that all genes that have the potential to be transcribed lie in open chromatin. As cells divide and differentiate, epigenetic marking acts as a cellular memory to decide which genes should be expressed, or repressed through chromatin condensation. These epigenetic signals are generated by polycomb (repression) and trithorax (activation) group proteins which mark genes by methylating hisone H3 on lysine residue 9/27 and 4 respectively. In an effort to understand how the muscle specific pattern of gene expression is established during differentiation, we set out to determine whether trithorax proteins play a role in establishing muscle specific gene expression. Initially, we used chromatin- immunoprecipitation (ChIP) to show that upon differentiation, several specific muscle specific genes are marked by H3K4 trimethylation. To determine how trithorax proteins are targeted to these promoters, we used co- immunoprecipitation and found that the Mef2 family of transcriptional regulators interact with the trithorax group protein Ash2L. In addition, pull-down studies suggest that in cellular extracts from muscle cells, only specific Mef2 isoforms (Mef2C and Mef2D but not Mef2A) interact with Ash2L. Importantly, this interaction is significantly increased by pre-treating Mef2C, or Mef2D with p38 kinase in the presence of ATP, suggesting that phosphorylation of Mef2 proteins enhances its binding to Ash2L. Finally, we demonstrate by ChIP that the timing of Histone H3K4 trimethylation (epigenetic mark established by Ash2L complex) during differentiation of muscle cells coincides with the binding of Mef2 to several muscle specific promoters. Thus, it appears that the transcriptional activator Mef2 is helping establish the muscle specific pattern of gene expression though epigenetic marking of specific genes.
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Ivana Djuretic Abstract P33 T-bet and Runx3 cooperate to activate Interferon gamma and silence Interleukin-4 in T helper-1cells
Ivana Djuretic1, Ditsa Levanon2, Varda Negreanu2, Yoram Groner2, Anjana Rao1 and K. Mark Ansel1
1Harvard Medical School and the CBR Institute for Biomedical Research, Boston, MA, USA 02115, U.S.A., 2The Weizmann Institute of Science, Rehovot, Israel 76100
Cell differentiation requires activation of lineage-appropriate genes and silencing of lineage- inappropriate genes. Naive CD4+ T cells can differentiate down one of the two effector pathways: T helper-1 (Th1) or T helper-2 (Th2). T-bet, a T-box family transcription factor, is a central regulator during Th1 differentiation because it directly activates Th1-specific genes such as Interferon gamma (Ifng), and silences Th2-specific genes, such as Interleukin-4 (Il4). Here we demonstrate that T-bet induces another transcription factor, Runx3, with which it cooperates in both the activation of Ifng and silencing of Il4. Although each factor was capable of functioning on their own, optimal gene activation and rtpression depended on the presence of both T-bet and Runx3. The mechanim of repression likely involves a direct co-operation on DNA, as the two factors were able to form a complex on the Il4 silencer- derived probe in vitro. Current studies are aimed in further defining the nature of T- bet/Runx3 cooperation, including their ability to cause context-dependent gene activation and repression. In addition, since Il4 silencing has been associated with the appearance of H3K27 methylation, an important goal of this study will be to dettermine if direct cooperation of T-bet and Runx3 mediates the process of H3K27-methylation dependent silencing in the Il4 locus as well as other relevant loci.
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Tom Donndelinger Abstract P34 Seeing cells in a new light: Improving resolution with a scientific approach to tissue processing
Tom Donndelinger and Elizabeth Oldenkamp
BI-Biomics, 1512 12th Ave Rd, Nampa, Idaho, U.S.A.
One of the greatest shortcomings of fluorescent microscopy is the lack of resolution. Fluorescence studies alone cannot be solid proof of sub-cellular localization. Although this problem will never be entirely overcome, our methods for tissue fixation combined with fluorescent technology return more conclusive results for localization studies with resolution up to 2400x. We also show that superimposition of fluorescent images with H&E stained images creates a much more informative and cohesive picture of cell biology than traditional images with DAPI counterstains. Further, our studies have revealed unprecedented nuclear and nucleolar detail as well as multiple novel cellular processes that have never been seen through light microscopy. This technology, while a simple upgrade to an existing procedure, is revealing data that could seriously alter currently accepted scientific paradigms.
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Bojan Drobic Abstract P35 Characterization of Histone H3 kinases, MSK1 and MSK2
Bojan Drobic and James R. Davie
CancerCare MB, Manitoba Institute of Cell Biology, 675 McDermot Ave., Winnipeg, Manitoba, R3E 0V9, Canada
Stimulation of the Ras-MAPK signal transduction pathway by growth factors (EGF) or phorbol esters (TPA) in parental (10T1/2) and oncogene (H-ras)-transformed (Ciras-3) mouse fibroblasts induces rapid phosphorylation of histone H3. Phosphorylation of H3 occurs on Ser10 and Ser28 in the NH2-terminal tail. This phosphorylation event is implicated in the regulation of immediate early genes such as c-fos and c-jun. Constitutive activation of the Ras-MAPK pathway in ras-transformed mouse fibroblasts increases phosphorylation of H3 at Ser10 and Ser28 and we have shown that this increase is due to enhanced activity of histone H3 kinase, mitogen- and stress-activated protein kinase 1 (MSK1). Characterization of the MSK complex will be undertaken in 10T1/2, Ciras-3 and HEK293. Preliminary results demonstrate that MSK1 is associated with SWI/SNF ATPase (Brg1) and histone H3 acetyltransferase (HAT) [PCAF], as well as with c-Fos/c-Jun, p65 sub-unit of NFKB, 14-3-3 proteins, but not with HDAC1. Furthermore, chromatin remodeling activity of the MSK complex will be investigated by isolating the MSK complex from 10T1/2, Ciras-3 and HEK293 (cycling, serum-starved, TPA/EGF treated) cells and performing HAT and mononucleosome disruption assays. Preliminary HAT assay results suggest that the MSK complex contains HAT activity. Dynamics of MSK1/2 association with c-fos and c-jun promoters will be investigated via ChIP assay. Since the Ras-MAPK signaling is frequently deregulated in cancer (30% of human cancers contain aberrant ras), characterization of the Ras-MAPK activated MSK complex and its associating activities (chromatin remodeling and modifying activities) could provide a basis for the assessment of MSK as a novel therapeutic target to treat cancer.
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Danielle Ellis Abstract P36 Histone acetylation of SRC and p21 promoters in response to histone deacetylase inhibitor treatment; implications of HDAC activity and SRC expression
Danielle Ellis1 and Keith Bonham2
1Department of Biochemistry, University of Saskatchewan. and 2Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, SK, Canada, S7N 4H4
Histone deacetylase (HDAC) inhibitors (HDIs), such as trichostatin A (TSA), are well documented for their ability to induce apoptosis, growth arrest and differentiation in a variety of neoplasms. These effects are mediated through the activation and/or repression of gene expression. Some classic examples of this phenomenon include the transcriptional activation of p21WAF1 and the transcriptional repression of SRC. Currently, it is believed that HDAC inhibitors act at the histone level to alter chromatin dynamics through the inactivation of HDACs thereby resulting in histone hyperacetylation and increased transcriptional activation. However, transcriptional repression of gene expression is not so easily explained by this model. Indeed, changes in the acetylation status of histones associated with genes repressed by HDAC inhibitors, such as SRC, have not been reported. Therefore, we carried out a systematic investigation of the changes in histone H3 and H4 acetylation status at the promoter regions of two genes differentially affected by HDAC inhibitors. Treatment of HT29 colon cancer cells with TSA led to similar changes in the acetylation of discreet H3 and H4 lysine residues at the SRC1A, SRC1alpha and p21 promoter regions. Differential promoter specific acetylation changes were also observed; whereby, the SRC1alpha and p21WAF1 promoter regions demonstrated differential changes in acetylation as compared to SRC1A. The observations that SRC is repressed by HDIs and demonstrates rapid changes in histone acetylation upon HDI treatment suggests that an HDAC(s) may be localized at the SRC promoter regions and may be involved in SRC activation. Through the use of RNA interference, we observed that the knockdown of each class I HDAC (HDAC 1, 2, 3 and 8) did not repress SRC but actually resulted in increased SRC expression. Taken together these results suggest that histone acetylation is not a simple predictor of promoter activity and class I HDACs have an inhibitory affect on SRC expression. Further study of class II HDACs may aid in elucidating the mechanism by which SRC is repressed by HDIs.
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Alexander Erkine Abstract P37 Differential mechanisms of nucleosome displacement at yeast heat shock gene promoters
T. Y. Erkina and A. M. Erkine
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, 414 E. Clark St., Vermillion, SD 57069-2390, U.S.A.
Chromatin remodeling at promoters of co-regulated and highly inducible heat shock genes HSP12, HSP82, and SSA4 is characterized by robust histone displacement during induction of transcription. These promoters reach their maximally histone stripped state via two distinct chromatin remodeling pathways: one associated (for the HSP12 and HSP82 promoters) and the other one not associated (for the SSA4 promoter) with robust histone H3 specific acetylation. This observation implies that a histone acetylated platform recognized by bromodomain containing chromatin remodeling complexes is not uniformly required for the robust histone displacement at gene promoters during induction of transcription. SNF2 deletion causes elimination of histone displacement from the HSP12 promoter but not from the HSP82 and SSA4 promoters. Out of three analyzed heat shock genes only HSP12 is characterized by inducible binding of HSF to the promoter, while HSP82 and SSA4 have HSF preloaded before heat shock. The SNF2 deletion prevents HSF binding to the HSP12 promoter. Knowing that HSF cannot bind to chromatinized DNA we speculate that in wild type cells the SWI/SNF complex determines sliding or relocation of nucleosomes along the HSP12 promoter allowing HSF to establish promoter binding, while in the snf2 mutant strain nucleosomes lose their dynamic behavior and block HSF loading, thus eliminating histone displacement.
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Ragnhild Eskeland Abstract P38 HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors
Ragnhild Eskeland, Anton Eberharter and Axel Imhof
Adolf-Butenandt Institut, University of Munich, Schillerstr. 44, 80336 Muenchen Germany
A large portion of the eukaryotic genome is packaged into transcriptionally silent heterochromatin. Several factors have been identified that play important roles during the establishment and maintenance of this condensed form. Methylation of lysine 9 within histone H3 and the subsequent binding of the chromo-domain protein HP1 is thought to initiate heterochromatin formation in vivo and to propagate a heterochromatic state through several cell divisions. Here we analysed the binding of HP1 to methylated chromatin in a fully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 only weakly binds to methylated chromatin. However, the addition of recombinant SU(VAR) proteins such as ACF1 or SU(VAR)3-9 facilitates HP1 binding to chromatin methylated at H3K9. We propose that HP1 has multiple target sites that contribute to its recognition of chromatin only one of them being H3K9me. These findings have implications for the mechanisms of how specific chromatin modifications are recognized in vivo.
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George Feehery Abstract P39 CpG methylated DNA standards and control primers for use in methyl sensitive PCR and bisulphite sequencing
George R. Feehery, Pierre-Olivier Esteve, Hang Gyeong Chin and Sriharsa Pradhan
New England Biolabs, 240 County Road, Ipswich, MA 01938-2723. U.S.A.
Methylation-specific PCR (MSP), is a technology for the sensitive detection of gene methylation in the vertebrate genome. The procedure employs an initial bisulfite reaction that modifies the genomic DNA, or converts unmethylated cytosines to uracils while 5- methylcytosines remain unaltered. The bisulphate modified DNA is amplified by PCR with specific primers designed to distinguish methylated from unmethylated sequences. Because this is a sensitive PCR-based assay, the use of DNA and primer controls are necessary to determine the quality of the bisulfite conversion, the identification of artifacts such as primer-dimer pairing, and mispriming to undesired target DNA that can cloud the interpretation of results. To create a methylation-positive DNA control, we have enzymatically methylated Hela, NIH- 3T3, and Jurkat genomic DNA with a prokaryotic CpG Methylase (M.SssI). All the cytosine residues (C) within the double-stranded dinucleotide recognition sequence 5?CG?3 are methylated cytosine (C5). To test the extent of CpG methylation, we challenged the various modified DNAs with [3H]AdoMet and an excess of bacterial methyltransferase M.SssI. No further incorporation of tritiated AdoMet was observed in the DNA substrates even after extensive overnight incubation. MSP-PCR using 10 different primer sets also revealed complete methylation of all CpG dinucleotides. Bisulphite sequencing of methylated DNA displayed ~99.5% of CpG methylation of candidate genes. We created a reduced methylation DNA control by incubating Jurkat cells with a 2?M concentration of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-Azadc) for eight days. The genomic DNA derived from cells treated with this drug exhibited some lower molecular weight smearing when visualized on a 0.8% agarose gel. Bisulfite conversion and sequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normally methylated revealed significant CpG demethylation. Used in conjunction CpG methylated and unmethylated genomic DNA controls may serve as powerful tools in the investigation of DNA methylation in the genome.
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Barna Fodor Abstract P40 Identification of novel pericentric proteins by their localization
Barna D. Fodor, Mario Richter, Manuela Scaranaro and Thomas Jenuwein
Research Institute of Molecular Pathology (IMP) Dr. Bohrgasse 7, A-1030 Vienna, Austria
The eukaryotic genome is organized into distinct euchromatic and heterochromatic subdomains. Heterochromatin has fundamental roles in the structural organization of chromosomes, genome stability, and controlling epigenetic programs. Genetic screens in S. pombe and Drosophila have identified a number of genes called suppressors of variegation [Su(var)s] most of which encode components of heterochromatin. These screens exploit the transcriptional silencing effect of heterochromatin on juxtaposed reporter genes, which is compromised in Su(var) mutants (Schotta et al. 2003). Another principle to discriminate heterochromatin components is their characteristic localization in cells. In mouse cells pericentric heterochromatin forms typical (DAPI dense) foci. Co-localization of epitope tagged factors to these foci can easily be detected. Localization screens based on this principle, were also successful in identifying heterochromatin associated proteins (Dellaire et al. 2003). However, the potential of this approach was not yet fully exploited. In this study we applied genetrapping to identify novel components of heterochromatin. The transduced genetrap constructs randomly integrate in the genome, generating translational fusions of endogenous loci and the GFP transgene. We observed characteristic localization patterns of the expressed GFP fusion product for a number of clones, proving the utility of our experimental design. The interesting clones were picked, and expanded. The identity of the trapped genes can be determined by 5’- and 3’-races. We are now extending our studies to a number of cell lines with a variety of genetrap constructs.
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Abstracts - Poster
Maria Fousteri Abstract P41 Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo
Anne Jensen and Leon H.F. Mullenders
Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 RC Leiden, The Netherlands
Restoration of UV-inhibited transcription requires removal of transcription-blocking DNA lesions by transcription-coupled repair (TCR), a specialized repair pathway that removes UV induced photolesions from transcriptional active genes. In mammals TCR is dependent on CSA and CSB proteins, however, their function and interactions are not well understood. CSA is a WD-40 repeat protein and a component of an E3-ubiquitin ligase, while CSB is a DNA-dependent ATPase that shares homology with SW2/SNF2 chromatin remodelers. Mutations in any of the two genes that encode for CSB and CSA lead to a rare recessive neuro-developmental progeroid-like disorder, Cockayne syndrome (CS). Currently, information is lacking on the exact composition and molecular interactions of an active TCR complex. Our aim was to improve understanding of TCR in vivo by isolation and analysis of lesion-stalled transcription elongation complexes. We have used in vivo crosslinking and ChIP and we examined the recruitment of specific repair and chromatin remodeling factors to a UV-stalled RNAPII in TCR-proficient and -deficient human cells. The protocol was modified in such a way that it enabled the analysis of co-immunoprecipitated proteins that reside in close proximity on damage containing chromatin fragments. Our study revealed that CSB and CSA display differential roles in recruitment of TCR-specific factors and that assembly for TCR in vivo occurs without disruption of the UV-stalled RNAPIIo. CSB fulfills a key role as coupling factor in the assembly of a chromatin-bound TCR complex that involves histone acetyltransferase p300, nucleotide excision repair (NER) proteins and CSA-DDB1 E3-ubiquitin ligase complex with the COP9 signalosome. CSA is dispensable for attraction of NER proteins, yet is required to recruit XAB2, the nucleosomal binding protein HMGN1 and transcription cleavage factor TFIIS. This approach highlights the essential roles of CS proteins in TCR complex formation and provides a molecular link between damage recognition, chromatin remodelling and NER.
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Robert Gillespie Abstract P42 Retinoid regulated association of transcriptional coregulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARß2, and Cyp26A1 in F9 embryonal carcinoma cells
Robert F. Gillespie and Lorraine J. Gudas
Molecular Biology Program of Weill Graduate School of Medical Sciences and Pharmacology Department of Weill Medical College of Cornell University, New York, New York 10021, U.S.A.
Hox gene expression is activated by retinoic acid (RA) binding to Retinoic Acid Receptor- Retinoid X Receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRα, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARß2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells during RA treatment. We demonstrate that the association of RARγ-RXRα with RAREs before and during RA treatment remains relatively constant. pCIP, p300, and RNA polymerase II levels at target RAREs also varied by gene, though RA increased the associaton of these proteins with RAREs. Conversely, SUZ12 was associated with all RAREs studied and this association was attenuated by RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of coregulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.
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Clara Goday Abstract P43 Chromatin modifications in germline chromosomes of sciarid flies
P.G. Greciano and C. Goday
Ramiro de Maeztu 9, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain
A classic example of programd chromosome elimination and genomic imprinting is found in sciarid flies (Diptera, Sciaridae), where whole chromosomes of paternal origin are selectively discarded from the genome during development. In early germ cells a single paternal X chromosome is eliminated in embryos of both sexes and in male meiotic cells the whole paternal complement is discarded. In sciarids, differential acetylation of histones H3 and H4 occurs between chromosomes of different parental origin, both in early germ nuclei and in male meiotic cells. We here investigated histone methylation modifications between chromosomes in germline cells of Sciara ocellaris. In early germ nuclei, maternal chromosomes show high levels of di- and trimethylated histone H3 at lysine 4, whereas this histone modification is not detected in paternal chromosomes. In male meiosis, only the eliminated paternal chromosomes exhibit high levels of di- and trimethylated histones H3 at lysine 4 and dimethylated H4 at lysine 20. In early germ nuclei, RNA polymerase II associates to maternally-derived chromosomes but lacks phosphorylation of the carboxy- terminal domain on serine 2. The results suggest that histone H3 methylation at lysine 4 does not correlates with transcriptional activity in early Sciara germline nuclei. Our results supports that specific covalent chromatin modifications such as histone acetylation/methylation are involved in the imprinted behaviour of germline chromosomes in Sciara.
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Aaron D. Goldberg Abstract P44 HIRA-dependent incorporation of histone H3.3 marks active genes in mouse embryonic stem cells
Aaron D. Goldberg1, Fyodor D. Urnov4, Ileana M. Cristea2, Chingwen Yang3, Jeffrey Miller4, Carlos Ramos5, Marco Seandel5,6, Daylon James5, Sandra B. Hake1,7, Peter J. Scambler8, Brian T. Chait2, Philip D. Gregory4, Shahin Rafii5,6,9 and C. David Allis1
1Laboratory of Chromatin Biology, 2Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, and 3Gene Targeting Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY, 10021, U.S.A. 4Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501, Canal Blvd, Suite A100 Richmond, California 94804, U.S.A. 5Department of Genetic Medicine, 6Department of Medicine, Division of Hematology-Medical Oncology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, U.S.A. 7Adolf-Butenandt-Institut, Molekularbiologie, Ludwig-Maximiliams-Universitaet, Schillerstr. 44, 80336 Muenchen, Germany. 8Molecular Medicine Unit, Institute of Child Health, London WC1N 1EH, U.K. 9Howard Hughes Medical Institute, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, U.S.A.
The histone H3 variant H3.3 has been shown to be associated with transcriptionally active genes in multiple organisms, and to be retained in some loci after transcription has ceased. (Ahmad and Henikoff 2002; Schwartz and Ahmad 2005) It is tempting to speculate that H3.3 associates with transcriptionally permissive chromatin in differentiating mammalian cells, and that this localization has functional importance in the establishment or maintenance of gene activation. However, it is difficult to address these questions due to the lack of available tools or antibodies to distinguish H3.3 from the other H3 variants H3.2 or H3.1. Here we describe a rapid and efficient method that uses engineered zinc finger protein nucleases and a short, selection-less, promoter-less targeting construct to introduce an enhanced yellow fluorescent protein (EYFP) sequence into the C-terminal coding exon of the endogenous histone H3.3B gene in mouse embryonic stem (ES) cells. In work in progress, we have used these heterozygous H3.3B-EYFP tagged ES cells to show that H3.3 is excluded from H3K9me3-rich pericentric heterochromatin on metaphase chromosomes, and partially but incompletely co-localizes with regions of H3K4me3. Through chromatin immunoprecipitation (ChIP) assays, we show that H3.3 is significantly enriched at the promoters, depleted at the transcriptional start sites, and enriched into the 3’ ends of pluripotency genes Oct4 and Nanog in undifferentiated ES cells. As a control for the specificity of H3.3 localization, we have also used our method to introduce point mutations in H3.3B, generating EYFP-tagged H3.2 expressed from the endogenous histone H3.3B locus. Unlike H3.3, H3.2-EYFP is diffusely localized throughout metaphase chromosomes, and is not excluded from pericentric heterochromatin. Finally, we have used our method to visualize the subcellular localization of H3.3 in ES cells homozygous for a knockout of the putative H3.3 chaperone HIRA, demonstrating that H3.3 incorporation into metaphase chromosomes is HIRA-dependent. These studies confirm and extend studies carried out in other non-mammalian models and provide a “proof-of-principle” of an approach permitting the tagging of key chromatin constituents for analyses in mammalian cells.
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Elizabeth Goneska Abstract P45 Phosphorylation of the SQ H2A.X motif is required for proper meiosis and mitosis in Tetrahymena thermophila
Xiaoyuan Song*1,3, Elizabeth Goneska*1,2, Qinghu Ren*1,4, Sean D. Taverna2, C. David Allis2 and Martin A. Gorovsky1
1Department of Biology, University of Rochester, Rochester, NY, U.S.A., 2Laboratory of Chromatin Biology, The Rockefeller University, New York, NY, U.S.A., 3School of Medicine, UCSD, La Jolla, CA, U.S.A., 4The Institute for Genomic Research, Rockville, MD, U.S.A.,*These authors contributed equally to this work
Phosphorylation of the H2A.X C-terminal SQ motif is required for efficient DNA double- strand break (DSB) repair in diverse organisms. Here we show that H2A.X, one of the two major H2As in Tetrahymena, is phosphorylated at serine 134 in response to DSBs induced by chemical agents and during prophase of meiosis I. Using strains containing a mutation (S134A) that abolishes this phosphorylation, we demonstrate that phosphorylation of the SQ motif is required for normal micronuclear meiosis and mitosis, and to a lesser extent, for normal amitotic macronuclear division. H2A.X phosphorylation is also important for Tetrahymena cells to recover from exogenous DNA damage, and its absence, while not lethal, leads to extensive accumulation of DSBs in both micro- and macronuclei.
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Susana Gonzalo Abstract P46 Telomere epigenetic modifications: a control of telomere length and a stop on recombination
Susana Gonzalo1,3, Isabel Jaco1, Roberta Benetti1, Gunnar Schotta2, Peter Klatt1, Thomas Jenuwein2 and María A. Blasco1
1Telomeres and Telomerase Group. Molecular Oncology Program. Spanish National Cancer Centre (CNIO). Madrid E-28029, Spain., 2Research Institute of Molecular Pathology (IMP). The Vienna Biocenter. A-1030 Vienna, Austria, 3Department of Radiation Oncology. Washington University School of Medicine. St. Louis, MO 63108, U.S.A.
Telomeres protect chromosome ends from being recognized as double strand breaks. Alterations in the binding of telomere proteins or erosion of telomeric DNA below a critical threshold lead to telomere instability, activation of the DNA damage response pathway, cell cycle arrest, and senescence or apoptosis. To avoid growth inhibition, cells maintain telomere length either by telomerase or alternative lengthening of telomeres mechanism (ALT). Recently, we and other investigators have demonstrated that telomere metabolism can also be regulated epigenetic modifications. Our studies identified a number of repressive chromatin-modifying activities that participate in the assembly of telomeric chromatin in mouse cells. In particular, the abrogation of histone methyltransferases (HMTases) Suv39h1 and h2, results in defective trimethylation of telomeric histone H3 at lysine 9 (H3K9me3), leading to telomere elongation. These observations, together with a report showing telomere erosion upon heterochromatin protein 1 (HP1) over- expression in human cells, was the first demonstration that epigenetic alterations of mammalian telomeres can lead to telomere length deregulation. Furthermore, loss of DNA methyltransferases Dnmt1 or Dnmt3a/3b, resulting in DNA hypomethylation of subtelomeres, or loss of Retinoblastoma (Rb) family function, affecting trimethylation of telomeric histone H4 at lysine 20 (H4K20me3) and global DNA methylation, lead to telomere elongation. Most recently, we have observed decreased telomeric H4K20me3 and telomere length deregulation in cells deficient for Suv4-20h1 and h2 HMTases, suggesting that these enzymes are responsible for this chromatin modification at telomeres. Based on the available data, we propose the hypothesis that the acquisition of a heterochromatic “condensed” structure at the telomere restricts the access of telomere elongating machineries in order to ensure telomere homeostasis. The “opening” of telomeric chromatin by loss of repressive chromatin marks or an excessive heterochromatinization by up-regulation of these marks could facilitate or further restrict the accessibility of telomere elongating activities, leading to telomere elongation or erosion, respectively. In fact, we have recently found an increased frequency of telomeric sister chromatid exchange (T-SCE) events in cells lacking Dnmt1 or Dnmt3a/3b, and also in cells lacking HMTases Suv39h1/h2 or Suv4- 20h1/h2, by the Chromosome Orientation FISH technique (CO-FISH). This technique is a read-out of recombination among sister telomeres, a hallmark of activation of ALT mechanism of telomere elongation. In summary, we conclude that modifications of telomeric chromatin serve to control telomere length and impose a stop on recombination.
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Tanya Gustafson Abstract P47 Epigenetic silencing of Singleminded-2 in breast cancer
Tanya Gustafson1, Keelan Anderson1, Mike Kladde2 and Weston Porter1
1Texas A&M College of Veterinary Medicine, Department of Integrative Biosciences, College Station, TX 77843, 2Texas A&M University, Department of Biochemistry and Biophysics, College Station, TX 77843, U.S.A.
Epigenetic gene regulation has been identified as a crucial event in breast cancer development. Epigenetic mechanisms of regulation include covalent modifications of histones and DNA methylation. These events occur throughout all stages of breast tumorigenesis and are a more common alternative to deletions or mutations for inactivating breast tumor suppressor genes. The human gene Singleminded-2 (Sim2) is a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of transcription factors, which includes genes responsible for maintenance of circadian rhythms (Per), sensors of hypoxia (Hif1α) and environmental contaminants (AhR). We have recently shown that Sim2 has tumor suppressor activity in the breast. Sim2 is expressed highly in normal breast cell lines, but expression is lost in invasive breast cancer cells. The purpose of this work is to elucidate the epigenetic mechanisms of Sim2 silencing in breast cancer cells. To determine whether Sim2 was silenced through epigenetic mechanisms, highly invasive breast cancer cells (MDA435) and normal breast epithelial cells (MCF10A) were treated with a demethylating agent and a histone deacetylase inhibitor. Sim2 expression was increased by treatment in MDA435 cells, but did not change in MCF10A cells. Bisulfite sequencing of the CpG islands in MCF10A, MCF7 (mildly invasive breast cancer cells) and MDA435 cells revealed that methylation of a large island in exon 1 correlates with Sim2 expression levels. Chromatin immunoprecipitation demonstrated that the Sim2 promoter is hyperacetylated in MCF10A cells and heterochromatic in MDA435 cells. The novel technique, MAP-IT, has been used to simultaneously characterize DNA methylation and chromatin structure on individual molecules. Overexpression of DNA methyltransferases has led to methylation, decreased expression and decreased histone acetylation of the Sim2 gene. The data support Sim2 silencing through epigenetic mechanisms during breast cancer progression.
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Soon-Ki Han Abstract P48 Role of plant CBP/p300-like genes in the regulation of flowering time
Soon-Ki Han1,3, Ju-Dong Song2,3 , Yoo-Sun Noh2,3 and Bosl Noh1,3
1Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. 2Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea. 3Global Research Laboratory for Flowering at SNU and UW, Seoul National University, Seoul 151-742, Korea
CREB-binding protein (CBP) and its homolog p300 possess histone acetyltransferase (HAT) activity and function as key transcriptional coactivators in the regulation of gene expression that controls differentiation and development in animals. However, the role of CBP/p300-like genes in plants is not yet elucidated. Here, we show that Arabidopsis CBP/p300-like genes promote flowering through affecting the expression of a major floral repressor FOWERING LOCUS C (FLC). Although animal CBP/p300 generally function as coactivators, Arabidopsis CBP/p300-like proteins are required for the negative regulation of FLC. This CBP/p300- mediated FLC repression might involve reversible protein acetylation independent of histone modification within FLC chromatin.
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Christin Hanigan Abstract P49 Identification of an HDAC2 mutation in colorectal cancer and its consequences
Christin L Hanigan, Manon van Engeland, James Eshleman and James Herman
Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, U.S.A., Department of Pathology, University Maastricht, 6200 MD Maastricht, The Netherlands
Histone deacetylases are an important component of chromatin remodeling machinery associated with transcriptional repression. Specifically, these proteins have been shown to aid in silencing tumor suppressor genes. While these proteins regulate a number of genes and cellular processes, we are only beginning to understand what genes and processes regulate them. One recent study showed mutations in the Wnt signaling pathway can lead to an upregulation of a histone deacetylase, HDAC2. Microsatellite instability can lead to increased mutation rates in genes that contain mono, di, and tri-nucleotide repeat tracts. We have found taht HDAC2 has a poly(A) tract in exon1, which in some MSI+ colon cancer cells is mutated leading to a frameshift and premature stop codon. We have observed the apoptotic resistance fo pharmacologically inhibiting HDACs in cell lines lacking HDAC2. We propose that HDAC2’s regulation of APAF-1 may play a role in this resistance. We have found HDAC2 on the APAF-1 promoter and see up-regulation of APAF-1 at the mRNA level only in cell lines that have functional HDAC2 and are responsive to HDAC inhibition. We suggest that SAHA treatment up-regulates APAF-1 as one mechanism of inducing cell death. Cell lines that have lost HDAC2 already have compensated for high levels of APAF- 1 and are resistant to HDAC inhibition induced cell death.
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Troy Harkness Abstract P50 Rsp5 is required for nuclear shuttling of the Snf1 kinase complex in yeast
Terra G. Arnason, Megan D. Dash, Gerald F. Davies and Troy A. A. Harkness
Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada
Chromatin assembly in yeast is regulated by a complex molecular network governed at least in part by the ubiquitin ligases Rsp5p, the Anaphase Promoting Complex (APC) and the SCF. We have shown that Rsp5p, localized exclusively to the plasma membrane and adjacent to vacuoles, triggers nuclear APC activity by blocking the activity of APC inhibitors, such as the SCF. The APC then initiates replication-independent, but CAF-I-dependent, chromatin assembly. Here, we demonstrate another mechanism leading to Rsp5p- dependent APC activity. Mutation to RSP5 leads to increased histone H3 phosphorylation and decreased histone H3 acetylation at elevated temperatures. We show that the histone H3 kinase, Snf1p, is required for the rsp5 phenotype. Interestingly, we previously demonstrated that the Snf1 kinase complex, which shuttles across the nuclear membrane, is required for APC activity. Thus, we propose that Rsp5p is required for the transit of Snf1p across the nuclear membrane. In support of this theory, we show that GFP-tagged Snf1p, Snf4p (activator subunit) and Gal83p (localizing subunit) all fail to localize to the nucleus upon carbon stress in rsp5 mutant cells. Similarly, the GFP-tagged Snf1p target, Mig1p, failed to exit the nucleus in rsp5 mutants. We next asked whether Snf4p, which requires ubiquitination for stability and function, requires Rsp5p or any of the Rsp5p associated E2 enzymes. In ubiquitin coimmunoprecipitation (CoIP) experiments, we recovered GST-Snf4p bound to ubiquitin, but not GST alone. We observed that carbon stress induced an increase in ubiquitinated GST-Snf4p in wild type cells. When ubiquitin was CoIPed from ubc4∆ ubc5∆ cells, GST-Snf4p was again recovered, but we failed to observe induction of ubiquitinated GST-Snf4p upon carbon stress. The influence of i) Rsp5p, ii) the Snf1p and Rsp5p interacting protein, Rod1p, and iii) Ubc7p, an E2 that physically interacts with Rsp5p, on Snf4p ubiquitination will be discussed.
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Tiffany Hung Abstract P51 ING4 recognition of histone H3 trimethylated at lysine 4
Tiffany Hung, Xiaobing Shi and Or Gozani
Stanford University, 371 Serra Mall, Stanford, CA 94305, U.S.A.
ING4 is a candidate tumor suppressor protein and member of the evolutionarily conserved ING (Inhibitor of Growth) family of chromatin-regulatory proteins. ING4 is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and is thought to link HAT activity and tumor suppression. Here we present in vitro and in vivo evidence that the PHD finger (plant homeodomain) module of ING4 specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). This modification is an epigenetic hallmark of active transcription, and we are testing the model that recognition of H3K4me3 by the ING4 PHD finger is important for gene activation via an increase in HBO1-dependent acetylation at nearby nucleosomes.
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David Johnson Abstract P52 E2F1 and GCN5 facilitate the recruitment of nucleotide excision repair factors to sites of UV-induced DNA damage
Ruifeng Gou, David L. Mitchell, Thomas R. Berton and David G. Johnson
The University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis, Science Park-Research Division, PO Box 389, 1808 Park Road 1-C, Smithville, TX 78957, U.S.A.
The E2F1 transcription factor regulates the expression of genes involved in cell cycle progression, apoptosis, differentiation, and DNA repair. In addition, emerging data suggests that E2F1 has a transcription-independent function in response to at least some forms of DNA damage. We find that E2F1 localizes to sites of DNA damage caused by ultraviolet (UV) radiation and that this requires the ATR kinase and phosphorylation of E2F1 at serine 31. In the absence of E2F1 the recruitment of nucleotide excision repair (NER) factors, such as XPC and XPA, to sites of UV damage is impaired. This correlates with a defect in DNA repair and an increased sensitivity to UV-induced apoptosis in cells lacking E2F1. The GCN5 histone acetyltransferase also accumulates at sites of UV-induced DNA damage. Moreover, GCN5 associates with E2F1 in response to UV exposure and knocking down E2F1 expression impairs co-localization of GCN5 with damaged DNA. Like E2F1, GCN5 is also important for the efficient recruitment of repair proteins to sub-nuclear regions containing UV damage. These findings indicate that phosphorylation mediated by ATR converts E2F1 into a DNA repair factor that localizes to UV damaged DNA. E2F1 then promotes DNA repair by recruiting GCN5, and in turn, the NER machinery to sites of damage.
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Paul Kalitsis Abstract P53 Nucleosome spacing analysis of repeat DNA regions in the mouse genome
Paul Kalitsis, Sheena Rigby and K.H. Andy Choo
Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne 3052, Australia
Mammalian genomes consist of highly repeated satellite DNAs that are found in gene-poor regions such as centromeres and telomeres. In the mouse genome the proximal telomere is within 11 kb of the centromeric minor satellite DNA. These chromosome structures have separate and unique roles in chromosome segregation and stability. To investigate the relationship between these two domains we have examined the primary chromatin structure by partially digesting mouse chromatin with micrococcal nuclease from a variety of cell lines and tissues. Each digested chromatin extract was run on an agarose gel and hybridised with a representative repeat probe from telomeric, centromeric and peri-centromeric domains. Furthermore, we examined the effects on nucleosomal spacing in cell lines that were treated with chemical agents that perturb chromatin structure and differentiate cells.
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Min-Jeong Kang Abstract P54 Role of a RPD3/HDA1 family histone deacetylase in the regulation of phytochrome-mediated light respases in Arabidopsis
Min-Jeong Kang1,3 , Soon-Ki Han2,3, Yoo-Sun Noh1,3 and Bosl Noh2,3
1Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea. 2Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. 3Global Research Laboratory for Flowering at SNU and UW, Seoul National University, Seoul 151-742, Korea
Posttranslational acetylation of histone N-terminal tail is well known to influence gene transcription by changing chromatin structures: it relaxes the association of histone proteins with DNA, displacing nucleosomes from the promoters of genes. Lately, histone acetylation has also been thought to provide surfaces which transcription activators or repressors recognize and bind to. Histone acetylation is reversibly regulated by histone deacetylases (HDACs) that are categorized into three major groups; the RPD3/HDA1 superfamily, the SIR2 family, and the HD2 family. Arabidopsis has 10 members of HDACs belonging to the RPD3/HDA1 superfamily. In order to address the biological roles of the RPD3/HDA1 family HDACs in plants, we isolated the loss of function mutants of Arabidopsis HDACs by a reverse genetics and have characterized their phenotypes. We found the mutation in one of the HDACs causes hypersensitivity to the light-mediated inhibition of hypocotyl elongation. Genetic analyses showed the mutation in the photoreceptor phyB is epistatic to the hdac mutation in the hypocotyl elongation. More data indicating the role of the HDAC in the regulation of light responses will be presented.
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Panagiota Karagianni Abstract P55 ICBP90, a putative link between histone ubiquitination and cell cycle progression
Panagiota Karagianni, Jun Qin and Jiemin Wong
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston TX 77030, U.S.A.
ICBP90 was initially identified as a transcription factor that can regulate Topoisomerase IIα expression. The primary sequence of ICBP90 contains a Ubiquitin like, a SET and RING Associated (SRA), a PHD and two RING domains. It was recently shown that ICBP90 exhibits auto-ubiquitination activity. Since a murine protein with high sequence identity with ICBP90, Np95, has been shown to bind chromatin through the SRA domain as well as ubiquitinate core histones in vitro, we wanted to examine if ICBP90 can also function as a ubiquitin ligase for histones. We have found that ICBP90 can bind to chromatin and ubiquitinate histone H3 both in vitro as well as in transfected cells, in a RING domain- dependent fashion. In fact, ICBP90 can poly-ubiquitinate H3 in vitro, which provides, to our knowledge, the first paradigm of histone H3 poly-ubiquitination. By using mass spectrometry we have mapped Lysine 79 of histone H3 as the site of covalent ubiquitin linkage. ICBP90 has been previously shown to be required for G1/S as well as G2/M transition. We are currently addressing the potential role of ICBP90-mediated histone ubiquitination in cell cycle progression.
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Emmanuel Kas Abstract P56 Altering the structure and functional properties of heterochromatin with satellite-specific minor-groove binders
Roxane Blattes, Guillaume Susbielle, Caroline Monod and Emmanuel Kas
LBME - UMR5099 CNRS/UPS, IBCG, 118 route de Narbonne, 31062 Toulouse cedex 9, France
Repeated AT-rich sequences constitute an enormous target for DNA minor-groove binders such as oligopyrrole polyamides and diamidines. These sequence arrays are highly enriched in pericentric heterochromatin, but also in the genomes of certain pathogenic parasitic microorganisms, or portions thereof as in the kinetoplast DNA of trypanosomes. We have previously shown that targeting of these sequences is easily achieved in vivo and is extraordinarily specific. The D1 protein of Drosophila melanogaster is associated with the 359-bp 1.688 g/cm3 satellite III (SAT III) repeats that are cleaved by topoisomerase II (topo II) in vivo. We show that synthetic minor-groove binders that selectively target SAT III sequences alter the nuclear localization of topo II or interfere with its enzymatic activity to induce modifications of position-effect variegation (PEV). P9, a satellite-specific polyamide, affects a D1-dependent pathway that directs topo II to the SAT III array and also perturbs the association of HP1 with heterochromatin. In contrast, synthetic diamidines uncouple the topo II/D1 interaction and cause a massive D1-independent relocalization of topoisomerase II to AT-rich heterochromatin. This mobilization in turn results in a suppression of PEV. We propose that synthetic and natural AT-specific minor groove binders act coordinately with topo II to effect assembly of specialized nucleoprotein structures such as heterochromatin. The antiparasitic diamidines used here also target AT-rich DNA sequences in their target microorganisms. Their effects on the localization of topo II provide keys to understanding their mechanism of biological action, which most likely reflects an anti-genome activity that interferes with essential nucleoprotein complexes involving AT-rich sequences.
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Chul Geun Kim Abstract P57 PIAS1 confers erythroid cell specific α-globin gene regulation by the CP2 transcription factor family
Ho Chul Kang, Kyung Sook Choi, Hyen Seok Heo, Tae Ho Jeon and Chul Geun Kim
Department of Life Science, Hanyang University, Seoul 133-791, Korea
CP2c, a member of CP2 transcription factor family genes, was discovered initially in mouse as a transcription factor that binds to and stimulates transcription from the α-globin promoter. We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of α-globin through interactions with CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon, and protein inhibitor of activated STAT1 (PIAS1) (Kang et al., 2005). Indeed, significant reduction of α-globin expression was observed in RNAi-mediated knockdown of CP2c, CP2b or PIAS1 in erythroid cells, indicating that these three factors are indispensable components for α-globin expression. However, the mechanisms by which how these three factors confer erythroid-specific activation of α-globin in vivo are unsolved. Here we report that PIAS1 confers erythroid cell specific α-globin gene regulation by the CP2 transcription factor family. We find that CP2c is mostly present in the cytoplasm, whereas both CP2b and PIAS1 are solely in the nucleus and CP2a is in the cytoplasm. Interestingly, overexpression of CP2b or PIAS1 induces nuclear translocation of CP2c. Furthermore, the strong DNA binding activity of CP2c which was destabilized by CP2b is restored by supplementation of PIAS1 into the EMSA reaction, suggesting that PIAS1 induces the DNA/CP2b/CP2c/PIAS1 quaternary complex formation in the nucleus. We confirmed that all three factors interact with each other using their two discrete binding domains, and proteins containing minimally two tethered CP2b/CP2c binding domains of PIAS1 are sufficient to maintain the high level of DNA binding and transcriptional activities of CP2b/CP2c. Taken together, our data suggest that PIAS1 commands the protein-protein interaction, subcellular localization, and DNA binding abilities of the CP2 transcription factor family to confer erythroid cell specific α-globin gene regulation.
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Keun Il Kim Abstract P58 A novel link between SUMO modification of a chromatin remodeling complex and cancer metastasis
Sung Hee Baek1, Jung Hwa Kim1, Hee June Choi1, Bogyou Kim1, Ji Min Lee1, Ik Soo Kim1 and Keun Il Kim2
1Department of Biological Sciences, Seoul National University, Seoul 151-746, South Korea, 2Department of Biological Sciences, Sookmyung Womens University, Seoul 140- 742, South Korea
Defining the functional modules with transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. We have reported the dynamic role of a b-catenin/reptin chromatin remodeling complex to regulate a metastasis suppressor gene KAI1, which is capable of inhibiting the progression of tumor metastasis, and further which signaling factors confer repressive function on reptin and hence maintain a repressed state of KAI1 (Kim et al., Nature 434, 921-6; Kim et al., Nature Cell Biol. 8, 631-9). Biochemical purification of a reptin-containing complex has revealed the presence of specific deSUMOylating enzymes that reverse the SUMOylation of reptin that underlies its repressor function. DeSUMOylation of reptin alters the repressive function of reptin and its association with HDAC1. Further, SUMOylation status of reptin modulates the invasive activity in cancer cells with metastatic potential. This provides a clear definition of the functional model and a novel insight for linking SUMO modification to cancer metastasis. As a follow-up study, we will address novel findings on the function of newly identified histone methyltransferase as a component of reptin, linking chromatin remodeling process and cancer metastasis.
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Abstracts - Poster
Sarah Kimmins Abstract P59 Methylation of Histone H3 at lysine 4 is dynamic and tightly regulated during male germ cell development
Véronik Auger, Maren Godmann and Sarah Kimmins
Departments of Animal Science and Pharmacology and Therapurtics, McGill University, Montreal, Canada
Idiopathic male infertility is associated with genetic and epigenetic abnormalities. Histones can undergo epigenetic modifications on the N-terminus including methylation, acetylation and phosphorylation, among others. These histone modifications are hypothesized to signal changes in chromatin structure leading to altered gene expression and recruitment of regulatory transcription complexes. To date there is little information on the distribution and significance of histone modifications in spermatogenesis. We have identified the cellular localization patterns and developmental regulation of histone H3 mono-, di-, and tri- methylation at lysine 4 (K4), and the epigenetic modifiers implicated in the, removal and reading of these epigenetic marks namely, LSD1, MBD2a/b, and HDAC1, during mammalian spermatogenesis. Testis were collected from the first wave of spermatogenesis in mice at postnatal days 6 (type A spermatogonia), 8 (type A and B spermatogonia), 10 (pre-leptotene and leptotene), 12, (zygotene), 14 (early pachytene), and 20 (late pachytene) for immunolocalization and Western blot analysis. Patterns of distribution were further confirmed using highly specific cell isolation and staging methods. Histone H3-K4 mono-, di- and tri-methylation is dynamic and widely distributed in spermatogenic cell types and is associated with euchromatic regions. This strongly suggests that as in other tissues, in the testis, methylation of histone H3 at lysine 4 serves key functions in the regulation of gene transcription. Expression of LSD1 is tightly regulated during germ cell differentiation. Remarkably, in comparison to somatic tissues, LSD1 is preferentially expressed in the testis. Interaction studies reveal unique transcriptional regulatory complexes associated with H3- K4 methylation in the testis including the association of LSD1 and MBD2b in a complex with HDAC1, presumably forming transcriptional repressor complexes. These studies serve to enhance our understanding of epigenetic control of the transcriptional program governing male germ cell differentiation in normal and pathological states.
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Robert Klose Abstract P60 JmjC-domain-containing proteins and histone demethylation
Robert J. Klose and Yi Zhang
Howard Hughes Medical Institute, Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, U.S.A.
Histone methylation plays important roles in regulation of gene expression, maintenance of genome integrity, and epigenetic inheritance. A wealth of understanding is available about the histone methyltransferase enzymes which place these covalent modifications, but, until recently, enzymes capable of reversing histone methylation remained elusive and histone methylation was thought to be a relatively static modification. The identification of lysine specific demethylase1 (LSD1) revealed that histone methylation could be dynamically regulated in a manner similar to histone acetylation and phosphorylation. Recently the Jumonji C (JmjC) domain has been shown to possesses Fe(II)/alpha-ketoglutarate dependent histone demethylase activity. Bioinformatic analysis of the extended JmjC- domain containing family of proteins has enabled us to categorize these proteins into seven evolutionarily conserved groupings based on homology within the JmjC-domain and overall protein domain architecture. By analysing the predicted co-factor binding sites within individual JmjC-domain groupings we have been able to utilize a targeted approach to characterize additional JmjC-domain containing histone demethylases. Based on our analysis we recently identified a novel histone demethylase, JHDM3A, which has the capacity to reverse the tri-methyl lysine modification mark on histone H3K9/36. Our continued functional analysis has revealed additional histone demethylases within the JmjC- domain containing family of proteins.
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Christoph M. Koch Abstract P61 The landscape of activating histone modifications across 1% of the human genome
Christoph M. Koch, Robert M. Andrews, Paul Flicek, Shane C. Dillon, Ulas Karaoz, Gayle K. Clelland, Sarah Wilcox, Dave M Beare, Joanna C. Fowler, Phillippe Couttet, Keith D. James, Gregory C. Lefebvre, Alexander W. Bruce, Oliver M. Dovey, Peter D. Ellis, Pawandeep Dhami, Cordelia F. Langford, Cordelia F. Langford, Zhiping Weng, Ewan Birney, Nigel P. Carter, David Vetrie and Ian Dunham
The Wellcome Trust Sanger Institute and European Bioinformatics Institute Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K., Bioinformatics Program and Biomedical Engineering Department Boston University, 24/44 Cummington St., Boston, MA 02215, U.S.A.
The NHGRI has established a pilot project (ENCODE) to explore computational and experimental methods to develop an encyclopaedia of DNA elements in the human genome. Initially the project targets 1% of the genome chosen according to the criteria outlined at http://www.genome.gov/10506161. We have constructed a microarray representing the 44 ENCODE regions consisting of 24005 PCR fragments with an average size of ~1 kb. It covers ~80% of the targeted regions including repetitive elements where possible. We are using this microarray to assay DNA samples enriched for sequences involved in specific biological processes and functions generated by chromatin immunoprecipitation (ChIP). ChIP experiments are being performed with a variety of antibodies for specific histone modifications in a lymphoblastoid cell line (GM06990), an erythroleukemia cell line (K562) , foetal lung fibroblastoid cell lines (IMR90, HFL-1), cervix carcinoma cell line (HeLaS3), a T-cell line (MOLT4) and a chimpanzee cell line PTR8. We correlate maps of histone modifications with a range of genomic DNA features including C+G content, genes/exons, repeat elements, SNP density and regions of conserved DNA sequence identified by comparative sequencing across multiple species as well as the expression profiles of the cell lines. Preliminary analysis reveals strong enrichments of acetylated histone H3 and di- and tri-methylated histone H3 (H3K4me2 and H3K4me3) at 5 ends of transcriptional start sites. Mono-methylated histone H3 (H3K4me1) was found to be widely distributed and not exclusive focussed to transcriptional start sites similar to acetylated histone H4 (H4ac). While comparing enrichments between different cell lines we found a correlation of the expression status of genes and the absence or presence of H3K4me3 at the transcriptional start site/promoter. Active promoters in each cell line show a robust enrichment of H3K4me3 while inactive promoter do not.
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Ryoki Kujiki Abstract P62 1alpha,25(OH)2D3-induced transrepression on 1alpha- hydroxylase gene promoter mediates chromatin remodeling through WINAC
R. Fujiki1, M. Kim1, Y. Sasaki1, H. Kitagawa1 and S. Kato1,2
1Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan, 2ERATO, Japan Science and Technology Agency
Vitamin D receptor (VDR) is a nuclear receptor (NR) regulating bone metabolism and calcium homeostasis. VDR modulates many target genes in a ligand-dependent manner. VDR-mediated gene expression requires a large number of co-regulator complexes. Recently, we identified WINAC as a novel VDR co-regulater complex that reconfigures nucleosomal array around vitamin D response element (VDRE) (Cell, 113, 905, 2003). While mechanism of transactivation by NRs is well understood, the molecular basis of the ligand-induced repression remains to be uncovered. To address this issue, we focused on the 1a,25(OH)2D3-induced transrepression mechanism of 25(OH) D3 1a-hydroxylase [1a(OH)ase] gene, encoding a key enzyme of vitamin D metabolism. The transactivation function by VDIR, a transcriptional factor recognizing negative VDRE in 1a(OH)ase promoter (1anVDRE) (EMBO J. 23, 1598, 2004), was found to be suppressed by liganded VDR and/or WSTF, a WINAC major component. In co-immunoprecipitation assay, WSTF physically interacts with both unliganded VDR and acetylated histones. ChIP assay showed that this interaction presumably allowed DNA- unbound VDR to associate with the 1anVDRE region prior to ligand binding. Using mouse embryonic fibloblasts from VDR knockout mice, we further found that VDR is required for the association of WSTF with 1a(OH)ase promoter, implying 1anVDRE-specific association of WSTF is defined by VDR. Next, we showed in vitro that the association of WSTF with acetylated histones is required for assembly of liganded VDR with VDIR bound to the 1anVDRE. WSTF bromodomain was then mapped as interaction surface to acetylated histone H3. Interestingly, WSTF with deleted bromodomain acts as a dominant negative mutant in the transrepression of 1a(OH)ase gene. All together, WSTF association with both unliganded VDR and acetylated nucleosomes, appears to be indispensable for this ligand- induced transrepression (EMBO J. 24, 3881, 2005). Thus, we have identified a novel mechanism of ligand-induced tranrepression by NRs that links transrepression and promoter histone acetylation.
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Sharmistha Kundu Abstract P63 SWI/SNF establishes transcriptional memory at the Saccharomyces cerevisiae GAL1 gene
Sharmistha Kundu, Peter J. Horn and Craig L. Peterson
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Biotech 2, Suite 210, Worcester, 01605, U.S.A.
Chromatin remodeling enzymes that chemically modify histones or DNA have long been linked with transcriptional memory in diverse eukaryotes. We show that the ATP-dependent chromatin remodeling enzyme, SWI/SNF is essential for establishing transcriptional memory in the yeast, Saccharomyces cerevisiae. We observe memory in the regulation of the yeast GAL1 gene expression. Though SWI/SNF is dispensable for inducing GAL1 transcription, its recruitment allows much rapid kinetics of GAL1 reinduction, after being transitorily repressed with glucose. This phenomenon requires the ATPase activity of SWI/SNF and is epigenetically inherited by daughter cells. Significantly, SWI/SNF appears to antagonize the ISWI complexes to establish transcriptional memory at GAL1. Upon further examination we find that deleting the ISW2 complex specifically rescues growth defect of swi2- cells in galactose and also rescues expression of another SWI/SNF dependent gene. Further examples of SWI/SNF - ISW2 antagonism are presented.
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Georg Kustatscher Abstract P64 Metabolite-sensitive and metabolite-insensitive chromatin surfaces through the human histone macroH2A
Georg Kustatscher, Judith Sporn, Michael Hothorn, Bjoern Fritz, Miriam Bortfeld, Klaus Scheffzek and Andreas Ladurner
Gene Expression and Structural Biology Unit, EMBL Heidelberg, Germany
Small-molecule metabolites play an important role in the regulation of gene expression. We discovered that human chromatin might be a receptor for NAD-metabolites produced by Sir2 deacetylases. We are now studying whether this histone might provide a regulatory connection between metabolism and vertebrate chromatin. Unlike other deacetylases, Sir2 and its metazoan homologues use NAD to carry out the chemically straightforward deacetylation reaction. Sir2 may thus respond to the redox state of a cell. In fact, evidence links Sir2 to metabolism, chromatin and aging by mediating the benefits of life-extension through caloric restriction in yeast, C. elegans and Drosophila. The mammalian orthologue, SirT1, is induced under caloric restriction and regulates fat storage and the insulin pathway through PPARgamma and FOXO deacetylation. Sir2/SirT1 might thus play a conserved role in the metabolic control of gene expression and in heterochromatin formation. We find that its metabolite, O-acetyl-ADP-ribose (AAR), binds the macro domain of the human histone macroH2A1.1. The novel crystal structure of the protein-nucleotide complex reveals how chromatin may be a direct target for endogenous metabolites. Further, macroH2A1 is subject to alternative splicing, where two mutually exclusive exons produce two distinct proteins. Crucially, these exons encode a region critical for ligand-binding. In fact, the splice variant cannot bind AAR. Structural plasticity between the isoforms thus results in proteins that adopt the same fold, but show binary sensitivity to AAR. Human chromatin may thus bear metabolite-sensitive and metabolite-insensitive surfaces. We present in vivo evidence, including human tumors, that the two macroH2A1 isoforms show cell-type specific expression, in particular with regard to proliferation. Could there be metabolic control of gene activity through macroH2A-containing chromatin and how is this linked to proliferation and gene repression in mammals?
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Hyockman Kwon Abstract P65 BAF53-dependent higher-order chromatin structure as the compartment of replication and repair foci
Ki Won Lee1, Su Jin Kwon1, Phan Kyu Park1, Jae Yong Kim1, Yunhee Kim Kwon2, and Hyockman Kwon1
1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea, 2Department of Biology, Kyunghee University, Seoul 130-701, Republic of Korea
It is becoming evident that higher-order chromatin structure plays a critical role in many aspects of gene regulation in interphase mammalian nuclei. The emerging view is that chromosomes are compartmentalized into discrete chromosome territories in which chromatins are packaged into compact chromosomal subdomains with diameters of 100 to 450 nm. Chromosomal subdomain may define the functional compartment as well as the structural compartment. In mammalian cell nucleus, DNA replication and DNA double- strand break repair occur at discrete sub-nuclear structure called replication foci and repair foci, respectively. Interestingly, the average sizes of replication and repair foci, ~1 Mb, are similar each other, suggesting that they could represent different functional states of chromosomal subdomains. However, this possibility has not been fully appreciated yet. Previously, we showed that BAF53 is required for the higher-order chromatin structure. BAF53 knockdown resulted in the expansion of chromosome territories and the remarkable increase in the micrococcal nuclease sensitivity of chromatin. Here we found that BAF53 knockdown suppressed the formation of replication foci and repair foci. Although DNA replication proceeded normally in the BAF53-knockdowned cells, the early S-phase replication foci were not observed. Instead, a diffused pattern of BrdU incorporation was found in the nucleoplasm. Interestingly, the mid and late S-phase replication foci remained intact. Reduction of H3-K9 dimethylation foci in the nucleoplasm in the BAF53- knockdowned cells supports the specific disappearance of the early S-phase replication foci. In addition, the formation of H2AX foci in response to DNA damage by adriamycin was largely reduced in the BAF53-knockdowned cells. Activation of ATM appeared unchanged in the BAF53-knockdowned cells. Taken together, these results raised the possibility that replication foci and repair foci are originated from the same structural entity such as chromosomal subdomain whose formation requires BAF53. We discussed our results based on the multi-loop subcompartment model for chromosomal subdomain.
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Monika Lachner Abstract P66 Studying lysine methylation in non-histone proteins
Monika Lachner1, Kristie L. Rose2, Jeffrey Shabanowitz2, Karl Mechtler3, Thomas Jenuwein3, Donald F. Hunt3 and C. David Allis1
1The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A., 2Department of Chemistry, University of Virginia, Charlottesville, VA 22901, U.S.A., 3Research Institute of Molecular Pathology, Dr. Bohrgasse 7, 1030 Wien, Austria
While the impact of histone lysine methylation on chromatin structure and function has been extensively studied, there is little known about the occurrence and function of this post- translational modification in non-histone proteins. This obvious lack of understanding sparked our interest in exploring lysine methylation in non-histone proteins. In order to identify novel lysine methylation sites in non-histone proteins, we employed an approach that combines immunoprecipitation with a pan-methyl-lysine antibody and mass- spectrometry. These experiments provided several interesting candidate proteins that are involved in a variety of cellular processes. For a more detailed analysis we chose two of these proteins (Eset and mAM), since they have a well-documented function. Eset and mAM form an enzymatic complex that specifically trimethylates histone H3 on lysine 9. Interestingly, immunoprecipitations with the pan-methyl-lysine antibody suggested that both complex members are lysine-methylated proteins. At this point, we have confirmed this finding and we have been able to map one methylation site in mAM. Experiments are currently underway that will address the identity of the respective methyltransferase as well as the potential functional implications of this post-translational modification (e.g. modulation of the enzymatic activity of the complex, regulation of protein-protein interactions). We anticipate that further investigation of lysine methylation in non-histone proteins will demonstrate the importance of this post-translational modification in numerous biological processes.
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Brian D. Larsen Abstract P67 Caspase 3 mediated DNA strand breaks contribute to genomic reorganization during skeletal muscle terminal differentiation
Brian D. Larsen1,2 and Lynn A. Megeney1,2
1Ottawa Health Research Institute, Centre for Stem Cell Research, Molecular Medicine Program, Ottawa Hospital General Campus, Ottawa ON, Canada. 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa ON, Canada
Skeletal muscle differentiation is dependent on chromatin remodelling and genome wide reorientation of gene expression programs. However, the factors controlling this transition have not been fully elucidated. One mechanism known to influence the differentiation process is the activity of pro-apoptotic proteins. The apoptotic process is itself associated with genomic reprogramming events i.e. DNA strand breaks, suggesting that similar mechanisms may influence the genomic transition during cell differentiation. Here we explored the role of strand break formation in skeletal muscle differentiation. In situ nick translation (ISNT) was used to detect DNA strand breaks during differentiation of an in vitro model system (C2C12 myoblast cell line). Transient DNA strand breaks were detected early during differentiation between 12 and 24 hours. The formation of DNA strands breaks coincided with caspase 3 activation, a requirement for skeletal muscle differentiation. Moreover, treatment of differentiating C2C12 myoblasts with the caspase 3 inhibitor (z- DEVD-fmk) blocked the detection of DNA strand breaks by ISNT coincident with an inhibition of differentiation/myotube formation. A well characterized function of caspase 3 is the activation of the caspase activated nuclease (CAD) through proteolytic cleavage of its inhibitor (ICAD). Western blot analysis revealed cleavage of the long isoform of ICAD in C2C12 cells at 12 and 24 hours following low serum induction of differentiation; these results suggest the stand breaks are mediated through caspase 3 activity. The formation of transient DNA strand breaks implicates the necessity of a DNA repair mechanism during the differentiation process. As such we monitored the phosphorylation status of the histone variant H2AX, a well characterized marker of DNA double strand breaks, during myoblast differentiation. Phosphorylation of H2AX coincided with the formation of DNA strand breaks during myoblast differentiation and also appeared to be dependent on caspase 3 activity. These results suggest a role for pro-apoptotic proteins in regulating gene expression and chromatin remodelling to promote myoblast differentiation.
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Richard Lawrence Abstract P68 Mechanisms controlling dynamic Swi6/HP1 binding in S. pombe facilitate de novo heterochromatin formation
Richard J. Lawrence and Thomas A. Volpe
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, U.S.A.
Heterochromatin plays a critical role in genome stability and organization, and has long been regarded as statically condensed and inert. Contrarily, recent evidence suggests that heterochromatin is maintained by dynamic binding of heterochromatin protein 1 (HP1). However, the mechanisms, if any, that mediate dynamic HP1 binding and the in vivo function of HP1 mobility are unknown. We are studying a jmjC domain protein that modulates binding of the HP1 homolog, Swi6, to heterochromatin in S. pombe thereby antagonizing epigenetic stability. Interestingly, the protein lacks histone demethylase activity. In the absence of this protein a reporter gene in centromere heterochromatin is more enriched with Swi6 and in vivo binding studies indicate that Swi6 is more tightly bound to heterochromatin. Paradoxically, this “hyper-heterochromatization” promotes epigenetic heterochromatin stability but inhibits de novo heterochromatin nucleation. These results ultimately suggest that Swi6 binding is actively antagonized by this protein; thus, it functions to mobilize a pool of Swi6 that targets heterochromatin nucleation, most likely through a novel mechanism that is independent of histone demethylase activity.
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Frederic Leduc Abstract P69 Presence of gamma-H2AX in elongating spermatids: involvement of NHEJ?
Frederic Leduc, Veronique Lauziere, Marilyne Joly, Leila Jaouad and Guylain Boissonneault
Faculte de Medecine, Departement de Biochimie, 3001, 12eme avenue Nord, Sherbrooke, Qc J1H 5N4, Canada
The histone variant H2AX is involved in early DNA damage response and controls the recruitment of DNA repair proteins at sites of double-stranded breaks. In testis, the active, phosphorylated form of H2AX (gamma-H2AX) is thought to be involved in two processes namely the inactivation of sex chromosomes (at the sex vesicle) and the control of genome integrity during meiotic recombination. We used confocal microscopy applied to both immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) on squash preparations of seminiferous tubules. Stage-specific sections were obtained according to their light absorption pattern. Here, we demonstrate the presence of gamma-H2AX in the whole population of elongating spermatids in mouse (steps 8-9), coincident with the onset of transient DNA strand breakage and chromatin remodeling as shown by the hyperacetylation of histone H4. These results strongly suggest that a complex DNA repair system is recruited and required during the chromatin remodeling steps in elongating spermatids. Given their haploid character, we hypothesize that the non- homologous end-joining (NHEJ) is responsible for the DNA repair during step 8 through 13. The presence of NHEJ-associated factors is being investigated. Funded by Canadian Institutes of Health Research (Grant# MOP-74500)
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Min Gyu Lee Abstract P70 Functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein
Min Gyu Lee1, Jessica Norman1, Anne llvarsonn2, Joel C Eissenberg2, Ali Shilatifard2 and Ramin Shiekhattar1
1The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 U.S.A., 2Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, Saint Louis, MI 63104, U.S.A.
Histone methylation is a post-transcriptional mark regulating chromatin structure and gene regulation. Once deemed irreversible, recent findings have identified two classes of enzymes capable of demethylating lysine residues. BHC110/LSD1, the catalytic heart of multiple co-repressor complexes, was the first of such demethylases shown to reverse dimethyl histone H3K4. However, due to intrinsic limitations of BHC110/LSD1 mode of action, it is unable to remove trimethyl H3K4 marks. Here we show that JARID1d, a member of a second class of histone demethylases containing JmjC-domain, can specifically demethylate trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjC- domain, the BRIGHT and zinc-finger-like C5HC2 domains are required for maximum catalytic activity. Importantly, isolation of native JARID1d complexes from human cells revealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR. Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymatic activity. We show that JARID1d occupies human Engrailed 2 promoter and regulates its expression and H3K4 methylation levels. Finally, we show that the single Drosophila homolog of JARID1d, little imaginal discs (Lid), is also a trimethyl H3K4 demethylase attesting to the cross-species conserved function for this family of histone demethylases.
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Niraj Lodhi Abstract P71 Histone acetylation (H3K9) and methylation (H3K4) of the nucleosome over core promoter are associated with the induction of tobacco PR-1a gene
Niraj Lodhi, C. P. Chaturvedi, Suraiya A. Ansari, Rakesh Srivastava, Samir V. Sawant and Rakesh Tuli
National Botanical Research Institute India, Rana Pratap Marg, Lucknow, India 226001
The PR-1a gene encodes one of the major defense related protein in tobacco (Nicotiana tabacum) and it is tightly regulated at the transcription level. Detailed studies on PR-1a promoter in tobacco leaves have shown that it is induced specifically on pathogen attack or after Salicylic Acid (SA) induction. Our work suggested that the core promoter region of PR- 1a gene plays an important role in determining SA induced transcription. We have mapped a nucleosome which spanned the core promoter of PR-1a gene. The nucleosome spanned the region between -103 to +55 relative to the transcription initiation site of the PR-1a gene. We carried out Chomatin Immunoprecitation (ChIP) with H3K9 acetylation specific and H3K4 methylation specific antibodies to identify the role of acetylation and methylation of nucleosome over the core promoter region. Our results revealed that the nucleosome over the core promoter results in a repressive chromatin architecture which is remodeled by histone modifications concomitant with the gene activation after SA induction. The SA induction leads to shifting of nucleosome from the core promoter which allows the assembly of the pre-initiation complex and initiation of transcription. The results related to our new findings will be presented at the conference.
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Mattias Mannervik Abstract P72 An HDAC3/SMRTER/Ebi complex required for Snail repressor function in Drosophila development
Mattias Mannervik, Dai Qi and Mattias Bergman
Stockholm University, Wenner-Gren Institute, Dept. of Developmental Biology, Arrheniuslaboratories E3, Stockholm, Sweden
The zinc-finger transcription factor Snail is critical for Drosophila embryo development by preventing expression of neuroectoderm-specific genes in the mesoderm. We found that in embryos devoid of maternal Ebi protein, Snail repressor function is impaired. Drosophila Ebi and its mammalian homolog TBL1 are WD40 proteins with a divergent F-box domain. Previous studies have linked Ebi and TBL1 to two different pathways; ubiquitin conjugation through SCF-type ligases, and N-CoR/SMRT/HDAC3-mediated transcriptional repression. In ebi mutant embryos, Snail target genes are de-repressed in the mesoderm. De-repression of a Snail-dependent reporter gene in ebi mutant embryos, and genetic interactions with snail support a requirement for Ebi in Snail function. Snail-mediated repression was previously shown to depend on another co-repressor, CtBP. We found that both CtBP and Ebi can interact with Snail protein in vitro, but through different interaction domains. A minimal Ebi-interaction domain that fails to bind CtBP constitutes a potent repression domain in both S2 cells and in transgenic embryos. This suggests that Snail uses Ebi as co- repressor independently of CtBP. The repression activity of this domain can be attenuated either by knockdown of HDAC3 or by TSA treatment, indicating an involvement of histone deacetylation. By contrast, inhibition of proteasome activity does not affect Snail-mediated repression. We suggest that Ebi as part of a SMRTER/HDAC3 co-repressor complex is required for Snail function in Drosophila, and that histone deacetylation is part of the mechanism by which Snail represses transcription.
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Abstracts - Poster
Robert Martin Abstract P73 Chromatin labeling and distribution in living cells
Martin, R.M., Leonhardt, H. and Cardoso, M.C.
Max Delbrueck Center for Molecular Medicine, Robert Roessle Str. 10, 13125 Berlin, Germany and Ludwig Maximilians University Munich,Department of Biology II, 82152 Planegg-Martinsried, Germany
Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission. It could be used in a variety of cells of different species including primary cultures after a few minutes incubation in the culture medium. Furthermore the effect on histones and other chromatin proteins will be discussed. DRAQ5 is a useful molecular tool for cell biology that allows a fast and non invasive labeling as well as microscopic visualization of DNA structures in living cells.
Ref: Martin, R. M., Leonhardt, H., and Cardoso, M. C. (2005) Cytometry A 67, 45-52
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Peter McKeown Abstract P74 Chromatin components of the Arabidopsis thaliana nucleolus
Peter C. McKeown, Alison F. Pendle and Peter J. Shaw
Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich, Norfolk U.K., NR4 7UH, John Innes Centre, Colney Lane, Norwich, Norfolk, NR4 7UH, U.K.
Eukaryote DNA is packaged into chromatin through association with highly basic histone proteins. The presence of histone variants and multiple covalent modifications engender a ‘histone code’ which controls the expression of the packaged DNA. This chromatin is not purely informational – it also has a structural existence within the nucleus. This is well- demonstrated in the largest nuclear structure, the nucleolus, which forms through the chromatin-regulated expression of rRNA genes and acts as the site of pre-ribosome formation and many RNA processing pathways. However, few distinctive chromatin marks have been identified in the nucleolus, in contrast to other structures such as centromeres, telomeres and sites of DNA breaks. We have now identified a group of nucleolar histones and histone modifications in the model plant, Arabidopsis thaliana thaliana, through a combination of techniques. Previously, we used mass spectrometry of extracted nucleoli to analyse the organellar proteome (Pendle et al., Mole. Biol. Cell, 16:260-269, 2005) identifying several nucleolar histones in the process. We have now used further MS techniques to determine how these proteins are modified, and demonstrated nucleolar specificity with GFP-fusion proteins. Immunofluorescence has identified other modifications, and confirmed the presence of a nucleolus-specific linker histone which may bind inactive rRNA genes and acetylated histone H2B which colocalises with sites of RNA polymerase I-mediated transcription. Such chromatin components may represent a source of nucleolus-specific information additional to the standard histone code. We conclude that the nucleolus is a good model for assessing the links between the informational content of the histone code and the its effects upon nuclear structures, and are currently determining the roles of the chromatin marks identified in both rRNA transcription and nucleolar structure.
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Rosalind Meldrum Abstract P75 Visualisation of DNA repair and chromatin dynamics
Rosalind Meldrum
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT U.K.
The area of research describing interactions between chromatin structure and the DNA damage response is receiving an increasing amount of attention. It is likely that DNA damage has a considerable influence on the physical structure and movements of chromatin. It is with out doubt that new high-resolution microscopy techniques, that can visualise detailed geometrical and spatial changes that take place in the cell in response to DNA damage, will play a critical role in this area of research. The most useful approaches to detecting and visualising chromatin dynamics are those where DNA damage can be induced in predetermined patterns and target specific structures in a cell nucleus. To be able to see the details of structural and dynamic changes it is desirable to induce and visualise DNA damage with very high resolution. UV lesions can be induced with nanoscale resolution in cell nuclear DNA by triple-photon infra-red absorption. The number or lesions induced is much smaller than when total cell irradiation by a mercury lamp is used or when localised damage is induced by irradiation of cells with UV light through a micro-filter. Because the UV photoproducts can be induced in a defined pattern, during a period of incubation following irradiation, distinctive movement of the damage DNA is seen to take place and the damage distributed over the cell nucleus forms individual clusters of lesions. The characteristics of the clustering of lesions are being investigated in relation to cell cycle, histone modification, ATP-dependent remodelling and incorporation of histone variants. A further interesting observation revealed that higher levels of damage immobilise and preserve the induced pattern for some hours following irradiation.
Ref: R.A.Meldrum, S.W.Botchway, C.W.Wharton, G.J.Hirst. (2003) Nanoscale induction of UV photoproducts in cellular DNA by 3-photon near infra-red absorption EMBO Reports 4, 12 1144-1149
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Brendon Monahan Abstract P76 Purification and characterization of the fission yeast Swi/Snf and RSC chromatin remodeling complexes
Brendon J. Monahan1, Judit Villen2, Samuel Marguerat3, Jurg Bahler3, Steve Gygi2 and Fred Winston1
1Department of Genetics, Harvard Medical School, Boston MA, U.S.A, 2Taplin Biological Mass Spectrometry Facility, Department of Cell Biology, Harvard Medical School, Boston MA, U.S.A., 3Wellcome Trust Sanger Institute, Cambridge, U.K.
The Swi/Snf type of ATP-dependent chromatin remodeling complexes are molecular motors that modify chromatin structure thereby regulating transcription and mediating other cellular processes such as DNA repair. Two evolutionarily conserved and distinct subclasses of Swi/Snf, named Swi/Snf and RSC, have been extensively studied in Saccharomyces cerevisiae. Here we present the purification and characterization of the Swi/Snf and RSC chromatin remodeling complexes from the fission yeast Schizosaccharomyces pombe. S. pombe, which is as closely related evolutionary to humans as it is to S. cerevisiae, is of interest as its chromatin shares several important similarities to mammalian chromatin that do not occur in S. cerevisiae and also provides a basis for valuable comparative analysis. The S. pombe Swi/Snf and RSC complexes were purified using tandem affinity purification (TAP) methodology and components identified by mass spectrometry. The S. pombe Swi/Snf complex is composed of 12 subunits, of which six are shared with the 14-member RSC complex. Deletion and tetrad analysis has shown that the core subunit genes in RSC are essential for growth whereas deletion mutants of the paralogous Swi/Snf genes were viable. Four of the six genes shared between the two complexes are essential for cell growth, the exceptions being the two arp (actin related protein) genes, arp4+ and arp9+. To investigate the global effect Swi/Snf has on S. pombe gene expression, whole genome expression analysis was done using deletion mutants of two core Swi/Snf complex subunit genes, snf22 and snf5. Overall, the expression levels of approximately 2.5% of S. pombe genes were altered greater than 2-fold in the swi/snf mutants. Interestingly, genes involved in sugar uptake and iron homeostasis were significantly enriched in the up-regulated gene set. This work has established the foundation for further detailed analysis into the role of these chromatin remodeling complexes in transcription activation and repression and other cellular processes in fission yeast.
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Antonin Morillon Abstract P77 Transcriptional co-suppression in S. cerevisiae
Julia Berreta, Benjamin Pernot-Cornu and Antonin Morillon
CGM-CNRS, Gif/Yvette, France
Cosuppression has been defined as high gene copy number-triggered, homology- dependent, gene silencing and may have evolved in eukaryotic cells as a defensive strategy against viral infections and to control activity of transposons. In S. cerevisiae, co- suppression was first unveiled in 2002 (Y. Jiang, 2002) and was shown to act at the transcriptional level controlling the Ty1 retrotransposon. As the main actors of Transcriptional Gene Silencing are not conserved in baker yeast, co-suppression must be mediated by an original pathway which we try to characterize in this work. We hypothesize that it is mediated by regulatory RNAs which may inhibit Ty1 expression. We show that the expression of Ty1 is dependent upon the presence of several proteins involved in RNA degradation, in particular the 5-3 exonuclease Xrn1. Furthermore, the drop of Ty1 expression correlates with the accumulation of an antisense non coding RNA corresponding to the 5 Long Terminal Repeat (LTR) region of Ty1. Through ChIP experiments, we show that the decrease of Ty1 RNA does not correlate to a reduction of RNAPII occupancy on the Ty1 gene suggesting that Ty1 RNA is affected at a post- transcriptional step. Finally, it has been shown that the retrotransposon is very sensitive to the histone levels and to Chromatin Remodeling Complex activity. We will discuss our preliminary results on nucleosome positions and histone modifications on Ty1 elements upon co-suppression conditions.
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Ashby Morrison Abstract P78 Mec1/Tel1-dependent phosphorylation of a chromatin remodeling complex influences the DNA damage checkpoint pathway
Ashby J. Morrison1, Jung-Ae Kim2, Maria D. Person3, Jessica Highland1, Tammy S. Wehr1, Sean Hensley1, Jianjun Shen1, Sean R. Collins5,Jeff Delrow4, Nevan J. Krogan5, James E. Haber2 and Xuetong Shen1
1Department of Carcinogenesis, Science Park Research Division, University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, 2Rosentiel Center and Department of Biology Brandeis University, Waltham, Massachusetts 02454, 3College of Pharmacy, Division of Pharmacology & Toxicology, University of Texas at Austin, Austin, Texas 78712, 4Division of Basic Sciences Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, 5Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94143 U.S.A.
The Mec1/Tel1 kinases in yeast, ATM/ATR in mammals, play central roles in coordinating the DNA damage response by phosphorylating proteins involved in DNA repair and checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as the yeast INO80 complex, which were originally characterized as transcriptional regulators, have also been implicated in the DNA damage response. Here, we show that the Ies4 subunit of the INO80 complex is phosphorylated in a Mec1/Tel1-dependent manner during exposure to DNA damaging agents. The phosphorylation status of Ies4 does not significantly affect transcription or DNA repair processes, such as homologous recombination. However, DNA damage checkpoint pathways are influenced by the phosphorylation status of Ies4. These findings establish a chromatin remodeling complex as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates checkpoint responses, and suggest that post-translational modification of chromatin remodeling complexes may regulate their involvement in distinct nuclear processes.
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Raul Mostoslavsky Abstract P79 Genomic instability and aging-like phenotype in the absence of mammalian SIRT6
Raul Mostoslavsky1, David B. Lombard1,2, Katrin F. Chua1, Jennifer Kim1, Lionel Gellon4, Bruce Demple4, George Yancopoulos3 and Frederick W. Alt1
1Howard Hughes Medical Institute, The Children’s Hospital, CBR Institute for Biomedical Research, and 2Department of Genetics, Harvard Medical School, Boston, Massachusetts 021151, 3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 021152, 4Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-67073, 5Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston Massachussets 021154
In yeast, the histone deacetylase/ADP-ribosyltransferase Sir2 inhibits DNA recombination, promoting longevity. Seven mammalian Sir2 homologs, termed SIRT1-SIRT7, have been described. Here we show that SIRT6 is a nuclear, chromatin-associated protein, expressed in multiple tissues. At the cellular level, SIRT6 promotes resistance to DNA damage and suppresses genomic instability, in association with a role in Base Excision Repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include acute lymphocyte depletion, loss of subcutaneous fat, lordokyphosis and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss in mice leads to defects in lymphocyte homeostasis and abnormalities that overlap with aging-associated degenerative processes. Recent progress in understanding the role of SIRT6 in DNA repair and metabolism will be discussed.
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Takahiro Nakayama Abstract P80 Drosophila GAGA factor promotes histone H3.3 replacement that prevents the heterochromatin spreading
Takahiro Nakayama, Kenichi Nishioka, Yi-Xin Dong, Tsukasa Shimojima and Susumu Hirose
Department of Developmental Genetics, National Institute of Genetics, and Department of Genetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan
Drosophila white gene that is normally located euchromatin region governs a red eye phenotype. When the white gene is juxtaposed with centromeric heterochromatin regions by chromosomal rearrangement, its expression is subject to variable in a heritable silencing manner, giving rise to white mottled eye color. This phenomenon termed position effect variegation (PEV) provides evidence for a crucial role of chromatin structure in gene expression. Previously, Karch group showed that the Trithorax-like gene encoding GAGA factor is a dominant enhancer of PEV, suggesting that the GAGA factor plays a role in the active maintenance of white under heterochromatin environment. However, little is known about the molecular mechanism. Here, we demonstrate that the GAGA factor binds to a site just downstream of the white gene and this binding site is necessary and sufficient to block heterochromatin spreading. Interestingly there are a dip of histone H3 Lysine 9 methylation and a peak of H3 Lysine 4 methylation at this site. Furthermore, the GAGA factor promotes chromatin remodeling and replacement of histone H3 with H3.3 through recruitment of HIRA at this site, and maintains white expression under the heterochromatin environment. Based on these findings, we propose that the GAGA factor- dependent replacement of Lysine 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin.
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Zuyao Ni Abstract P81 The tumor suppressor BRG1 silences the distal silencers at interferon-responsive genes
Zuyao Ni*, Mohamed A I Abou El Hassan*, Zhaodong Xu, Tao Yu, and Rod Bremner
Toronto Western Research Institute, University Health Network, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Departments of Lab Medicine and Pathobiology and Ophthalmology and Vision Science, University of Toronto, Ontario, Canada
Communication between distal regulatory DNA elements is essential for control of many nuclear processes in higher eukaryotes. Long-range gene regulation has been associated primarily with step-wise activation of differentiation genes over days. Recently, our group implicated long-range effects in rapid IFNgamma (IFNg)-mediated gene induction. However, the mediators of such long-range effects are unknown. Previously, we showed that the tumor suppressor and chromatin remodeling factor BRG1 has a primary role in regulating IFNg-responsive genes, so we hypothesized it may act through remote elements. Here, we show that BRG1 constitutively binds distal regulatory sites and binding increases upon IFNg treatment. BRG1 is required for transcription factor (STAT1 and IRF1) binding, histone acetylation, and chromatin remodeling at the distal IFNg sensitive sites at several target loci. At both CIITA and SOCS1 loci distal elements loop and interact with target promoters and/or with each other in a BRG1 dependent manner. To determine the functional relevance of these events in the appropriate context, we constructed a 200 kb bacterial artificial chromosome (BAC) reporter vector containing the entire CIITA locus. Remarkably, deleting any one of several distal elements induced basal CIITA activity to levels similar to those seen in IFNg-treated cells, even in the absence of BRG1. Thus, multiple remote elements cooperate to silence CIITA, and IFNg-overcomes their effect, derepressing CIITA in a BRG1-dependent manner. Negative regulation of distal silencing complexes is a novel mechanism of action of BRG1.
(* Equal contributors)
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Olivia Osborn Abstract P82 Transcriptional targets of Af4
Osborn O, Oliver P.L., Bitoun E. and Davies K.E.
Dept of Physiology Anatomy & Genetics, Oxford University
The putative transcription factor Af4, known to be translocated in acute leukaemia, is also expressed in the Purkinje cells of the cerebellum. The robotic mouse mutant has an ataxic gait, is smaller than its littermates and shows distinct and progressive pattern of Purkinje cells loss from 8 weeks of age. This phenotype is caused by a gain of function mutation (V280A) in Af4 which prevents its normal rapid turnover by the proteosome and consequently its accumulation in numerous tissues as well as the brain. The cause of the cell loss in robotic and the normal function of Af4 is, however, unknown. Chromatin immunoprecipitation coupled with microarrays have been used with the aim of finding the direct targets of this transcription factor along with expression analysis to determine the downstream targets that contribute to the ataxic phenotype.
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Julia Pagan Abstract P83 A novel corepressor, BCOR-L1, functions through CTBP and class 2 HDACs
Julia K. Pagan1,2, Jeremy Arnold1, Kim J. Hanchard1, Mathew J.K. Jones1,2, Derek J. Richard1, Alistair Forrest3, Amanda Spurdle1, Eric Verdin4, Merlin Crossley5, Georgia Chenevix- Trench1, David B. Young*1 and Kum Kum Khanna*1
1Queensland Institute of Medical Research, 300 Herston Rd, Herston 4029, Queensland, Australia, 2School of Medicine, Central Clinical Division, University of Queensland, Royal Brisbane Hospital, Herston 4029, Queensland, Australia, 3Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia, 4Gladstone Institute of Virology and Immunology, University of California San Francisco, San Francisco, California, United States of America, 5School of Molecular and Microbial Biosciences, G08, University of Sydney, New South Wales 2006, Australia
Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR corepressor. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Most corepressors function by associations with histone deacetylase (HDAC) activity. BCoR-L1 coprecipitates with Class II HDACs; HDAC4, HDAC5 and HDAC7, suggesting they are involved in its role as a transcriptional repressor. BCoR-L1 interacts with the CtBP corepressor through a CtBP-interacting motif in its amino-terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-Cadherin promoter, a known CtBP regulated promoter, and is involved in repression of E-Cadherin.
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Maria V. Panchenko Abstract P84 Role of Jade-1 in the HAT HBO1 complex
Rebecca L. Foy, Vipul C. Chitalia, Herbert T. Cohen and Maria V. Panchenko
Boston University School of Medicine, Evans Biomedical Research Center, MA 02118- 2393, U.S.A.
Regulation of global chromatin acetylation is important in processes requiring chromatin remodeling, including DNA replication. We demonstrated that PHD zinc finger protein Jade- 1 is localized to the nucleus, activates rates of transcription when tethered to a heterologous promoter, and is associated with endogenous HAT activity. It has been recently reported (J. Coté’s lab, 2006) that Jade-1 co-purifies with a novel HAT complex, consisting of three additional proteins, including HBO1, ING4/5 and Eaf6. In mammalian cells, HBO1 provides the basal level of global histone H4 acetylation, is required for DNA replication, and its activity is regulated during the cell cycle. We thus investigated a functional role for Jade-1 in the HBO1 complex. We demonstrated that while overexpression of HBO1 did not alter levels of endogenous histone H4 acetylation, co-transfection of even low sub-sufficient amounts of Jade-1 resulted in a dramatic, up to a 50-fold upregulation of histone H4 acetylation, strongly suggesting that Jade-1 plays a crucial role in HBO1-mediated acetylation of nucleosomal histones. Interestingly, while PHD fingers were indispensable for Jade-1 to synergize with HBO1, they were dispensable for Jade-1-HBO1 physical interactions. We proposed that Jade-1 might promote histone acetylation by binding chromatin via its PHD fingers and targeting the HAT HBO1 complex to the proximity of histone substrates. Because Jade-1 partner HBO1 is involved in DNA replication, we investigated an effect of cell cycle progression on endogenous Jade-1 expression and nuclear distribution. We found that Jade-1 nuclear distribution and presumably chromatin association is strongly regulated by cell growth arrest and during cell cycle progression. In addition, Jade-1 undergoes phosphorylation followed by dephosphorylation in synchronized cycling cells. The data suggest a role for cdks in this posttranslational modification of Jade- 1 and implicates a potential mechanism for the regulation of HBO1 HAT activity during the cell cycle and DNA synthesis.
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Tej Pandita Abstract P85 Mammalian ortholog of Drosophila MOF is critical for embryogenesis and DNA repair
Arun Gupta1, Geraldine Guerin-Peyrou2, Girdhar G. Sharma1, Manjula Agarwal1, Raj K. Pandita1, Raju Kucheralapati3, Thomas Ludwig2 and Tej K. Pandita1
1Washington University School of Medicine, Saint Louis, MO 63108; 2College of Physicians and Surgeons, Columbia University, New York, NY 10032, 3Harvard Medical School, Boston, MA 02115, U.S.A.
Human ortholog (hMOF) of the Drosophila MOF gene (males absent on the first) is histone H4 lysine K16-specific acetyltransferase. It is involved in transcription as it is a component of a functional dosage compensation complex required for male killing in Drosophila and DNA damage response. Cellular exposure to ionizing radiation enhances hMOF-dependent acetylation of its target substrate, lysine 16 of histone H4. Depletion of hMOF results in abrogation of ATM autophosphorylation, ATM kinase activity and DNA repair as well as increases cell killing after IR exposure. Based on these preliminary studies, we hypothesize that hMOF is involved in the regulation of DNA damage-induced ATM activation. In addition, MOF function is indispensable for development, because Mof-deficiency in mouse embryos results in early embryonic lethality which cannot be overcome by inactivation of p53 or ATM. Mice with haploinsufficiency of mMof in Atm null background are smaller in size and die early mostly because of leukemia. MOF over expression results in enhanced oncogenic transformation and is over expressed in most of the human tumors. These results demonstrate that ‘MOF’ is not only required for early embryonic development but plays a critical role in cell growth during oncogenic transformation in absence of Atm. We will discuss the role of MOF in regulation of DNA double strand break repair.
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Maëlle Pannetier Abstract P86 Imprinting perturbation in mouse hepatocarcinoma: link between DNA methylation and histone methylation
Maëlle Pannetier, Katia Delaval, Alexandre Wagschal and Robert Feil
Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535 et Université Montpellier II, 1919 route de Mende, 34293 Montpellier cedex 5, France
Genomic imprinting is an essential mechanism in development that gives rise to mono-allelic expression of genes depending on the parental origin of the allele. This mono-allelic expression is controlled by imprinting control regions (ICRs), short CpG-rich sequences that are differentially methylated. In addition to differential DNA methylation, allelic histone modifications, like lysine methylation and acetylation, are observed. We found that trimethylation of H3K27, H3K9 and H4K20 are associated with the DNA-methylated allele, whereas dimethylation of H3K4 and acetylation of H3 are associated with the unmethylated one. Cancer cells are characterized by prominent epigenetic dysregulation, including altered DNA methylation patterns, chromatin modifications and loss of genomic imprinting. Using a murine model of hepatocarcinoma, we study such epigenetic alterations at two imprinted domains located on chromosome 7: the Kcnq1 and Igf2/H19 domains. Transgenic mice showing liver-specific expression of a c-myc transgene were crossed with P53 knock-out mice. Thus, c-myc overexpression, in a hemizygous state for P53, gives rise to liver tumours at about 9 months of age. Moreover, these mice have a paternal chromosome 7 from M. Spretus and a maternal one from M. Domesticus, allowing us to discriminate the maternal allele from the paternal one. In this context, we determine if imprinting is perturbed in thus- induced hepatocarcinomas, studying DNA methylation and histone modifications. 1/4 of tested tumours showed a gain of DNA methylation at the Igf2/H19 ICR, whereas 1/16 showed a loss of DNA methylation at the Kcnq1 ICR. Analysis of histone modifications by ChIP will allow us to determine how DNA and histone modifications are correlated during epigenetic perturbation.
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Janet Partridge Abstract P87 Establishment and maintenance of centromeric heterochromatin in fission yeast are functionally separable
Janet F. Partridge, Jennifer L. DeBeauchamp, Michael Hadler, Dagny L. Ulrich and Victoria J.P. Noffsinger
Dept. Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN. U.S.A.
Both the establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1) and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, it remains unclear if mechanisms governing RITS- dependent establishment of centromeric heterochromatin differ from its maintenance. Here, we generate a Tas3 protein, mutated in a highly conserved GW-rich Argonaute-binding domain, which cannot bind Ago1. This mutant exhibits near normal maintenance of centromeric heterochromatin, but cannot support its establishment. We show that Ago1 can be maintained at centromeres through binding siRNA, but to establish centromeric heterochromatin, Ago1 must bind Tas3-Chp1. Our results support a model whereby recruitment of RITS to centromeres is initiated by Chp1 binding to K9-methylated histone H3, with the RNAi pathway responsible for maintenance of RITS at centromeres.
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Kelly Perkins Abstract P88 Activated HIV-1 provirus forms a gene loop, connecting viral transcriptional initiation with termination
Kelly J. Perkins, Marina Lusic2, Mauro Giacca2 and Nick J. Proudfoot1
1Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford OX1 3RE, U.K., 2Laboratory of Molecular Medicine, International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99-34012 Trieste, Italy
We are investigating high order chromatin structure and factor recruitment to integrated HIV- 1 provirus following re-activation from latency. Both chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) techniques are being utilized. 3C analysis of pro- monocytic cell line U937 cell line U1/HIV-1 (U1) chromatin induced by TPA treatment or exogenous viral Tat expression indicated that activated provirus exists in a transcription- dependent loop conformation that juxtaposes the 5 long terminal repeat (LTR) promoter and 3LTR terminator regions. Based on ChIP analysis, RNA polymerase II (Pol II) and factors involved in transcriptional initiation and elongation (Cdk9 and USF) were detected at both ends of the HIV-1 provirus, supporting the existence of LTR-LTR interaction upon activation of proviral reservoirs. To determine whether proviral loop formation occurs before or after Cdk9-mediated Pol II CTD phosphorylation, we are currently performing 3C and ChIP analysis on chromatin treated with flavopiridol, a specific inhibitor of Cdk9 kinase activity. We propose that when latent integrated HIV-1 proviral DNA is transcriptionally activated in reponse to cellular or Tat-mediated stimulation, a structural formation is created where the flanking LTRs reside in close spatial proximity. This permits “cross-talk” between initiation and termination factors and so may allow efficient recycling of transcriptional machinery. This is the first study to identify the spatial arrangement of active integrated HIV-1 provirus and may provide valuable insight into the physical properties of HIV-1 proviral reservoirs upon transcriptional activation.
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David Picketts Abstract P89 SNF2L-mediated control of cell number in the developing brain
Darren J. Yip1,3, Stephen Rennick1,3, Adriana de Maria1, Josée Coulombe1, Michael Rudnicki1,2 and David J. Picketts1,3
1Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON, 2Department of Cellular and Molecular Medicine and 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON
Epigenetic modification of the genome is becoming recognized as a major point of regulation governing many developmental processes. Certainly, defective epigenetic regulation is already implicated in a wide range of genetic disorders including mental retardation, autoimmune disorders, and cancer. We have shown that the ISWI family of chromatin remodelling proteins are abundantly expressed during mammalian brain development and thus, may be key participants in establishing chromatin domains that are characteristic of neurons. Here we used a conditional gene-targeting approach to inactivate the murine Snf2l gene in order to assess its role in neurodevelopment. Heterozygous females (Snf2lf/x) were crossed to mice expressing Cre-recombinase under the control of the GATA-1 promoter. SNF2L-null male mice were viable and born at classic Mendelian ratios. These mice displayed no overt developmental or behavioral abnormalities; however, the loss of Snf2l resulted in a 2-fold increase in the brain weight to body weight ratio. This was accompanied by a concomitant increase in cell number in the hippocampus and cerebral cortex ranging from 0.5-2-fold in the six distinct cortical layers and a 2-fold increase in all hippocampal strata. Using a combination of in situ BrdU labeling experiments and primary neuroprogenitor cultures we demonstrate that the increased cell number results from a delay in terminal differentiation. Indeed, neuroprogenitor cultures isolated from knockout animals continue to incorporate BrdU twice as long as wild type cells when induced to differentiate. As a result, fewer cells stain positive for neuronal or astrocytic markers at earlier times (4 days) but are equivalent by the end of the 7-day differentiation time course. Taken together, our results suggest that Snf2l has an important role in regulating the switch from proliferation to differentiation as a mechanism to control cell number and brain size during neuronal development. Moreover, we propose that Snf2l regulation of brain size occurs through direct effects on genes involved in cell cycle exit and/or terminal differentiation.
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Romina Ponzielli Abstract P90 Optimization of experimental design parameters of ChIP-on- chip studies
Romina Ponzielli1 Paul C. Boutros1,2,3 , Sigal Katz1 , Igor Jurisica1,3,4 and Linda Z. Penn1,2
1Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, Toronto, Canada M5G 2M9, 2Department of Medical Biophysics, University of Toronto, Canada, 3Division of Signaling Biology, Ontario Cancer Institute, University Health Network, Toronto, Canada M5G 2M9, 4Department of Computer Science, University of Toronto, Canada
Chromatin immunoprecipitation (ChIP) is an antibody-based technique for determining transcription-factor binding in vivo. ChIP-chip technology combines the sensitivity and specificity of ChIP with high-throughput analysis, by exploiting microarray platforms spotted with promoter region DNA. In yeast, this technique has been used to map transcription- regulatory networks; in mammals, ChIP-on-Chip has determined transcription-factor binding in normal and cancerous cells and tissues. While ChIP-on-Chip studies are yielding useful results, their bioinformatic analysis is not yet fully realized. In this study we characterize both the statistical and experimental-design features of ChIP-on-Chip. To profile the signal-to-noise characteristics of ChIP-on-Chip data we performed a series of validation studies. First, we compared the performance of different amplification methods. Second, we determined optimal sample-to-array allocation. Third, we profiled the effect of dye-bias. Fourth, we evaluated effects of array batch variability. Finally, we determined the importance of antibody purity for successful ChIP-on-Chip studies. These studies encompass over 100 arrays, the data from which was exploited in a large-scale empirical study of statistical pre-processing methods. We assessed 84 distinct analysis methods for sensitivity, stability, and selectivity. Through these analyses we have optimized the major design parameters of ChIP-on-Chip studies. Our rigorous characterization of ChIP-on-Chip data is a key step towards exploiting this important technology for the rapid elucidation of regulatory networks.
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Abstracts - Poster
Ryan Raisner Abstract P91 Single nucleosome resolution mapping of the histone variant H2A.Z in a developing organism
Ryan M Raisner and Hiten D Madhani
UCSF Department of Biochemistry and Biophysics, U.S.A.
The conserved histone variant H2A.Z is an essential protein in higher organisms that is required early in development. It is poorly understood where H2A.Z is localized in metazoans, and furthermore what it’s essential biological function is. It has been shown by several groups that in the budding yeast Saccharomyces cerevisiae, H2A.Z is present throughout the genome, and more specifically it marks the promoter regions of the majority of genes, regardless of their transcription rate. More recently, there has been evidence that H2A.Z can selectively mark active genes in vertebrates. We have chosen the model organism Danio rerio (zebrafish) to study the distribution of H2A.Z using chromatin immunoprecipitation along with high resolution tiling microarrays at a range of genes, including the developmentally important Hox gene clusters at several stages of the developing animal. In addition to H2A.Z, we have also profiled the chromatin modifications H3 tri-methyl lysine 27 and tri-methyl lysine 4 at the same regions at single nucleosome resolution. These modifications have been shown recently to be present predominantly at transcription factor promoters in pre-differentiated cells. Accordingly, their coincidence with H2A.Z, along with the dynamics of all three chromatin marks at the Hox gene clusters throughout development is of great general interest. Our localization studies of H2A.Z in the developmentally tractable vertebrate system of zebrafish will allow us to better understand the nature of the activity of H2A.Z in the context of a complex developing organism.
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Rama Natarajan Abstract P92 Genome-wide analysis of histone lysine methylation variations caused by diabetic conditions in human monocytes
Feng Miao1, Xiwei Wu2, Lingxiao Zhang1, Yate-Ching Yuan2, Arthur D. Riggs3 and Rama Natarajan1
Departments of Diabetes1, Biomedical Informatics2 and Biology3, Beckman Research Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, U.S.A.
Histone modifications in chromatin, particularly histone lysine methylations, play key epigenetic roles in gene expression and constitute a new layer of transcription regulation. Aberrant methylation patterns that change chromatin structure can promote dysregulated gene expression associated with disease progression. Current approaches for acquiring genome-wide information of histone modifications involve the use of chromatin- immunoprecipitation linked to DNA arrays (ChIP–arrays). This method generates data sets of chromatin status and provides snapshot views of the cell at the layer of histone methylation. Diabetic conditions such as high glucose (HG) can alter key pathologic pathways and genes. However, their impact on cellular histone lysine methylation is not known. We hypothesized that chronic HG can induce aberrant changes in histone H3 lysine-4 and lysine-9 dimethylation (H3K4me2 and H3K9me2), key histone marks normally associated with active and repressed genes respectively. We used antibodies to H3K4me2 and H3K9me2 in ChIP-arrays to compare their profiles and variations in human THP-1 monocytes cultured in normal glucose (NG) and HG separately. We used human 12K cDNA and 12K CpG arrays representing coding and promoter regions respectively. After statistical analyses of the ChIP-microarray data, we identified key candidate genes relevant to diabetes that displayed changes in H3K4me2 and H3K9me2 in HG relative to NG and also validated them with follow-up conventional ChIPs. Relevance to diabetes was further demonstrated by examining these modifications by ChIPs in peripheral blood monocytes isolated from patients with type 1 diabetes relative to normal controls. In addition, regular mRNA profiling with the cDNA arrays revealed both anticipated and unanticipated correlations between mRNA expression, H3K4me2 and H3K9me2 levels. These results showing HG-and diabetes-induced variations in histone methylation genome-wide suggest that the diseased cells may have distinct epigenomes and also provide new insights into diabetic changes in the context of histone methylation. This may lead to an epigenetic memory of transcription history responsible for the “metabolic memory” and sustained clinical complications of diabetes.
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Abstracts - Poster
Edward Ramos Abstract P93 Global characterization and function of Gypsy-like endogenous insulators in Drosophila melanogaster
Edward Ramos and Victor Corces
Johns Hopkins Univ, Dept of Biol Mudd Hall, 3400 N Charles St, Baltimore, MD 21218, U.S.A.
Higher-order chromatin organization is essential for the proper expression of eukaryotic genes. The chromatin domains established through this organization are thought to regulate gene expression by controlling intra-chromosomal domain communication. The gypsy chromatin insulator proteins of Drosophila melanogaster have been implicated in the establishment of these domains. However, the mechanism governing the genomic organization and maintenance of these elements is not well understood. To better understand the role of insulators in chromatin organization, we investigated the function of the insulator protein Su(Hw) at the global level. To this end we used a combination of traditional molecular, biochemical and genetic approaches along with novel computational tools for our studies. We have thoroughly characterized a number of endogenous Su(Hw) binding sites and determined, through gel shift binding assays, direct interaction between predicted Su(Hw) binding sites and Su(Hw) insulator protein. In addition, in vivo experiments demonstrated that these predicted endogenous Su(Hw) binding sites co-localize with Su(Hw) protein on polytene chromosomes. Finally and most importantly, these endogenous Su(Hw) binding sites function as bona fide insulators as they can prevent communication between enhancers and promoters as demonstrated in an enhancer blocking assay. Using computational analysis we are in the process of mapping these Su(Hw) binding site to the entire Drosophila genome to determine the global function of the Su(Hw) protein during fly development and everyday function. It is our hope to find a functional correlation between our endogenous insulator binding sites and global genomic regulation
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William Renthal Abstract P94 Class II histone deacetylases regulate the behavioral adaptations to chronic cocaine and stress
William Renthal1, Arvind Kumar1, Vaishnav Krishnan1, Scott J. Russo1, David E. Theobald1, Kwang-Ho Choi1, Nadia Tsankova1, Rachel Neve2, Eric N. Olson3 and Eric J. Nestler1
1Department of Psychiatry, UT Southwestern Medical Center, 2Department of Psychiatry, McLean Hospital, Harvard Medical Center, 3Department of Molecular Biology, UT Southwestern Medical Center, U.S.A.
Addiction and depression are chronic psychiatric disorders in which long-lasting changes in gene expression are thought to contribute to the behavioral pathologies. Previous studies from our lab have identified alterations in histone acetylation in rodent models of addiction and depression that may contribute to the longevity of these disorders. Since neurotransmission is highly dependent on calcium signaling, we focused on the calcium- regulated class II histone decacetylases (HDACs) as potential mediators of the addiction and depression-induced changes in chromatin structure. Calcium influx results in the rapid phosphorylation and nuclear exportation of these HDACs, which in turn leads to a subsequent increase of histone acetylation on target genes. Mice lacking the class II HDACs, HDAC5 or HDAC9, are completely normal in a variety of behavioral tests from learning and memory to acute cocaine reward. However, after chronic cocaine treatments or chronic stress, they demonstrate increased sensitivity to both stimuli. These mice seem to hyper-adapt, suggesting that proper balance of histone acetylation is important in the behavioral adaptations to chronic but not acute stimuli. We have also found that cocaine and stress regulate HDAC5 phosphorylation in wild type mice, which corresponds with changes in histone acetylation of HDAC5 target genes that were identified by gene expression microarrays and ChIP on chip technology. We are currently investigating the role of these HDAC5 target genes individually using viral-mediated gene transfer into specific nuclei in the brain. These studies together with analogous findings in models of chronic cardiac stress, implicate class II HDACs in the molecular machinery needed for adapting to a variety of chronic environmental stimuli.
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Karsten Rippe Abstract P95 Activities of histone chaperone NAP1: Association states and interactions with histones, nucleosome assembly and effect on the chromatin fiber conformation
Karsten Rippe, Jacek Mazurkiewicz, Felix Kepert and Katalin Fejes Toth
Kirchhoff-Institut fur Physik, Molecular Biophysics Group, Ruprecht-Karls-Universitat Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany
The nucleosome assembly protein 1 (NAP1) is a histone chaperone that functions as a carrier of histones during nuclear import, nucleosome assembly and chromatin remodeling. We have examined (i) the association states of NAP1 alone and in complexes with histones [1], (ii) the process of mononucleosome assembly mediated by NAP1 on DNA fragments of 146 and 207 bp length containing a 5 S rDNA nucleosome positioning sequence [2], and (iii) how the interaction of NAP1 with core and linker histones affects the chromatin fiber organization [3]. Taken together these results provide complementary insight into the mechanisms, by which NAP1 exerts its different biological activites.
Refs: 1. Fejes Toth, K., Mazurkiewicz, J. & Rippe, K. (2005). J. Biol. Chem. 280, 15690-15699. 2. Mazurkiewicz, J., Kepert, J.F. & Rippe, K. (2006). J. Biol. Chem. 281, 16462-16472. 3. Kepert, J.F., Mazurkiewicz, J., Heuvelman, G., Fejes Toth, K. & Rippe, K. (2005). J. Biol. Chem. 280, 34063-34072.
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Charles Roberts Abstract P96 The Swi/Snf chromatin remodeling complex regulates lineage specific transcription programs during development and impairment of this activity causes cancer
Xi Wang, Miriam B. Werneck, Courtney G. Sansam, Michael S. Isakoff and Julia A. Evans
Harvey Cantor and Charles W. M. Roberts. Dana-Farber Cancer Inst., Boston, MA, U.S.A.
Chromatin remodeling complexes control nucleosome positioning and are dynamic regulators of transcription. While the biochemical role of these complexes in nucleosome mobilization is beginning to be elucidated, little understanding exists with respect to the role of the complexes in coordinated control of gene expression programs such as those required for lineage specific development. Snf5/Ini1/Baf47/Smarcb1 is a core member of the Swi/Snf chromatin remodeling complex and is a potent tumor suppressor that is inactivated in aggressive childhood cancers. As many transcription factors involved in oncogenic transformation are master regulators of lineage specific development, we hypothesized that the Swi/Snf complex, which also regulates transcription, may serve a similar role and that this activity may underlie its role in tumor suppression. We therefore sought to identify the developmental function of Snf5 and to elucidate the mechanistic basis of its tumor suppressor activity. Since the progressive stages of T cell development are well characterized and because Snf5 loss gives rise to T cell lymphomas, we chose to investigate the role of Snf5 in T cell development. We found that deletion of Snf5 in mice leads to aberrant gene expression in T cells, abnormal developmental progression with imbalanced CD4:CD8 bifurcation and culminates in a specific developmental block. This dysfunction and imbalance is coupled with the extremely rapid formation of mature CD8+ T cell lymphomas in 100% of mice. This developmental role is highly specific for alpha-beta T cells as we show that Snf5 is largely dispensable for T cells of the gamma-delta lineage and that it possesses no tumor suppressor activity within the gamma-delta or B cell lineages. Lastly, we show that the tumor suppressor activity is likely derived from the role of Snf5 in restricting cell cycle progression/proliferation and promoting differentiation. Loss of Snf5 leads to aberrant stimulation of the cell cycle by causing down-regulation of p16Ink4a and activation of E2F targets which in turn directly drive replication. These changes promote cancer formation. Collectively, our data demonstrate a role for the Swi/Snf chromatin remodeling complex in controlling lineage specific developmental programs and provide insight into how loss of this control leads to oncogenic transformation.
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Paul Sadowski Abstract P97 Post-translational modification of the insulator protein, CTCF
Melissa MacPherson, Linda Beatty, Wenjing Zhou and Paul Sadowski
Department of Medical Genetics and Microbiology, University of Toronto, Toronto M5S 1A8 Canada
The CTCF protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. It acts as a repressor of some genes such as the c-myc gene but it activates others such as the ß-amyloid precursor protein gene. It also plays a key role in regulating genomic imprinting of the IGF2 and H19 genes by binding to a differentially methylated insulator sequence that lies between the two genes. The CTCF protein may be over expressed or mutated in some cancers. It is post- translationally modified by poly(ADP) ribosylation and is phosphorylated by casein kinase II. We now report that CTCF is also post-translationally modified by the ubiquitin-like proteins, SUMO. We have co-transfected HEK293 cells with plasmids encoding tagged versions of CTCF and SUMOs 1, 2 or 3 followed by immunoprecipitation and western blotting to reveal that CTCF is SUMOylated by SUMOs 1, 2 and 3. The reaction is dependent upon the C- terminal diglycine amino acids of SUMOs 1 and 2 and is sensitive to the SUMO proteases SENP1 and SENP5 (vectors supplied by Dr. E. Yeh). The SUMOylation reaction is insensitive to 3-aminobenzamide, an inhibitor of poly (ADP) ribose polymerase. Thus far, we have identified one strong SUMO acceptor site in the C-terminus of CTCF. SUMOylation of CTCF also occurs efficiently in vitro and SUMOylation does not affect CTCF’s ability to bind to DNA that contains CTCF-binding consensus sequences from the H19 insulator. We are studying the interrelationship of SUMOylation of CTCF with other post-translational modifications of the protein and its influence upon the multitude of transcriptional activities of this protein. (Supported by the Canadian Institutes of Health Research)
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Teresa Sanchez Alcaraz Abstract P98 Role of USP7 and GMP synthetase in deubiquitination of human histone H2B
Teresa Sanchez Alcaraz1, Melissa Holowaty1, Yi Sheng2, Cheryl Arrowsmith2 and Lori Frappier1
1Department of Medical Genetics and Microbiology and 2Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada
One of the post-translational modifications that histones can undergo is monoubiquitination. This modification occurs predominantly on H2A and H2B but is poorly understood in terms of its effect on chromatin structure and function, as well as the enzymes controlling the ubiquitination/deubiquitination process. In mammalian cells, histone H2B has been shown to be ubiquitinated by Mdm2 and RFN20/40 but the ubiquitin-specific protease (USP) that reverses this modification is not known. We have been studying the structure and function of USP7 in human cells (also called HAUSP), which is the target of herpes simplex and Epstein-Barr virus proteins (ICP0 and EBNA1) and plays roles in p53 regulation (2005 Mol. Cell 18, 25; 2006 Nature Struc. Mol. Biol. 13, 285). To more completely determine how USP7 affects cellular processes, we investigated protein interactions of USP7 using an affinity column approach. We found that the most predominant interaction of USP7 is with human GMP synthetase. Interestingly, recent studies in Drosophila indicate that H2B is deubiquitinated by USP7 in complex with GMP synthetase (2005 Mol Cell 17, 695). We found that the USP7-GMP synthetase interaction occurs through the USP7 N-terminal domain, which is also responsible for binding p53, Mdm2 and EBNA1. We have shown that purified USP7 has some capacity to cleave ubiquitin from monoubiquitinated H2B isolated from human cells, but that this activity is greatly stimulated by purified GMP synthetase. This effect was specific for H2B, as deubiquitination of polyubiquitinated p53 by USP7 was not affected by GMP synthetase. Consistent with these observation, cellular levels of ubiquitinated H2B were decreased when USP7 was silenced in Hela cells by a hairpin RNA. The results suggest that a complex of USP7 and GMP synthetase is responsible for deubiquitination of H2B in higher eukaryotes.
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Annette Scharf Abstract P99 Dynamics of histone modifications during chromatin assembly
Annette ND Scharf, Ana Villar-Garea and Axel Imhof
Adolf Butenandt Institute, Schillerstrasse 44, 80336 Munich, Germany
Histone tails undergo a variety of posttranslational modifications that change their interaction with DNA and nuclear proteins. Combinations of modifications are thought to serve as a ‘histone code’. Changes in combinatorial histone modifications mark unique downstream events such as gene expression and DNA repair. Moreover histone modifications are thought to be transmitted through mitosis and therefore serve as mediators of cellular memory. However the way histone modifications are passed on from one cell generation to the other remains elusive. Using well-established MALDI-TOF and ESI MS/MS techniques our goal is to describe the changes in histone modifications during chromatin assembly. Therefore we use a Drosophila melanogaster assembly extract derived from preblastoderm embryos to assemble chromatin in vitro. Here, we find that there is a gradual deacetylation of H4K5 and H4K12 during the assembly, and that the deacetylation is dependent on ATP. We also confirm that the deacetylation is sensitive to HDAC inhibitors. We are currently investigating these findings in vivo by means of stable isotope labeling with amino acids in cell culture (SILAC).
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Stefan Schoeftner Abstract P100 Screening for miRNAs regulating mammalian telomeres
Stefan Schoeftner1, Susana Gonzalez1, Manuel Serrano1, Gregory J. Hannon2 and Maria A. Blasco1
1Spanish National Cancer Center (CNIO), 28029 Madrid, Spain; 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, U.S.A.
Mammalian telomeres are specialized chromatin structures at the ends of chromosomes that are essential for genomic stability. Telomeres shorten witch age due to the so called “end-replication problem”, a process underlying ageing. Telomere length is regulated by proteins binding directly to telomeric TTAGGG tandem repeats but also by chromatin modifying enzymatic activities such as histone lysine methyltransferases (HMTases) and DNA methyltransferases (DNMTs). Recently, microRNAs (miRNAs) were identified to play a central role in regulating animal development and physiology. miRNAs are ~21-26 nucleotide RNAs that regulate their target RNA by triggering endonuclease driven cleavage or translational repression. Mammalian miRNAs are generated from PolII transcribed precursor RNAs and processed into an active form by cleavage by the RNase III enzymes Drosha and Dicer. Importantly, mouse ES cells lacking Dicer show dramatically elongated telomeres with altered chromatin structure*. This finding suggests that miRNAs could be directly or indirectly involved in the regulation of telomere function and contribute to telomere associated diseases. In order to identify miRNAs involved in the regulation of telomeres we are taking advantage of a miRNA library comprising a set of 150 predicted miRNAs. The miRNA containing vectors were transiently transfected in to a Hela cell line carrying a luciferase reporter gene in close proximity to a telomere. miRNA induced changes in telomeric or subtelomeric chromatin result in altered expression of the reporter gene, a phenomenon known as “telomere position effect” (TPE). Additionally, the miRNA library was transfected into a standard Hela cell line to study changes in telomere length and global chromatin structure using high throughput quantitative fluorescence hybridisation (Q-FISH) and quantitative immunofluorescence (Q-IF). The poster outlines the primary screening as well functional validation of candidate miRNAs regulating telomere length and telomeric chromatin.
*Benetti et al., unpublished data
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Gunnar Schotta Abstract P101 A genome-wide transition to H4K20 mono-methylation impairs stress-induced and programd DNA damage response in the mouse
Gunnar Schotta, Roopsha Sengupta, Stefan Kubicek, Stephen Malin, Michaela Pagani, Monika Kauer, Alexsandra Espejo, Mark Bedford, Meinrad Busslinger and Thomas Jenuwein
Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, A-1030 Vienna, Austria; University of Texas, M.D. Anderson Cancer Center, Science Park Research Division, PO box 389, Smithville, Texas 79857, U.S.A.
H4K20 methylation is a broad epigenetic modification that has been linked with gene silencing, heterochromatin formation and response of the chromatin template to environmental signals. To analyze its function during mammalian development, we have disrupted the two Suv4-20h HMTases in the mouse. Whereas Suv4-20h1 null mutants display perinatal lethality, Suv4-20h2 deficient mice develop normally. Using conditional alleles, we generated Suv4-20h double null (dn) mice, in which nearly all H4K20me3 and H4K20me2 states are lost, resulting in a genome-wide transition to H4K20me1 chromatin. Suv4-20h dn mouse embryonic fibroblasts (MEFs) display impaired proliferation, have reduced S-phase ratios and enter crisis at early passage numbers. The DNA damage checkpoint protein 53bp1 selectively associates with H4K20me2 nucleosomes, and recruitment of 53bp1 to DSBs is significantly impaired in Suv4-20h dn cells. Importantly, Suv4-20h dn B-cells are also defective in class switch recombination, which reflects a developmentally programd pathway for DSB-mediated antibody isotype diversification. Thus, an H4K20me1 chromatin is insufficient to ensure mammalian genome function and H4K20me2 is a major signal in processing stress-induced and programd DNA damage response in the mouse.
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David Schrump Abstract P102 Brother of the Regulator of Imprinted Sites (BORIS) recruits Sp1 to modulate NY-ESO-1 expression in lung cancer cells
Yang Kang, Julie A. Hong, G. Aaron Chen, Dao M. Nguyen and David S. Schrump
Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, U.S.A.
Epigenetic alterations during malignant transformation facilitate de-repression of a variety of germ cell-restricted genes, such as NY-ESO-1, which encodes a cytoplasmic protein that is immunogenic in cancer patients. Recently we reported that the paralogous zinc finger proteins- CTCF and BORIS, directly contribute to transcriptional regulation of NY-ESO-1 in lung cancer cells. To further examine mechanisms that mediate NY-ESO-1 expression, we performed software-guided analysis of the NY-ESO-1 promoter region, which revealed several potential Sp1 binding motifs. Promoter-reporter assays demonstrated that deletions, which sequentially eliminated the putative BORIS/CTCF recognition sequence and a prototypic Sp1 binding site markedly decreased NY-ESO-1 promoter activity. Transient transfection experiments using promoter-reporter constructs, electromobility shift assays, and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activity coincided with increased occupancy of the proximal Sp1 binding site in lung cancer cells. Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to this motif in vitro. siRNA-mediated inhibition of Sp1 expression coincided with markedly diminished NY-ESO-1 promoter activity in lung cancer cells. In contrast, abrogation of CTCF expression resulted in pronounced augmentation of NY-ESO-1 promoter activity. Co- immunoprecipitation experiments indicated that Sp1 physically interacts with BORIS but not with CTCF in vivo. Collectively, these findings suggest that NY-ESO-1 expression is governed by distinct transcriptional complexes during pulmonary carcinogenesis, and that BORIS recruits Sp1 to augment NY-ESO-1 expression in lung cancer cells.
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Bonnie Scott Abstract P103 Evolution of centromere-binding proteins and their interactions with centromere DNA in Arabidopsis
Bonnie Scott, Song Luo, Sarah Hall and Daphne Preuss
Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology, GCIS W519, University of Chicago, 929 E. 57th Street, Chicago, IL 60637, U.S.A.
During cell division, proper chromosome segregation involves assembling a kinetochore protein complex at the centromere region of every chromosome. A fundamental yet unresolved question is how inner kinetochore proteins initially choose the site for kinetochore assembly and subsequently maintain its identity as a centromere throughout the cell cycle. The challenge in understanding these phenomena is that inner kinetochore proteins, namely centromere-binding proteins (CENP) A and C, share high sequence similarity between plants, yeast, and animals, yet the centromere DNA to which they associate lacks a conserved sequence and is instead composed of repetitive DNA, including rapidly evolving satellite DNA. These observations have led to the hypothesis that CENPs evolve adaptively across species to maintain protein-DNA interactions at the centromere for chromosome inheritance. The goal of this study is to identify structural features within plant centromere-binding proteins that are required for species-specific centromere function in vivo. To accomplish this, we have first cloned CENP-A and CENP-C sequences from increasingly diverged Arabidopsis species and used phylogenetic tools to understand their evolutionary history and identify putative regions important for species- specificity. Second, we are expressing these CENP sequences into corresponding Arabidopsis thaliana mutants to define the degree of natural variation able to rescue CENP function at the centromere.
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David Shechter Abstract P104 Histone H2A arginine3 is mono- and symmetrically-di methylated by a complex of PRMT5 and the WD-repeat protein MEP50 in Xenopus laevis eggs
David Shechter1, Raghu Chitta2, Eileen Woo3, Brian Chait3, Jeffrey Shabanowitz2, Donald F. Hunt2 and C. David Allis1
1The Laboratory of Chromatin Biology, Rockefeller University, New York, NY 10021; 2Chemistry Department, University of Virginia, Charlottesville, VA 22904; 3The Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY 10021, U.S.A.
A wealth of emerging literature demonstrate that post-translational modification of histones, the protein components of nucleosomes which are the fundamental repeating unit of chromatin, serve to regulate a wide variety of DNA-templated processes, notably gene regulation. Methylation of arginines in histones has been observed in many contexts, including gene activation and repression [1]. Histone arginine methylation has been correlated with events during development, especially in germ cells. Here we report the identification of the predominant histone methyltransferase in extracts of the large eggs of Xenopus laevis. The activity is composed of, at minimum, a complex of the methyltransferase PRMT5 (also known as Hsl7 in Xenopus and yeast) and the WD-Repeat Protein MEP50. It specifically catalyzes mono- and symmetric-di methylation on free histone H2A on arginine 3, as determined by mass spectrometry and by immunoblotting with H2A/H4R3me-specific antibodies. The complex methylates H2A and H4 in nucleosomes at a much lower activity. The methylation activity towards H2A is inhibited by H2A Serine-1 phosphorylation, although we did observe H2AS1phos and H2AR3Me on the same protein molecule by mass spectrometry. AMI-1, a general small molecule inhibitor of PRMTs, inhibits this complex at approximately 100µM. This activity and H2AR3Me2S modification is abundant in the egg and disappears rapidly during development. In conclusion, H2AR3 methylation is a major histone modification in the early development of Xenopus laevis. Experiments are in progress to gain insight into the biological function(s) of PRMT5/MEP50 complex in the context of early development. We also are exploring whether MEP50 serves to present H2A to PRMT5, analogous to the function for the WD-Repeat Protein WDR5 in MLL (mixed lineage leukemia) complex isolated from human cells as recently described by our laboratory [2, 3].
Refs 1. Bedford, M.T. and S. Richard, Arginine methylation an emerging regulator of protein function. Mol Cell, 2005. 18(3): p. 263-72. 2. Dou, Y., et al., Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat Struct Mol Biol, 2006. 13(8): p. 713-9. 3. Ruthenburg, A.J., et al., Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex. Nat Struct Mol Biol, 2006. 13(8): p. 704-12.
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Yoichi Shinkai Abstract P105 H3K9 methylation and germ cell development
Makoto Tachibana1, Masami Nozaki2, Naoki Takeda3 and Yoichi Shinkai1
1Institute for Virus Research, Kyoto University, 2Research Institute for Microbial Disease, Osaka University, 3Center for Animal Resources and Development, Kumamoto University, Japan
It is known that epigenetic cellular memories are dynamically reprogramd during germ cell development. G9a is one of major histone H3 lysine 9 (H3K9) methyltransferases at euchromatin. To elucidate how G9a and G9a-mediated H3K9 methylation are crucial for germ cell development, we established germ-lineage specific G9a deficient mice. In the absence of G9a, male and most female mice are sterile. Even TNAP (tissue non-specific alkaline phosphatase) promoter-driven Cre enzyme supposedly inactivates a G9a gene (and G9a-mediated H3K9 methylation) before the migration stage into testis, spermatogenesis in G9a-deficient mice until a gonia type stage seems to be intact. However, spermatocytes later a pachytene stage are missing. Furthermore, abnormal meiotic-prophase progression and synaptonemal complex formation are frequently observed in G9a-deficient spermatocytes. Oogenesis is also impaired around the pachytene stage. DNA microarray analysis described that multiple genes are activated or up-regulated in G9a-deficient testicular cells. These results emphasize a crucial function for G9a (and G9a-mediated H3K9 methylation) in controlling germ cell development and meiotic-prophase progression.
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Krishna Sinha Abstract P106 Inhibition of the transcriptional activity of osterix by interactions with NO66, a jumonji family chromatin protein
Krishna M Sinha, Xin Zhou, Chi Zhang, Lingna Zhang and Benoit de Crombrugghe
Department of Molecular Genetics, UT M. D. Anderson Cancer Center, Houston, TX 77030, U.S.A.
Osterix (Osx) is a critical osteoblast-specific transcription factor required for bone formation. Genetic inactivation of Osx in mice leads to complete arrest of osteoblast differentiation, although chondrocyte differentiation and cartilage formation are normal. However, the mechanism of Osx function during osteoblast differentiation is not well understood. In the present study, the Jumonji (JmjC) domain containing protein, NO66, was identified as an Osx-interacting protein using an Osx expressing stable osteoblast cell line by tandem immunoaffinity chromatography and mass spectrometry. Co-immunoprecipitation and GST- pulldown assays showed that NO66 physically interacts with Osx. NO66 interacts through its JmjC domain with the transcription activation domain of Osx. In situ RNA hybridization experiments revealed that NO66 and Osx are co-expressed in skeletal elements of hind limbs and forelimbs, in vertebrae, ribs, and craniofacial bones during mouse embryonic development. In DNA transfection assays, Osx strongly stimulates the activity of a 1kb osteocalcin promoter and that of an osteoblast specific 2.3-kb Col1a1 promoter. Co- transfection of NO66 results in a strong inhibition of the Osx-dependent activity of these promoters, whereas the activity of several other promoters was not affected by NO66. In addition to this, transfection of NO66 in osteoblasts also inhibits the activity of the endogenous osteocalcin gene. Furthermore, knockdown of Obelix expression in osteoblasts by specific siRNAs results in increased expression of several osteoblast-specific marker genes including Col1a1, Osteocalcin and Bone Sialoprotein (BSP) without apparent changes in the level of Osx expression. Overall our data suggest the hypothesis that NO66 is a negative regulator of Osx and, hence, of osteoblast differentiation or function. The presence of a JmjC domain in NO66 raises the intriguing possibility that inhibitory effect of NO66 is mediated by demethylation of specific lysine residues of histones within the chromatin environment of osteoblasts.
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Abstracts - Poster
Karen Smith Abstract P107 Identification and characterization of novel HDAC-associated proteins that regulate cancer cell growth
Karen T. Smith, Skylar A. Martin-Brown, Laurence Florens, Michael P. Washburn and Jerry L. Workman
The Stowers Institute for Medical Research, Kansas City, MO 64110 U.S.A.
Histone deacetylase (HDAC) inhibitors are one class of chemotherapeutic drugs. However, these inhibitors exhibit undesirable side effects in cancer patients due to their inability to discriminate among the several structurally similar HDACs in humans. HDACs reside in multi-subunit protein complexes and some of these HDAC-associated proteins are needed for full activity of the HDACs themselves. We propose that a single HDAC can be inhibited in the cell by targeting HDAC-associated proteins rather than the HDACs themselves. We have taken a proteomics approach to identify proteins differentially associated with human HDAC1 and HDAC3 in 293T cells. Preliminary experiments have identified several known members of HDAC-containing complexes and many novel HDAC-interacting proteins. Importantly, many of these proteins differentially associate with HDAC1 or HDAC3. Several of these newly identified HDAC1 and HDAC3-interacting proteins have a connection to chromatin or cell growth, while others have not yet been described. We are currently confirming these interactions in 293T cells and testing the conservation of these interactions in cancer cell lines. We then will test if these HDAC-interacting proteins are important for their associated HDAC’s activity. Proteins that have a significant effect on HDAC1 or HDAC3 activity will then be abrogated in cancer cell lines to determine if loss of these HDAC-associated proteins can inhibit cancer cell growth.
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Matthew Smith Abstract P108 Chromatin - mediated silencing of immune response genes
Matthew Smith, Jian Wu and Kenneth Wright
University of South Florida College of Medicine, Department of Molecular Medicine and Moffitt Cancer Center Tampa, FL, U.S.A.
The transcriptional repressor PRDI-BF1 (Blimp-1) is required for terminal differentiation of plasma cells, T-cell homeostasis and silencing of interferon beta expression upon viral induction. The repressive functions of PRDI-BF1 are mediated through interactions with additional co-factors, such as Groucho, HDAC1/2 and G9a. Here, we demonstrate a role for PRDI-BF1 in the maturation of dendritic cells (DCs). Considerable phenotypic differences exist between immature and mature DC populations. Immature DCs function in immune surveillance via antigen uptake and processing, while mature DCs present antigen in the context of MHC Class II molecules to immune effector molecules. This phenotypic transition involves an orchestrated change in gene expression profiles, exemplified by the down- regulation of CIITA and MRC1 upon maturation. CIITA, encoded by MHC2TA, is the master regulator of MHC Class II transcription which is known to be silenced upon maturation. MRC1 (CD206) recognizes extracellular pathogens via oligosaccharide domains and is capable of activating the innate immune response. PRDI-BF1 is induced upon DC maturation (antigen encounter) and inversely correlates with expression of MHC2TA and MRC1 mRNA. Using in vivo genomic footprinting and chromatin immunoprecipitation experiments, we show that transcriptional co-activators are displaced by PRDI-BF1 at the promoter regions of MHC2TA and MRC1. Upon maturation, these regions demonstrate hallmarks of silent chromatin, such as decreased acetylation of histones H3 and H4, decreased methylation of histone H3 lysine 4 and increased di-methylation of histone H3 lysine 9. Furthermore, this silencing is mediated by the G9a histone methyltransferase and the heterochromatin protein, HP1 gamma. These results provide novel insight into the molecular mechanisms underlying the maturation process in DCs.
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Hae-Ryong Song Abstract P109 Coordination of transcriptional regulation and chromatin modification of Arabidopsis circadian clock genes
Hae-Ryong Song1,3, Ju-Hee Jeong1,3, Bosl Noh2,3 and Yoo-Sun Noh1,3
1Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea., 2Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea. 3Global Research Laboratory for Flowering at SNU and UW, Seoul National University, Seoul 151-742, Korea
Circadian clock genes are regulated through a transcriptional-translational feedback loop. In Arabidopsis, LHY and CCA1 transcripts are highly expressed in the early morning. Translated LHY and CCA1 proteins repress the expression of TOC1 transcript which peaks in the evening. TOC1 protein elevates the expression of LHY and CCA1 mRNAs, forming a negative feedback loop that is believed to constitute the oscillatory mechanism of the clock. Recently the rhythmic oscillation of mouse clock genes, mPER1 and mPER2, was shown to be correlated with the regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the chromatin modification- mediated transcriptional regulation of Arabidopsis circadian clock genes. Here we propose a possibility that Arabidosis clock-associated genes, LHY, CCA1, and TOC1 might be regulated by rhythmic histone modifications. Our results show that certain type of histone modifications either has positive or negative correlations with the expression of LHY, CCA1, and TOC1 transcripts. Therefore, the rhythmic transcription of these clock genes might depend on regular histone modifications within their chromatin and the fine-tuning of the feedback loop comprising an oscillator in plants might be accomplished by an ordered modification of histones.
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Stacey Southall Abstract P110 Structural studies of histone methyltransferases
Stacey M. Southall and Jonathon R. Wilson
Chromatin Regulation Team, Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB
Histone lysine methylation is an important epigenetic marker that can profoundly influence delineation of chromatin regions determining patterns of gene expression and consequently defining cell state. This precisely targeted modification is catalysed by a family of histone methyltransferases (HMTs) containing the evolutionarily conserved SET domain. Our focus is on the methylation of lysine 20 of histone H4. Multiple SET-containing methyltransferases are able to specifically mono-, di- and/or tri-methylate this lysine residue. Although the precise downstream effects of these modifications have not been determined, it is clear that each methylation state has a different physiological role. Structural analysis of SET domain methyltransferases has given insight into the molecular mechanism of methyltransfer, however to fully understand epigenetic regulation both in the normal cell and in disease we need to obtain a better understanding of how different HMTs are targeted to the same residue and how their activity is regulated at the molecular level. Our strategy is to apply biochemical, biophysical and structural techniques to different H4- K20 HMTs such as nuclear receptor-binding SET domain-containing proteins (NSD family) and the Suv4-20h family. It is now understood that many cancers have an epigenetic component and so modifying enzymes such as HMTs are likely to become drug targets. Our studies will therefore help to inform the rational design of such drugs.
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Maike Stam Abstract P111 Molecular analysis of chromatin changes involved in b1 paramutation, an allele-dependent transfer of epigenetic information
Maike Stam, Max Haring, Rechien Bader and Marieke Louwers
SILS, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
We investigate the molecular mechanism underlying paramutation, a mitotically and meiotically heritable change in gene expression induced by allele interactions in trans. Paramutation is observed in a wide variety of plants and more recently in fungi and mammals, emphasizing its significance. We study paramutation at the b1 locus in maize, where it affects the pigmentation phenotype. The low expressed B’ epiallele imposes its low transcription rate onto the high expressed B-I epiallele in trans. This change correlates with changes in DNA methylation and chromatin structure. A regulatory element, containing seven tandem repeats located ~100 kb upstream of the b1 coding region, is essential for b1 paramutation and functions as an enhancer for the B-I epiallele. We hypothesize a physical interaction between the repeats and the b1 promoter, and are currently using 3C technology (Dekker et al., 2002) to identify spatial, long range, in cis interactions within the 100 kb b1 chromatin domain. The regions spanning the repeat junctions show differential DNA methylation (only methylated in B’) and nuclease sensitivity (nuclease hypersensitive in B-I), suggesting differential binding of chromatin factors. DNA methylation analyses of mutants preventing paramutation indicate that the repeat junctions play an important role in trans-inactivation, while the regions flanking the junctions play a role in enhancement of b1 expression. ChIP experiments using an anti-H3K4me2 antibody indicates that both the B’ and B-I coding regions are transcription competent throughout development. Upon transcriptional enhancement of b1, the repeat region shows H3K9ac/14ac in high expressing B-I, and H3K9me2 in low expressing B’ tissue, indicating a developmentally regulated crosstalk between the transcription activation machinery and the heritable B-I and B’ epigenetic states. Intriguingly, B’ is H3K27 dimethylated at the promoter and coding region throughout development, suggesting a role for H3K27me2 in the heritability of the B’ expression state. We are currently using mutants to dissect the role of histone modifications in paramutation.
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Sean Taverna Abstract P112 Connecting H3 methylation and acetylation : The role of Yng1 in transcription
Sean D. Taverna1, Serge Ilin3, Richard S. Rogers4,5, Jason C. Tanny1, Heather Lavender6, Haitao Li3, Lindsey Baker1, John Boyle4,5, Lauren P. Blair6, Brian T. Chait2, Dinshaw J. Patel3, John D. Aitchison4,5, Alan J. Tackett6 and C. David Allis1
1Laboratory of Chromatin Biology, The Rockefeller University, New York, NY 10021, U.S.A., 2Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10021, U.S.A., 3Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, U.S.A., 4Institute for Systems Biology, Seattle, WA 98103, U.S.A., 5Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada, 6Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, U.S.A.
Post-translational histone modifications participate in modulating the structure and function of chromatin. Promoters of transcribed genes are enriched with K4 trimethylation and hyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like the Yng1 component in the NuA3 HAT complex, were shown to interact with H3K4me3, indicating a biochemical link between K4 methylation and hyperacetylation. Using a combination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD- H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14. Furthermore, genome-wide ChIP-Chip analysis demonstrates co-localization of Yng1 and H3K4me3 in vivo. Disrupting the K4me3-binding capacity of Yng1 altered K14ac and transcription at certain genes, thereby demonstrating direct in vivo evidence of sequential trimethyl-binding, acetyltransferase activity, and gene regulation by NuA3. Our data support a general mechanism of transcriptional control through which histone acetylation upstream of gene activation is promoted partially through availability of H3K4me3, “read” by binding modules in select subunits.
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Tage Thorstensen Abstract P113 The Arabidopsis SUVR proteins define a novel subgroup of SET domain proteins associated with the nucleolus
Tage Thorstensen, Andreas Fischer1, Silje V. Sandvik, Sylvia S. Johnsen, Paul E. Grini, Gunter Reuter1 and Reidunn B. Aalen
Department of Molecular Biosciences, University of Oslo P.O. Box 1041 Blindern, N-0316 Oslo, Norway, 1Institute of Genetics, Biologicum, Martin Luther University Halle Halle, Germany
The methylation pattern of the different lysine residues on histone tails has been shown to be part of the so called “histone code” and to be important in the regulation of eukaryotic gene expression and chromatin structure. The proteins responsible for the majority of this methylation contain the evolutionary conserved SET domain. SET domain proteins related to the Drosophila SU(VAR)3-9 protein have been associated with gene repression and hete- rochromatinization. There are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9 in Arabidopsis, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel WIYLD domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far which are associated with heterochromatin. The subnuclear localization of SUVR proteins is regulated by alternative splicing and we found that the SUVR4 protein is a histone lysine methyltransferase (HKMTase) with preference for monomethylated histone H3K9 in vitro.
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Christopher Topp Abstract P114 Unusually-sized centromeric RNAs associate with maize centromeric chromatin
Christopher N. Topp1 and R. Kelly Dawe1,2
1Department of Plant Biology and 2Department of Genetics, University of Georgia, Athens, GA 30602
Centromeres control the strict inheritance of genetic information, yet are themselves genetically ill-defined. The repetitive DNA sequences of most eukaryotic centromeres are disposed to rapid evolutionary change, differing among even recently diverged species. Currently the best definition for a functional centromere is epigenetic: the presence of a centromere-specific histone variant, CENH3. When highly overexpressed, CENH3 can apparently nucleate de novo kinetochore formation at ectopic locations (Heun et al 2006). However the normal mechanisms that specify CENH3 deposition and the composition of centromeric chromatin remain unclear. Here we demonstrate that unusually-sized small, non-coding centromeric RNA species are physically associated with maize centromeric chromatin, as assayed by chromatin immunoprecipitation with CENH3. We discuss the implications of these data for centromere function and evolution.
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Martin Tribus Abstract P115 Molecular mechanisms of histone variant H3.3 assembly by the motor protein CHD1
Martin Tribus1, Alexander Konev2, Valerie Podhraski1, Dmitry Fyodorov2 and Alexandra Lusser1
1Division of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Insbruck, Austria, 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A.
The Drosophila Snf2-related ATPase CHD1 functions as an ATP-dependent chromatin assembly factor. We have previously shown that CHD1 mediates the assembly of extended periodic nucleosome arrays in a purified system containing recombinant histone chaperone NAP-1, purified Drosophila core histones and relaxed circular DNA. We have begun to examine the biological functions of CHD1. To this end, we generated three mutant alleles of Chd1 in Drosophila. Two mutations result in a loss of CHD1 protein, while the third allele gives rise to a C-terminally truncated polypeptide. We discovered that all three alleles are maternal effect embryonic lethal mutations as embryos laid by homozygous mutant females die before hatching. Intriguingly, we discovered that the absence of maternal CHD1 blocks the incorporation of the histone variant H3.3 into the paternal chromatin and thus results in the exclusion of the paternal genome from zygote formation. Furthermore, CHD1 is required for H3.3 deposition in transcriptionally active chromatin of late syncytial embryos. It was recently shown that the histone chaperone HIRA is required for delivery of H3.3 into the male pronucleus in Drosophila. Here we present evidence that CHD1 can utilize HIRA as a chaperone to mediate the assembly of H3.3 containing nucleosomes in vitro. We could not detect direct or histone mediated interactions between recombinant CHD1 and HIRA. However, co- immunoprecipitation from Drosophila embryonic extracts revealed a weak association of CHD1 with HIRA. Thus, our in vivo and in vitro data uncover a CHD1 and HIRA-dependent pathway for the assembly of the histone variant H3.3. CHD1 contains two chromodomains that have recently been shown to be H3-methylK4 binding modules. Binding of CHD1 to methylated H3 is thought to be critical for the function of CHD1 as an elongation factor. To examine the contribution of the chromodomains of CHD1 to its transcription-independent function in chromatin assembly during early embryonic development, we have generated transgenes that carry point mutations at conserved amino acid residues critical for H3-methylK4 binding. We will present data that show that the chromodomains are required for some but not all in vivo functions of CHD1.
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Christopher Vakoc Abstract P116 A profile of histone lysine methylation generated by mammalian gene transcription
Christopher R. Vakoc, Mira M. Sachdeva, Hongxin Wang, and Gerd A. Blobel
Children’s Hospital of Philadelphia,Division of Hematology, Philadelphia, PA 19104, and the University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.
Complex patterns of histone methylation encode distinct functions within chromatin. Lysine methylation displays the highest degree of complexity among known covalent histone modifications, with each site of methylation regulating the association of different effector molecules. We and others previously reported that tri-methylation of lysine 9 of histone H3 occurs at both silent heterochromatin and at the transcribed region of active mammalian genes, suggesting that the extent of histone lysine methylation involved in mammalian gene activation is not completely defined. To identify additional sites of histone methylation that respond to mammalian gene activity, we describe here a comparative assessment of all six known positions of lysine methylation and relate them to gene transcription. For our studies we used the highly expressed housekeeping gene PABPC1 that spans more than 19 kb thus permitting high-resolution comparison of histone lysine methylation landscapes. In addition, we used genes that can be transcriptionally induced or repressed. We observed high trimethylation of H3K4, H3K9, H3K36, andH3K79 in actively transcribed regions, consistent with previous findings. H4K20 mono-methylation, a modification previously linked with repression, we identified as a mark of transcription elongation in mammalian cells. In contrast, H3K27 monomethylation, a modification enriched at peri-centromeric heterochromatin, was observed broadly distributed throughout all euchromatic sites analyzed, with selective depletion in the vicinity of the transcription start site at active genes. Together, these results underscore that similar to other described methyl-lysine modifications, H4K20 and H3K27 mono-methylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle.
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Claudius Vincenz Abstract P117 Visualizing polycomb group protein interactions with histones in vivo
Claudius Vincenz and Tom Kerppola
1150 West Medical Ctr. Dr., 4574 MSRBII, Ann Arbor, MI 48109, U.S.A.
The Polycomb Group proteins consist of about three dozen structurally unrelated proteins that were originally identified by their ability to produce a common phenotype in Drosophila. More recent mechanistic studies have identified chromatin as the target for the activity of these proteins. Specifically, these proteins both methylate lysine 27 of H3 as well as bind to this post-translational modification to repress transcription of the underlying DNA. This repression is maintained through cell division producing a molecular memory that enables the cell to have a stable phenotype. In Drosophila the Polycomb protein has been shown to be the anchor that recruits a whole complex of Polycomb Group proteins to the methylated lysine. In mammalian cells a gene expansion has produced five homologues of this anchor protein. Here we show that these structural homologues, termed CBX proteins in vertebrates, display a nuclear distribution pattern characteristic for each protein. This suggests that methylation of lysine 27 cannot be the only determinant of their binding to chromatin. Furthermore, we use Bimolecular Fluorescence Complementation (BiFC) to document that homologous domains have different roles in mediating Histone H3 binding of each CBX protein. For example, deletion of the chromodomain of CBX4 reduces interaction with H3 greatly, confirming in vitro studies. However, the homologous mutation in CBX6 and CBX7 does not affect H3 binding. Both, CBX6&7 are highly expressed in Embryonic Stem cells. These results are discussed in the context of the unusual distribution of H3K4 and H3K27 methylation in the genome of ES cells. We postulate that this atypical binding mode has evolved to accommodate the rapid genome wide changes in transcription that are induced upon differentiation in ES cells.
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Vikki Weake Abstract P118 The SAGA histone acetyltransferase complex functions in the development of neuronal connectivity in the Drosophila compound eye
Vikki M Weake, Kenneth K. Lee, Sebastian Guelman and Jerry L. Workman
Stowers Institute for Medical Research, Kansas City, Missouri, U.S.A.
The SAGA histone acetyltransferase complex is involved in transcriptional regulation via the covalent modification of histones. A proteomics approach is being used in our lab to identify and characterize the components of Drosophila SAGA. This approach has identified two novel components of the Drosophila SAGA complex: nonstop and CG13379. Nonstop is homologous to yeast Ubp8, which catalyzes removal of the ubiquitin group from histone H2B. A critical balance in the level of ubiquitylated H2B is required for correct transcription of SAGA regulated genes in yeast, and crosstalk between histone ubiquitylation and methylation has been observed in previous studies. CG13379 is homologous to yeast Sgf11, which together with Ubp8 constitutes a functional module within yeast SAGA. We have confirmed that nonstop and Sgf11 are stable components of Drosophila SAGA by co- immunoprecipitation, and a pupal-lethal mutation in sgf11 has been isolated. Furthermore, we show that nonstop can complement the ubp8 deletion strain in yeast and incorporates into yeast SAGA. The deubiquitiylation activity of nonstop is currently being characterized both in yeast and Drosophila. Independently of our studies, a mutation in nonstop was isolated during a screen for mutations disrupting neuronal connectivity in the Drosophila visual system. In order to determine whether the neuronal defect of the nonstop mutant is due to its role in SAGA, neuronal connectivity of other Drosophila SAGA mutants has been analyzed. Mutation of other components of SAGA results in an identical phenotype to that of nonstop, indicating that SAGA function is required for correct axon targeting in the developing fly eye. The eventual aim of this work is to identify the underlying genetic pathways regulated by SAGA that are required for the establishment of correct neuronal connectivity in the developing Drosophila eye.
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Stephanie Williams Abstract P119 Mechanistic insights into promoter chromatin disassembly
Stephanie Williams, Melissa Adkins and Jessica Tyler
UCHSC, Dept. of Biochemistry, RC-1S, Rm 10403, 12801 E. 17th Ave., Aurora, CO 80010, U.S.A.
The disassembly of chromatin from promoter regions is a recently discovered mechanism for regulation of Eukaryotic gene expression. This process is best understood at the budding yeast PHO5 gene, where the histone H3/H4 chaperone Anti-silencing function 1 (Asf1) is essential for removing 3-4 nucleosomes from the PHO5 promoter upon phosphate depletion - the signal for activation of the PHO5 gene (Adkins et al., Molecular Cell 2004). We now show that Asf1-mediated promoter chromatin disassembly is also required for the activation of additional yeast genes. To understand the mechanism whereby promoter chromatin disassembly mediates transcriptional activation, we examined factor occupancy in vivo. Somewhat surprisingly, promoter chromatin disassembly is not required to enable access of the transcriptional activators to the promoter, but is required to enable access by the general transcription machinery, including TBP and RNA polymerase II. We will also present our recent analyses of the involvement of chromatin remodelers and histone acetyltransferases in promoter chromatin disassembly during transcriptional activation.
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Jon Wilson Abstract P120 Structural studies of SET domain methyltransferases
Jon Wilson
Institute of Cancer Research, Section of Structural Biology, 237 Fulham Road, Chelsea, London, SW3 6JB, U.K.
The methylation of lysine residues on histone tails is a potent signalling mechanism for defining patterns of gene expression via variation of chromatin organisation. Only a limited number of the lysines in histone tails are subject to modification but the possibility of the discreet addition of either one, two or three methyl groups allows for a high combinatorial potential. Correctly regulating the activity of histone methyltransferase enzymes is crucial as the consequence of mis-regulation can effect the expression of a large number of genes downstream. It is becoming increasingly apparent that epigenetic processes are involved in disease especially cancer. Whether lysine methylation leads to activation or repression is context specific. Disease can be caused by either activation of oncogenes or repression of tumour suppressors. Building upon previous analysis on Set7/9[1] and PR-Set7[2] we are establishing a better understanding of the molecular mechanisms involved in SET domain catalysed methyl transfer. The major area of interest is targeting of a particular lysine residue substrate and the determination of activity in terms of how many methyl groups a particular enzyme can add. This is a complex area given that some SET domain methyltransferase enzymes seem to have varying specificity depending on interactions within the multi-protein complexes of which they are components.
Refs: [1] Xiao, B., Jing, C., Wilson, J.R., Walker, P.A., Vasisht, N., Kelly, G., Howell, S.A., Taylor, I.A., Blackburn, G.M. & Gamblin, S.J. (2003) Structure and catalytic mechanism of the human histone methyltransferase SET7/9. Nature 421, 652-656. [2] Xiao,B., Jing, C., Kelly, G., Walker, P.A., Muskett, F.W., Frenkiel, T.A., Martin, S.R., Sarma, K., Reinberg, D., Gamblin, S.J. and Wilson, J.R. (2005) Specificity and Mechanism of the Histone Methyltransferase Pr-Set7. Genes & Development 19, 1444-1454.
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Zhaodong Xu Abstract P121 Remote elements critical for cytokine induced gene expression
Zhaodong Xu, Zuyao Ni, Mohamed Abou El Hassan, Tao Yu, Monika Sangwan, Mohamad Ahmad and Rod Bremner
Toronto Western Research Institute, Toronto Western Research Institute, 399 Bathurst Street, Mc6-424,Toronto, Ontario M5T 2S8, Canada
The role played by long-range elements in the regulation of gene transcription is still underappreciated. Hunting for these elements is a daunting task and developing a rapid and easy procedure is essentially important. Towards this end, we utilized histone acetylation to precisely mark interferon gamma (IFNgamma) specific regulatory elements throughout megabase (Mb) regions around 56 known IFN-responsive genes. IFNgamma induced STAT1 and IRF1 binding specifically at promoter proximal and remote sites from gene starts. Remarkably the vast majority of IFNgamma-induced STAT1 and IRF1 binding at promoters as well as remote sites was flagged by acetylated histones, verifying the role of acetylated chromatin in the prediction of promoters and most importantly of long-range elements. The activity at novel long-range elements, residing at as far as 70 kb of gene starts, were confirmed at the CIITA and SOCS1 loci using ChIP, reporter and looping assays. Thus, long- range elements are a novel aspect of IFNgamma-mediated gene induction and mapping acetylated chromatin is an excellent tool to find them, implying the specificity of the present method in finding remote regulatory elements in large genomic scales.
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Xiaofang Yang Abstract P122 Dissecting SWI/SNF ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae
Xiaofang Yang1, Roser Zaurin2, Sharmistha Kundu1, Miguel Beato2 and Craig Peterson1
1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605. 2Centre de Regulacio Genomica, Universitat Pompeu Fabra, Passeig Maritim 37- 49, E-08003 Barcelona, Spain
Yeast SWI/SNF is the founding member of an ATP-dependent chromatin remodeling superfamily involved in transcriptional regulation for a subset of genes. Most SWI/SNF-like chromatin remodeling enzymes have been purified as multiprotein complexes that contain an ATPase catalytic subunit highly homologous to yeast Swi2/Snf2. The fact that ATPase subunit like human Brg1 alone is active for chromatin remodeling raised the question on the role of other subunits for chromatin remodeling. Interestingly, mutations of core subunits of hSWI/SNF such as hSwi2/BRG1, hSnf5/INI1, or hSWI3 are found in many types of cancers. In this study, we investigated the role of different subunits for yeast SWI/SNF function through a partial deletion of the SANT domain of Swi3. Genome-wide transcriptional profile suggested that SWI3deltaSANT was basically a weak null allele of SWI3. SWI3deltaSANT crippled the recruitment of SWI/SNF to target promoter of CDC6, SIC1 and HO. In the absence of an intact SANT domain, tethering SWI/SNF via LexA-Swi2 was insufficient for transcriptional activation of a LexAop-GAL1TATA-LacZ reporter in vivo. Surprisingly, SWI3deltaSANT, SWI3R564E or swi3 caused dissociation of SWI/SNF into at least 4 stable subcomplexes, indicating that the SANT domain of Swi3 is critical for SWI/SNF assembly. Intriguingly, a triplex of Swi2/Arp7/Arp9, the minimal complex of SWI/SNF, was completely competent for ATP hydrolysis, generating superhelical torsion, inducing restriction enzyme accessibility and catalyzing nucleosome mobility change in vitro. However, the minimal complex was defective to catalyze histone H2A/H2B dimer loss from MMTV mononucleosomes. Finally, we found that the minimal SWI/SNF was functional to regulate SRG1 transcription in vivo, based on Northern blot and ChIP. We propose that SWI/SNF is an integration of at least 4 functional modules that are responsible for multi-step chromatin remodeling process. Our data help to understand both structural and functional organization of SWI/SNF ATP-dependent chromatin remodeling complex.
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Juan I. Young Abstract P123 Post-transcriptional functions of MeCP2
Mauricio A. Saez, Matias Alvarez-Saavedra, Huda Y. Zoghbi and Juan I. Young
Centro de Estudios Cientificos and Universidad Austral de Chile, Valdivia, 905-9100, Chile. Department of Molecular and Human Genetics, Department of Neuroscience and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, U.S.A.
Epigenetic modifications of DNA provide an extra level of genetic information. The main epigenetic modification of mammalian genomes is cytosine methylation, commonly associated with gene silencing. The importance of this process for chromatin function has been underscored by the discovery that Rett syndrome, a disabling neurodevelopmental disease, is caused by mutations in MeCP2. MeCP2 (methyl-CpG-binding protein 2) is a methylation-dependent transcriptional repressor. We found that MeCP2 is a multifunctional- multilevel regulator of gene expression; interacts with proteins involved in RNA processing and regulates alternative splicing in addition to its role as a transcriptional repressor.
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Veronica Yu Abstract P124 Over-expression of Cks proteins causes gene derepression in Saccharomyces cerevisiae
Roman Holic and Veronica Yu
MRC Clinical Sciences Centre, Imperial College Hammersmith Campus, Du Cane Road, London, W12 0NN, U.K.
Cks (cyclin-dependent kinase interacting) proteins are evolutionarily conserved and are frequently over-expressed in multiple cancers of high grade and stage. It has previously been demonstrated that the Saccharomyces cerevisiae cell cycle regulatory cyclin-dependent kinase interacting protein, Cks1 and its kinase partner, Cdc28 (Cdk1); are involved in the control of transcription at multiple gene loci. This transcriptional role is mediated through a requirement of Cdc28/Cks1 for recruiting proteasomes to coding regions. However, it is independent of the protein kinase activity of Cdc28. We over-expressed yeast and mammalian Cks proteins in S. cerevisiae in order to mimic the protein over-expression pattern in tumour cells. We found that over-expression of Cks proteins caused derepression of a wide-range of genes under repressive conditions. Possible mechanisms and implications for this observation will be discussed.
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Rebekah Zinn Abstract P125 hTERT is expressed in cancer despite promoter DNA methylation by preservation of unmethylated DNA and active chromatin around the transcription start site
Rebekah L. Zinn1,2, Kevin Pruitt1, Sayaka Eguchi1, Stephen B. Baylin1,2 and James G. Herman1,2
1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 21231 2The Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
hTERT, which encodes the catalytic subunit of telomerase and is expressed in most immortalized and cancer cells, has been reported to have increased DNA methylation in its promoter region in many cancers. This pattern is inconsistent with observations that DNA methylation of promoter CpG islands is typically associated with gene silencing. Here we provide a comprehensive analysis of promoter DNA methylation, chromatin patterns, and expression of hTERT in cancer and immortalized cells. Methylation specific PCR (MSP) and bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells shows that all cancer cell lines retain alleles with little or no methylation around the transcription start site, despite being densely methylated in a region 600 bp upstream of the transcription start site. By real-time RT-PCR, all cancer cell lines express hTERT. Chromatin immunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9, dimethyl-H3K4) and inactive (trimethyl-H3K9, trimethyl-H3K27) chromatin marks are present across the hTERT promoter. However, using a novel approach combining methylation analysis of ChIP DNA, we show that active chromatin marks are associated with unmethylated DNA, while inactive marks of chromatin are associated with methylated DNA in the region around the transcription start site. These results demonstrate that DNA methylation patterns of the hTERT promoter (-150 to +150 around the transcription start) are not inconsistent with the usual dynamics of gene expression in that the absence of methylation in this region and the association with active chromatin marks allow for the continued expression of hTERT.
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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 Additional poster submissions Yoshimitsu Takahashi Abstract P126 Degree of SUMO modification as a differential tag for targeting to specific chromosomal domains
Yoshimitsu Takahashi and Alexander Strunnikov
NIH, NICHD, LGRD, Bethesda, MD, U.S.A.
SUMO is a ubiquitin-like modifier that regulates many proteins by direct conjugation. E1, E2 and E3 enzymes are needed to transfer SUMO to a specific target. Recently, numerous potential SUMO substrates were identified by proteomic approaches. However, functional roles of individual modification are obscure, as only a small fraction of a given protein is sumoylated. In order to overcome this technical difficulty, we designed a novel technique Constitutive SUMO Modification (CSM), which allows to track only the modified form of the protein. We validated this method using Top2p as a model substrate. The advantages of Top2p for application of this technique are: (1) sumoylation sites are in a non-essential C- terminal domain. (2) Top2p expression is abundant. (3) Top2 modification is relatively strong both in vivo and in vitro. We have evidence that sumoylation of Top2p to different degrees changes intranuclear targeting of this essential protein.
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Marna S. Costanzo Abstract P127 The evolutionary conservation of chromatin modifying proteins in malaria
Marna S. Costanzo1, Szymon Kaczanowski2, Thanat Chookajorn3, and Daniel L. Hartl1
1Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA; 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02- 106 Warsaw, Poland; 3Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
The malaria parasite is responsible for most of the deaths related to infectious disease in the world today. This single cell eukaryote has a complex life-cycle consisting of two hosts and many morphologically different forms. The majority of genes expressed during the pathogenic bloodstages correlate to the cell cycle. Transcription of these genes is achieved monocistronically despite the apparent lack of specific transcription factors and covalent histone modification provides a possible mechanism of expression control. We investigate the apparent conservation of histone lysine methyltransferase function in malaria, despite an evolutionary history that is far diverged from most eukaryotes. The implications of these findings are discussed in context of the parasite biology and evolutionary implications.
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Philippe Prochasson Abstract P128 Functional characterization of the HIR corepressor complex
Philippe Prochasson, Laurence Florens and Jerry L. Workman
The Stowers Institute for Medical Research, Kansas City, Missouri 64110, U.S.A.
The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Previously, we showed that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR corepressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, we demonstrated that the HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription. We are now pursuing the functional characterization of the HIR complex to better understand its role in gene regulation and its interplay with the SWI/SNF chromatin remodeling complex. We will present data showing that the HIR complex is directly involved in the transcriptional regulation of the SUC2 gene and that the presence of the HIR proteins at the SUC2 promoter renders its transcriptional activation swi2/snf2 dependent. In a second part, we will focus on the identification of post-translational modifications (PTMs) of the HIR proteins during the cell cycle and their role on the histone genes transcriptional regulation.
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Chromatin Structure & Function Punta Cana, Dominican Republic, 5 - 8 December 2006 Occidental Allegro Hotel Map
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Sponsor of 2006 Chromatin Structure & Function Keynote Presentation
Chroma Therapeutics (Oxford, UK) is a privately-held clinical stage company focused on the discovery and development of novel small molecule drugs based upon chromatin biology. Chroma's highly experienced management team has built a broad and innovative product pipeline of first-in-class and best-in-class targeted approaches to treat cancer and inflammatory disorders.
Chroma's lead program, CHR-2797, is an aminopeptidase inhibitor that has demonstrated strong anti-tumour activity in a number of models of cancer, both as a single agent and in synergy with cytotoxic agents. The safety and tolerability of CHR-2797 is currently being assessed in a Phase I study in patients with treatment refractory metastatic cancer. Phase I studies of CHR-2797 as monotherapy in haematological cancer patients and in combination with paclitaxel in patients with treatment- refractory solid tumours have also been initiated. Following this, Chroma has a best-in-class HDAC inhibitor and a Aurora kinase inhibitor in preclinical development.
In parallel to its leadership position in chromatin modifying enzyme inhibitors, Chroma has developed a proprietary chemistry approach that enchances the accumulation and retention of a therapeutic agent inside cells leading to significant improvements in potency and pharmacokinetics properties. Usings this technology, Chroma has rapidly expanded its preclinical pipeline in both oncology and inflammation.
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