Nassau Prog03

Programme & Abstract Book CHROMATIN STRUCTURE & FUNCTION Nassau, Bahamas 15th - 18th November 2005

Organised By: Tony Kouzarides and Abcam Programme & Abstract Book

The second CHROMATIN STRUCTURE & FUNCTION Nassau, Bahamas 15th - 18th November 2005

Organizers: Tony Kouzarides (University of Cambridge) and Abcam

Table of contents

Timetable ...... Page 2

Conference Programme ...... Page 3

Poster Index ...... Page 8

Abstracts - Oral ...... Page 18

Abstracts - Poster ...... Page 57

Resort Information ...... Page 173

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.

1 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005 Timetable Tuesday 15th November 18:00 Keynote Speaker 19:00 Poolside dinner reception

Wednesday 16th November 09:00 - 10:30 Oral presentations Drinks break 11:00 - 12:30 Oral presentations Lunch and free time 16:00 - 17:30 Oral presentations Drinks break 18:00 - 21:00 Poster session and evening buffet

Thursday 17th November 09:00 - 10:30 Oral presentations Drinks break 11:00 - 12:30 Oral presentations Lunch and free time 16:00 - 17:30 Oral presentations Drinks break 18:00 - 19:30 Oral presentations 20:00 Beach Barbeque

Friday 18th November 09:00 - 10:30 Oral presentations Break 11:00 - 12:15 Oral presentations Lunch and departures

Keynote Speaker Sponsors:

2 Conference Programme Conference Programme Tuesday 15th November Chair: Tony Kouzarides

Keynote Speaker 18:00 - 19:00 Danny Reinberg ...... Page 18 Histone marks and chromatin states

Wednesday 16th November Chair: Ali Shilatifard

09:00 - 09:30 Shelley Berger ...... Page 19 Factor and histone covalent modifications in genome regulation

09:30 - 09:45 Brad Bernstein ...... Page 20 Genomic studies of chromatin modifications in normal and malignant cells

09:45 - 10:00 Hendrik Stunnenberg ...... Page 21 Histone modification patterns associated with X-inactivation and escape

10:00 - 10:30 Sandra Hake ...... Page 22 Beyond the double helix: Understanding the functional importance of histone H3 and its variants

Break

11:00 - 11:30 Yang Shi ...... Page 23 A new kid on the chromatin block: identification of the first histone demethylase LSD1 and its regulation

11:30 - 11:45 Roland Schuele ...... Page 24 LSD1 demethylates repressive histone marks to promote androgen receptor dependent transcription

11:45 - 12:15 Tony Kouzarides ...... Page 25 Proline isomerisation of histone H3 regulates gene expression

12:15 - 12:30 Victoria Lunyak ...... Page 26 ESET dependent H3-K9 methylation upon restructuring of chromosomal domains during development.

Lunch and free time

3 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Wednesday 16th November (continued) Chair: Craig Peterson

16:00 - 16:30 Yi Zhang ...... Page 27 Histone modifications in polycomb silencing and cancer

16:30 - 16:45 Patrick Grant ...... Page 28 Generation and recognition of histone modifications by the SAGA and SLIK HAT complexes in health and disease

16:45 - 17:00 Vincent Geli ...... Page 29 Structure and function of the Set1 RNA recognition motif

17:00 - 17:30 Ali Shilatifard ...... Page 30 Yeast COMPASS points the way to human MLL and its role in pathogenesis of leukemia

Break

18:00 - 21:00 Posters

Thursday 17th November Chair: Shelley Berger

09:00 - 09:30 Kevin Strule ...... Page 31 Abstract title to be confirmed

09:30 - 09:45 Oliver Rando ...... Page 32 Genome-scale characterization of chromatin structure

09:45 - 10:00 Rolf Sternglanz ...... Page 33 Histone mutations that suppress the silencing defects of a yeast sir3 Ala2Gly mutation

10:00 - 10:15 Antonin Morillon ...... Page 34 Histone variant HT2A-Z promotes promoter-proximal nucleosome dissociation upon transcription activation in S. cerevisiae.

10:15 - 10:30 Sharon Dent ...... Page 35 New functions for histone modifying enzymes

Break

4 Conference Programme

Thursday 17th November (continued) Chair: Shelley Berger

11:00 - 11:30 Jerry Workman ...... Page 36 Set2 methylates histone H3 K36 providing transcriptional memory that signals Rpd3S to deacetylate histones in transcribed regions to suppress spurious intragenic transcription

11:30 - 11:45 Nevan Krogan ...... Page 37 Protein complexes and pathways involved in chromatin function

11:45 - 12:00 Bruno Amati ...... Page 38 Epigenetic determinants of Myc binding to the human genome

12:00 - 12:15 Francois Fuks ...... Page 39 A direct mechanistic link between the Polycomb Group protein EZH2 and DNA methyltransferases

12:15 - 12:30 Julie Ahringer ...... Page 40 Chromatin regulation and sumoylation in the inhibition of Ras induced vulval development in C. elegans

Lunch and free time

Chair: Sharon Dent

16:00 - 16:30 Peter Becker ...... Page 41 Site-specific acetylation defines an embryonic form of ISWI that associates with mitotic chromatin

16:30 - 16:45 Stephen Rea ...... Page 42 hMOF complex is required for histone H4 lysine 16 acetylation in mammalian cells

16:45 - 17:00 Susana Gonzalo ...... Page 43 DNA methyltransferases control telomere length in mammalian cells

17:00 - 17:30 Craig Peterson ...... Page 44 Chromatin remodeling: Regulation of chromatin higher-order folding and DNA repair

Break

5 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Thursday 17th November (continued) Chair: Sharon Dent

18:00 - 18:30 Bob Kingston ...... Page 45 Remodeling chromatin without covalent modification

18:30 - 18:45 Lianna Johnson ...... Page 46 DNA and histone methylation in Arabidopsis

18:45 - 19:00 Eric Miska ...... Page 47 Short RNAs in C. elegans development and human cancer

19:00 - 19:30 Ramin Shiekhattar ...... Page 48 Integrator, a multiprotein mediator of small nuclear RNA processing associates with the C-terminal repeat of RNA polymerase II

Friday 18th November Chair: Kevin Struhl

09:00 - 09:30 Kami Ahmad ...... Page 49 Abstract title to be confirmed

09:30 - 09:45 Jacques Cote ...... Page 50 Dissection of the NuA4 HAT complex and crosstalk with other chromatin modifications and remodelers

09:45 - 10:00 Benoit Guillemette ...... Page 51 H2A.Z is globally localized to the promoters of inactive genes and regulates nucleosome positioning

10:00 - 10:30 Karolin Luger ...... Page 52 Structure and function of yeast nucleosome assembly protein 1

Break

11:00 - 11:30 Wolf Reik ...... Page 53 Regulation of imprinting and epigenetic reprogramming in mammals

11:30 - 11:45 Aidan Doherty ...... Page 54 Structure and function of the Jumonji C Domain

6 Conference Programme

Friday 18th November (continued) Chair: Kevin Struhl

11:45 - 12:00 Andreas Ladurner ...... Page 55 Human chromatin as a molecular target of cellular NAD metabolites

12:00 - 12:15 Jef Boeke ...... Page 56 Comprehensive mutagenesis of sites of histone modification

7 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005 Poster index Abstract 1 Ted Abel ...... Page 57 The role of CBP and histone acetylation in memory storage and synaptic plasticity

Abstract 2 Melissa Adkins ...... Page 58 Spt6-mediated nucleosome reassembly is required for transcriptional repression of the PHO5 gene

Abstract 3 Adriana Alejandro-Osorio ...... Page 59 The role of the histone deacetylase Rpd3p in coordinating the environmental stress response in yeast

Abstract 4 Erik Andersen ...... Page 60 MET-1 and MET-2, two putative histone methyltransferases, are redundantly required for proper vulval cell fate specification

Abstract 5 Anthony Argentaro ...... Page 61 Structure/function studies of the PHD-like domain of the chromatin-remodelling protein, ATRX

Abstract 6 Koh Meng Aw Yong ...... Page 62 The presence of EBV oriP within a transcription unit inhibits replication as well as transcription

Abstract 7 Slobodan Barbaric ...... Page 63 Two nucleosomes at the yeast PHO84 promoter demonstrates differential requirement for chromatin remodelling activities

Abstract 8 Vivian Bardwell ...... Page 64 Polycomb group and SCF ubiquitin ligases are found in a BCL6 corepressor complex

Abstract 9 Ralph Bash ...... Page 65 AFM recognition imaging: using antibodies to track proteins during in vitro remodeling

Abstract 10 Juraj Bies ...... Page 66 Pc2, the polycomb group protein recruits the c-Myb into PcG bodies and inhibits its activity

Abstract 11 Caroline Bouchard ...... Page 67 c-Myc induces localized H2A.Z exchange in the promoters of target genes

8 Poster Index

Abstract 12 Ray Camahort ...... Page 68 Genome-wide localization of the budding yeast histone variant Cse4

Abstract 13 Kendra Cann ...... Page 69 Multiple regions of the DNA-damage response protein TLS/FUS can regulate its relocalization to the nucleolei following transcriptional inhibition

Abstract 14 Rafael Casellas ...... Page 70 Effect of DNA double-stranded break repair on gene expression

Abstract 15 Beverley Chilton ...... Page 71 It Takes Two to Tango: autorepression by RUSH-1α/β

Abstract 16 Sung Hee Baek ...... Page 72 A chromatin remodeling complex in regulation of a metastasis suppressor gene

Abstract 17 Pierre Close ...... Page 73 Elongator depletion causes defective transcript elongation of genes that regulate cell motility

Abstract 18 Megan Cole ...... Page 74 Genome-wide map of nucleosome acetylation and methylation in yeast

Abstract 19 Jean-Francois Couture ...... Page 75 Structural insights into lysine multiple methylation by SET domain methyltransferases

Abstract 20 Qi Dai ...... Page 76 Snail-mediated repression in Drosophila requires Ebi and histone deacetylation

Abstract 21 Ana D’Alessio ...... Page 77 The kinetics of chromatin-driven active DNA demethylation in living cells

Abstract 22 Jan-Hermen Dannenberg ...... Page 78 Role of mSin3A in development, regulating transcriptional networks and tumorigenesis

9 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Abstract 23 Gregory David ...... Page 79 Functional specialization of the mSin3 complex in development and oncogenesis

Abstract 24 James Davie ...... Page 80 Recruitment of phosphorylated HDAC2 to promoters

Abstract 25 Cecile de la Cruz ...... Page 81 Developmental regulation of Suz12 localization

Abstract 26 Stephan Diekmann ...... Page 82 Functional complementation of human centromere protein A (CENP-A) by Cse4p

Abstract 27 Ivana Djuretic ...... Page 83 Silencing of a mammalian gene, Interleukin-4 in T helper lymphocytes

Abstract 28 Tom Donndelinger ...... Page 84 Asymmetric eukaryotic cell division and cellular dimorphism

Abstract 29 Yannick Doyon ...... Page 85 ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation

Abstract 30 Katherine Dunn ...... Page 86 Independent phosphorylation events on the amino-terminus of histone H3 associated with transcription

Abstract 31 Christine English ...... Page 87 ASF1 binds to a heterodimer of histones H3-H4: a two step mechanism for the assembly of H3-H4

Abstract 32 Alex Erkine ...... Page 88 Nucleosome displacement at promoters of yeast heat shock genes is proportional to the degree of transient histone H3 acetylation

Abstract 33 Laure Escoubet-Lozach ...... Page 89 Application of genome-wide location analysis to study chromatin modifications in a model of signal-dependent gene regulation

10 Poster Index

Abstract 34 Yuhong Fan ...... Page 90 A new link between linker histone H1 and DNA methylation

Abstract 35 Barna Fodor ...... Page 91 The full complement of Su(var) gene function in Drosophila

Abstract 36 Alexey Fomenkov ...... Page 92 Role of p63 in development disorders

Abstract 37 Benjamin Freedman ...... Page 93 Linker histone dynamics, structure, and function in interphase and mitotic egg extract chromosomes

Abstract 38 Jennifer Gallagher ...... Page 94 Exploring establishment of Sir1-dependent silencing using comparative genomics

Abstract 39 Nicolas Gévry ...... Page 95 Implication of histone variant H2A.Z in the regulation of the p53/p21CIP1/WAF1 pathway

Abstract 40 Anja Groth ...... Page 96 The role of human Asf1 in histone metabolism during replicational stress

Abstract 41 Matthew Guenther ...... Page 97 The active and Polycomb-repressed genome in human embryonic stem cells

Abstract 42 Shweta Hakre ...... Page 98 Growth factor mediated gene regulation by TFII-I

Abstract 43 Melissa Harrison ...... Page 99 The MBT repeat-containing protein LIN-61 regulates C. elegans vulval cell-fate specification

Abstract 44 Michael Hendzel ...... Page 100 The regulation of histone H1 binding in vivo

Abstract 45 Wilma A.Hofmann ...... Page 101 Specific roles for nuclear actin and nuclear myosin I in transcription by RNA Polymerase II

11 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Abstract 46 Sui Huang ...... Page 102 Upstream binding factor association induces large scale chromatin decondensation

Abstract 47 Dana Huebert ...... Page 103 Global analysis of MLL and MLL-fusion mediated histone modifications

Abstract 48 Christina Hughes ...... Page 104 Linking Menin mutations to defects in histone methylation and altered expression of target genes

Abstract 49 Irena Ivanovska ...... Page 105 Histone code in meiosis: histone kinase required for chromosomal architecture in Drosophila oocytes

Abstract 50 Danika Johnston ...... Page 106 Role of Drosophila ecdysone receptor and histone methyltransferase TRR in gene regulation

Abstract 51 Sarah Johnstone ...... Page 107 Embryonic stem cell transcription factors regulate developmental transcription factors

Abstract 52 Paul Kaufman ...... Page 108 Replication-independent histone deposition by the HIR complex and Asf1

Abstract 53 Ik Soo Kim ...... Page 109 A chromatin remodeling complex in regulation of crosstalk between the Wnt and the NF-kappaB pathway

Abstract 54 Elena Kisseleva-Romanova ...... Page 110 Identification of new mutants that lead to transcription internal entry in S. cerevisiae

Abstract 55 Christoph Koch ...... Page 111 A microarray based approach to develop high resolution maps of histone modifications in 1% of the human genome

Abstract 56 Hyockman Kwon ...... Page 112 BAF53 is essential for maintenance of chromosome territories and higher-order chromatin structure

12 Poster Index

Abstract 57 Eric Lam ...... Page 113 Chromatin charting in living plants: a global study

Abstract 58 Robert Lane ...... Page 114 Does chromatin inaccessibility contribute to mutually exclusive olfactory receptor transcription?

Abstract 59 Martin Law ...... Page 115 Mutations in the ATRX chromatin remodelling protein cause changes in histone H3 lysine 9 methylation

Abstract 60 Kenneth Lee ...... Page 116 Functional analysis of the ADA histone acetyltransferase complex

Abstract 61 Young Han Lee ...... Page 117 Epigenetic regulation of the tumor suppressor Egr-1 gene by oncogenic Ras

Abstract 62 Lin Li ...... Page 118 SP1 and SP3 dynamic association with estrogen regulated promoters

Abstract 63 Karen Lower ...... Page 119 ATRX acts in a localised manner to down regulate the expression of alpha globin genes in ATR-X syndrome

Abstract 64 Malik Lutz ...... Page 120 Geminin binding to Cdt1 controls, but does not necessarily block, Cdt1 function during replication origin licensing

Abstract 65 Dale Mackay ...... Page 121 Identification of an acetyltransferase of the DNA binding protein

Abstract 66 Asoke Mal ...... Page 122 Linking HDAC1 and MyoD function in Rhabdomyosarcoma

Abstract 67 Roberto Mantovani ...... Page 123 Epigenetic changes to NF-Y controlled genes upon ER induction

13 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Abstract 68 Jotin Marango ...... Page 124 The multiple myeloma SET domain (MMSET) protein is a histone H3 and H4 methyltransferase with properties of a transcriptional co-repressor

Abstract 69 Philip Marsden ...... Page 125 The cell-specific and hypoxia-regulated expression of endothelial nitric oxide synthase (eNOS) is controlled by chromatin-based mechanisms

Abstract 70 Darin McDonald ...... Page 126 Polymeric nuclear actin in chromatin modification and double- strand break repair

Abstract 71 Dominik Mojzita ...... Page 127 Pdc2, the yeast homologue of CENP-B, is a part of the centromere-kinetochore complex

Abstract 72 David Mosser ...... Page 128 Chromatin remodeling across the proximal promoter region of the interleukin-10 gene in macrophages

Abstract 73 Toshinori Nakayama ...... Page 129 Long range histone modification of the Type2 cytokine gene loci in developing Th2/Tc2 cells

Abstract 74 James Nicholson ...... Page 130 Structural studies of the oxidised histone octamer

Abstract 75 Barbara Nikolajczyk ...... Page 131 The IL-1 beta gene is transcribed from a poised promoter architecture in monocytes

Abstract 76 Keisuke Nimura ...... Page 132 Preferential association of Dnmt3l with Dnmt3a2 on chromatin in ES cells

Abstract 77 Esperanza Nunez ...... Page 133 Repetitive elements, transcriptional control and cell diversity

Abstract 78 Laura O’Neill ...... Page 134 Distinctive histone modifications mark specific chromosomes in both human and mouse models

14 Poster Index

Abstract 79 Hisanobu Oda ...... Page 135 Generation and characterization of Set9 and PR-Set7 conditional gene knockout mice

Abstract 80 Søren Ottosen ...... Page 136 Bromodomain protein 7 (Brd7) promotes nucleolar disruption in response to DNA breaks

Abstract 81 Mahadeb Pal ...... Page 137 The role of TFIIB and the transcription bubble during the earliest stages of transcription by RNA polymerase II

Abstract 82 Maria V. Panchenko ...... Page 138 pVHL partner and transcriptional co-activator Jade-1 is a novel substrate for HAT TIP60

Abstract 83 Tej Pandita ...... Page 139 Mammalian ortholog of Drosophila MOF is critical for embryogenesis and essential for DNA damage responses

Abstract 84 Biranchi Patra ...... Page 140 Genomewide localization of histone modifications during meiosis

Abstract 85 Rushad Pavri ...... Page 141 A fully reconstituted chromatin transcription system for the detailed study of epigenetic mechanisms

Abstract 86 Inka Pawlitzky ...... Page 142 A novel regulatory region 5’ of the mouse IgH locus

Abstract 87 Laura Perez-Burgos ...... Page 143 Domain organization at the chicken β-globin locus

Abstract 88 Yuri Postnikov ...... Page 144 Modulation of histone modifications by chromatin-binding architectural proteins

Abstract 89 Brendan Price ...... Page 145 The Tip60 histone acetyltransferase is essential for the acetylation and activation of the ATM protein kinase

Abstract 90 Michael Rehli ...... Page 146 Genome-wide profiling of CpG-methylation by methyl-CpG iimmunoprecipitation

15 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Abstract 91 Kristen Riley ...... Page 147 Involvement of the TAC1 complex in transcription elongation

Abstract 92 Flavio Rizzolio ...... Page 148 Chromatin alteration on POF associated X/A balanced translocation

Abstract 93 Orit Rozenblatt-Rosen ...... Page 149 Paf1 complex is associated with mRNA processing machinery.

Abstract 94 Ingemar Rundquist ...... Page 150 Analyses of linker histone - chromatin interactions in situ in different cell systems

Abstract 95 Michael Scher ...... Page 151 SirT3 is A nuclear histone deacetylase that translocates to mitochondria upon stress

Abstract 96 Gunnar Schotta ...... Page 152 H4-K20 methylation: a mark important for mouse development?

Abstract 97 Bernd Schuttengruber ...... Page 153 Effect of long-range chromosomal interactions mediated by the Fab-7 element on its target chromatin

Abstract 98 Alexandra Schulmeister ...... Page 154 Histone H3.3 phosphorylation during mitosis and meiosis in the urochordate Oikopleura dioica

Abstract 99 Jill Schumacher ...... Page 155 The Tousled kinase functions in mitosis as a substrate and activator of the Aurora B kinase

Abstract 100 David Schrump ...... Page 156 Gene expression profiling of primary lung cancers exposed to 5 aza 2’ deoxycytidine (DAC), depsipeptide FK228 (DP), or sequential DAC/DP

Abstract 101 Brian E. Schwartz ...... Page 157 Nucleosome assembly pathways discriminate between sites in the H3 and H3.3 histone tails

Abstract 102 Kristin Scott ...... Page 158 A heterochromatin barrier partitions the fission yeast centromere into discrete chromatin domains

16 Poster Index

Abstract 103 Judith Sharp ...... Page 159 XIST functions independently of BRCA1 in X chromosome inactivation Abstract 104 Shivendra D. Shukla ...... Page 160 Histone modifications by ethanol in liver

Abstract 105 Robert Sims ...... Page 161 Regulation of mRNA biogenesis by the coordinator complex, which specifically recognizes methyl H3K4

Abstract 106 Jeffrey Smith ...... Page 162 Genetic identification of chromatin-related regulators of ribosomal RNA synthesis in Saccharomyces cerevisiae

Abstract 107 Yee Sun Tan ...... Page 163 Skp1 regulates transcription via mono-ubiquitylation

Abstract 108 Hannah Tims ...... Page 164 Spontaneous and catalyzed nucleosome conformational changes

Abstract 109 Patrick Trojer ...... Page 165 A functional interplay between MLL and LSD1

Abstract 110 Patrick Trojer ...... Page 166 L3MBTL1 - the chromatin lock?

Abstract 111 María Isabel Tussié Luna ...... Page 167 Pro-proliferative function of the long isoform of PML-RARα involved in acute promyelocytic leukaemia

Abstract 112 Wim Vanden Berghe ...... Page 168 Differential interleukin-6 gene expression dynamics in benign and metastatic breast cancer cells reflects distinct chromatin signatures at the promoter region

Abstract 113 Adam Wood ...... Page 169 The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1

Abstract 114 Dag H. Yasui ...... Page 170 MeCP2 recruits SNF2h to the 15q11-13 imprinting control region

17 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005 Abstracts – Oral Danny Reinberg Histone marks and chromatin states

Danny Reinberg

Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, UMDNJ/RWJMS, RWJMS Research Building, 683 Hoes Lane, Piscataway, Middlesex 08854, U.S.A.

18 Abstracts - Oral

Shelley Berger Factor and histone covalent modifications in genome regulation Shelley Berger

Gene Expressia and Regulation Program, The Wistar Institute, 3601 Spruce Street, 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). Our research focuses on patterns and temporal sequences of PTMs and the interrelationship of factor PTMs and histone PTMs in S. cerevisiae and mammalian cells. In yeast current projects focus on dynamic PTMs during transcriptional regulation and sporulation. We are working to unravel the molecular outcomes of these PTMs. In transcriptional regulation our recent studies focus on the roles of histone H3 Ser10ph, H2B K123ub, and H2B/H4su. We find that, on one hand, H3 Ser10ph prevents the binding of certain corepressor complexes from binding to histones, and on the other hand, it promotes the binding of several novel transcriptional coactivator molecules. Thus, Ser10ph has an interesting dual role in transcriptional regulation. Secondly, we find that ub/deub of H2B K123 regulates transcriptional elongation by directly regulating histone binding of a RNA polymerase II kinase, Ctk1. Ctk1 binding, in turn, establishes a balance of H3 K4me vs. K36me in the ORF. Hence, K123ub has a unique attribute among histone PTMs: it must be both added and then removed for full transcriptional activation. Finally, we identify H2B/H4su as the first known negative histone modification in S. cerevisiae. The sumo mark appears to block certain positive marks such as acetylation and ubiquitylation. We are also studying H4 Ser1ph as a histone PTM that regulates gametogenesis. During both S. cerevisiae sporulation and metazoan spermatogenesis, H4Ser1ph persists after the decline of H3 Ser10ph, the well-known mitotic/meiotic modification involved in chromosome condensation. Our results suggest that H4 Ser1ph is involved in chromatin compaction during late stages of sporulation. We are currently analyzing whether Ser1ph directly compacts nucleosomes, or whether it promotes association of other molecules that carry out the compaction. Each of these PTMs (and many others!) appears to be conserved through evolution. Thus, mechanistic studies in S. cerevisiae, a genetically and biochemically tractable model organism, help us to understand the molecular outcomes of these complex patterns and temporal sequences of modifications, which can then be addressed in higher eukaryotes.

19 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Brad Bernstein Genomic studies of chromatin modifications in normal and malignant cells

Brad Bernstein1,2, Dana Huebert1,2, Mike Kamal2, Phil Kapranov3, Tom Gingeras3, Stuart Schreiber2,4 and Eric Lander2

1Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston MA, U.S.A. 2Broad Institute of Harvard and MIT. 3Affymetrix, Inc. 4Howard Hughes Medical Institute, Harvard Chemistry and Chemical Biology, Boston MA, U.S.A.

To gain insight into the role of chromatin in regulating genome structure and function, we mapped the patterns of histone H3 Lys4 and Lys27 methylation across several dozen genomic loci in human and mouse. The majority of loci contain punctate methylated sites that coincide with gene starts or regulatory elements, and appear to reflect their activation status. A smaller number of loci, including the HOX clusters, contain much broader regions that are continuously associated with Lys4 or Lys27 methylated histones. These ‘chromatin domains’ are cell type specific and correlate with the expression of underlying genes. Insulator elements and highly conserved sequences define the boundaries of these domains, while associated trithorax and polycomb proteins appear to function in their epigenetic maintenance. The domains are also affected by leukemogenic MLL fusion proteins, and we propose that this activity underlies the pathogenesis of MLL rearrangements.

20 Abstracts - Oral

Hendrik Stunnenberg Histone modification patterns associated with X-inactivation and escape

Arie B. Brinkman, Thijs Roelofsen, Sebastiaan W.C. Pennings, Joost H.A. Martens, Thomas Jenuwein, Hendrik G. Stunnenberg

Radboud University, NCMLS, Geert Grooteplein 28, Nijmegen, Gelderland 6525 GA, The Netherlands.

X-inactivation is associated with chromosome-wide removal of euchromatic histone modifications and deposition of heterochromatic histone modifications. While the resulting chromatin is classically viewed upon as facultative heterochromatin that is uniform in nature, the human Xi has been shown to be microscopically segregated into two distinct types of heterochromatin, characterized either by tri-methylated H3K27 or H3K9. However, the functional consequences of such segregation of these modifications at the single-gene level are unclear. Here we have analyzed the allelic distribution of histone modifications at two non-contiguous X-chromosomal regions in human somatic cells. Segregation of H3K27me3 and H3K9me3 is also evident at the single-gene level, and transitions between these chromatin types are marked with local H3K4 tri-methylation and H3/H4 acetylation around 5 ends of active genes. While these modifications are well-documented euchromatic marks, they do not necessarily mark ongoing transcription. Surprisingly, H3K27me3 is not exclusive for inactive genes: although it marks X-linked intergenic regions and inactive gene coding regions, it similarly marks some escape genes. At the latter genes, H3K27me3 appears as a dominant silencing mark over active modifications. Most remarkably, we find that H3K9me3 is not a hallmark of heterochromatin per se, since it is predominant in coding regions of active genes, a phenomenon that is not restricted to the X-chromosome. These results argue against the exclusiveness of individual marks for heterochromatin or euchromatin, but rather suggest that composite patterns of multiple interdependent or mutually exclusive modifications together signal gene expression status.

21 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Sandra Hake Beyond the double helix: Understanding the functional importance of histone H3 and its variants

Sandra B. Hake1, Benjamin A. Garcia2, Monika Kauer1, Elizabeth M. Duncan1, Rob Diaz1, Judith Recht1, Graham Dellaire3, Stephanie Morris4, Jeffrey Shabanowitz2, David P. Bazett-Jones3, Brian D. Strahl4, Donald F. Hunt2 and C. David Allis1.

1The Rockefeller University, New York, USA, 2University of Virginia, Charlottesville, USA, 3The Hospital for Sick Children, Toronto, Canada, 4University of North Carolina School of Medicine, Chapel Hill, USA

Chromatin, the repeating polymer of DNA and associated histone proteins, is the physiological template of our genome. As such, elaborate mechanisms have evolved to introduce meaningful variation into chromatin for purposes of altering gene expression and other important biological processes, including the repair of damaged DNA and chromosomal dynamics. Introduction of covalent histone modifications, chromatin remodeling by ATP-dependent complexes, and utilization of histone variants are three major mechanisms by which variation can be introduced into the chromatin fiber. Together, this variation might form a “histone code” that remains poorly understood. We favor the general view that histone proteins are major carriers of epigenetic information. The fundamental structure of chromatin suggests that all DNA-templated processes, including a wide range of epigenetic phenomena, are influenced by chromatin alterations with far-reaching implications for human biology and disease. We are particularly interested in the biological significance and function of histone H3 variants and their post-translational modifications.

22 Abstracts - Oral

Yang Shi A new kid on the chromatin block: identification of the first histone demethylase LSD1 and its regulation

Yu-Jiang Shi, Fei Lan and Yang Shi

Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, U.S.A.

Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined both by acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (alias KIAA0601/BHC110/p110/nPAO), a homolog of nuclear amine oxidases, functions as a histone demethylase and transcriptional co-repressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant de-repression of target genes, suggesting that LSD1 represses transcription via histone demethylation. Further analyses reveal that LSD1 stability, chromatin accessibility and demethylase activity are regulated by multiple factors associated with LSD1, suggesting that LSD1-mediated histone demethylation is regulated dynamically in vivo. Taken together, these results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.

23 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Roland Schuele LSD1 demethylates repressive histone marks to promote androgen receptor dependent transcription

Eric Metzger, Melanie Wissmann, Na Yin, Judith M. Mller, Robert Schneider, Antoine H. F. M. Peters, Thomas Gnther, Reinhard Buettner, and Roland Schuele

Universitt Freiburg, 66 Zentrale Klinische Forschung, Breisacherstrasse, Freiburg, Baden- Wrttemberg79106,Germany

Gene regulation in eukaryotes requires the co-ordinate interplay of chromatin modulating proteins with specific transcription factors such as the androgen receptor (AR)1. Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues2. Here we show that the lysine specific demethylase 1 (LSD1)3 co-localises with AR in normal human prostate and prostate tumour. LSD1 interacts with AR in vitro and in vivo and stimulates AR-dependent transcription. Conversely, LSD1 knockdown abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation (ChIP) analyses demonstrate that AR and LSD1 form chromatin- associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of AR target genes. Importantly, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently, AR-dependent transcription. Thus, modulation of LSD1 activity offers a novel strategy to regulate AR functions. Here, we link for the first time demethylation of a repressive histone mark with AR-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.

24 Abstracts - Oral

Tony Kouzarides Proline isomerisation of histone H3 regulates gene expression

Chris Nelson and Tony Kouzarides

The Gurdon Institute, Tennis Court Road, Cambridge, Cambridgeshire CB2 1QN, U.K.

Prolines can exist in a –cis or -trans conformation. Enzymes exist, (peptidyl prolyl isomerases) which can convert prolines from one conformation to the other. In an effort to establish if histones are modified in this way, we have analysed peptidyl prolyl isomerases in S. cerevisiae We find that FPR4 is an enzyme that can isomerise specific prolines in histone H3 and that this isomerisation affects lysine methylation of H3. The cross-talk between proline isomerisation and lysine methylation has a consequence in the regulation of transcription of certain genes in yeast. These data define a novel enzymatic activity that modifies histones and regulates gene expression.

25 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Victoria Lunyak ESET dependent H3-K9 methylation upon restructuring of chromosomal domains during development.

V.V. Lunyak1, Cramer T.2, Zhu X.1, Nunez E. 1, Hutt K.1, Roy R.3, García- Díaz A.3, Sze S.H.4, Montoliu L.3, and Rosenfeld M.G.1

1School of Medicine, USCD, San Diego, USA. 2Charite University, Virchow Klinikum, Berlin, Germany. 3Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain. 4Departments of Computer Science and Biochemistry and Biophysics, Texas A and M University, U.S.A.

A central question in development is the spatial and temporal order of molecular events by which genes silenced in precursor cells are ultimately activated in the mature organ in the correct cell- restricted fashion. Our study of the dynamics of chromatin modifications within the growth hormone (GH) gene locus in the developing pituitary gland demonstrate that di- or trimethylation of K9-H3 is a property of silenced GH gene, however the changes in the degree of H3-K9 methylation are tightly linked to the development of the gland. Our results suggest that di- and tri- H3-K9 methylation partitioning serves unique roles in the structural and functional organization of chromosomal domains. We will present data suggesting that chromatin remodeling could be one of the essential components of the molecular events controlling restructuring of the chromosomal domains within the cells traversing from the precursor to differentiated entity. Cumulative data obtained by analysis of nuclear positioning of the GH gene locus within developing cells point to the existence of HMTase activity capable of providing differential degrees of H3-K9 methylation, as well as to a developmental window during which the enzymatic activity could be expressed or assembled into the functional complex(es). Our data demonstrate a new in vivo function of ESET in the restructuring of chromosomal domains during development. Transgenic over-expression of the ESET protein earlier in development (in contrast to its normal temporal pattern of appearance) results in premature relocation of the developmentally regulated gene from condensed DAPI- stained chromatin to euchromatic compartments, observed by Immuno-FISH analysis. The relocation of the GH gene to euchromatic compartment coincides with the exchange of tri-H3-K9 for di-H3-K9 methylation. This event is not only targeted to nucleosomes in the immediate locale of the GH promoter, but rather responsible for the establishment of a specialized chromatin domain in the GH-gene locus. Furthermore, we have mapped the developmentally regulated boundary/insulator element involved in establishing the differential domains of tri-H3-K9 and di- H3-K9 methylated chromatin. We speculate that the boundary /insulator function can be, in part, provided by transcriptional activation of SINE B2 repeat embedded in the DNA segment, which is sufficient to provide boundary activity in an enhancer-blocking assay. By using a bioinformatic approach, we have designed an algorithm which predicts putative boundary/insulators within the mouse genome and will present some data on a functional assessment of their action. Our data support the hypothesis that the boundary/insulators elements are unlikely to be as static as previously thought, but rather act as a dynamic infrastructure adapted to the transcriptional and/or developmental state of the cell, providing the plasticity required to respond to developmental and environmental cues.

26 Abstracts - Oral

Yi Zhang Histone modifications in polycomb silencing and cancer

Yi Zhang

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A.

Increasing evidence suggest epigenetic modifications, particularly covalent histone modifications play an important role in regulating gene activity. Malfunction of histone modifications can cause serious problems including cancer. Previously, we have demonstrated that methylation of H3-K27 by the EED-EZH2 Polycomb group complex plays an important role in Polycomb silencing. Using a biochemical approach, we have purified a PRC1-like Polycomb complex that mediates histone H2A ubiquitination linking Polycomb silencing to histone H2A ubiquitination. Although both H3-K27 methylation and H2A ubiquitination are involved in PcG silencing, the relationship between these two modifications is not known. We are setting-up assay systems to address this issue. Of the histone methyltransferases characterized so far, the yeast Dot1 and its human counterpart, hDOT1L, are unique due to their lack of a SET domain, and that they methylate a lysine residue (K79) located in the globular domain of histone H3. To understand the biological function of hDOT1L, we set out to look for its functional partners. We found that hDOT1L is capable of interacting with a number of MLL (mix lineage leukemia) fusion partners involved in acute myeloid leukemia. The domains of the fusion partners involved in the interaction are sufficient for mediating leukemogenesis when fused to MLL. Myeloid transformation assay demonstrated that the enzymatic activity of hDOT1L is required for MLL-hDOT1L fusion to transform bone marrow cells in vitro. These observations in combination with the recent demonstration that MLL is a histone H3-K4 methyltransferase suggest that change from H3-K4 methylation to H3-K79 methylation of key MLL target genes may be a major mechanism in leukemogenesis. Current work is focused on identification of these key target genes and examination of their expression in normal and leukemia states.

27 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Patrick Grant Generation and recognition of histone modifications by the SAGA and SLIK HAT complexes in health and disease.

Marilyn G. Pray-Grant, Stacey J. McMahon, Michael S. Torok, Jeremy A. Daniel, Stephen P. Baker, Sharon R. Dent1 and Patrick A. Grant.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, U.S.A., 1Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, University of Texas, Houston, Texas 77030, U.S.A.

In eukaryotes, the condensation of DNA into chromatin provides a considerable obstacle to the nuclear machinery driving processes such as DNA replication, transcription or repair. Importantly the structure of chromatin is a dynamic one, which permits the localized decondensation and remodeling that facilitates the progress of these processes. Over the past few years considerable progress has been made into how chromatin remodeling occurs and in the identification of the enzymatic machines that mediate these processes. An emerging theme in the field of chromatin research has been the significant role that posttranslational modifications of histones play in regulating nuclear function. The conserved SAGA and SLIK transcriptional coactivator complexes modulate chromatin function via histone H3 and H2B acetylation and H2B deubiquitination. These complexes both generate specific epigenetic marks, but also respond to others, thus orchestrating histone modification patterns that dictate expression of protein coding genes. The chromodomains of Chd1 and bromodomain of Gcn5 regulate the enzymatic activity and recruitment of SAGA/SLIK to chromatin. We have also identified other novel protein modules as components of these complexes, which confer additional capability in the recognition of histone modifications. Furthermore, we have recently reported the yeast homologue of the human ataxin-7 as a component of SAGA and SLIK, which in its polyglutamine expanded form is responsible for the neurodegenerative disease spinocerebellar ataxia type7 (SCA7). Here we identify the mechanism by which this polyQ protein abrogates SAGA-dependent nucleosome acetylation and present evidence that Gcn5 inactivation causes SCA7 phenotypes.

28 Abstracts - Oral

Vincent Geli Structure and function of the Set1 RNA recognition motif

Lionel Trsaugues, Pierre-Marie Deh, Alfonso Rodriguez-Gil, Raphal Gurois, Isabelle Varlet, Sebastian Chavez, Herman van Tilbeurgh and Vincent Geli

CNRS,UPR9027, IBSM,CNRS, 31 chemin Joseph Aiguier, Marseille 13402, France

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains in addition to its catalytic SET domain, a conserved RNA Recognition Motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected RRM-fold, it lacks the typical RNA binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast destabilizing RRM1 reduces the total amount of Set1 within the cell. The destabilization of RRM1 leads to a concomitant increase of dimethylation of H3K4 at the 5’-coding region of active genes at the expense of trimethylation, whereas both, di- and tri-methylation, decrease at the 3 coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA binding activity is not linked to H3K4 methylation. We will bring new insights about the function of Set1 RRMs.

29 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Ali Shilatifard Yeast COMPASS points the way to human MLL and its role in pathogenesis of leukemia

Ali Shilatifard

Saint Louis University Cancer Center, Saint Louis University School of Medicine, Saint Louis, MO 63104, U.S.A.

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. We demonstrated previously that MLL translocation into the ELL gene result in the development of leukemia and that the C-terminal domain of ELL is required for the leukemic function of the chimera. 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. We have demonstrated that the yeast MLL homologue, Set1 exist in a complex we call COMPASS, which is a histone methyltransferases capable of mono- di and trimethylating the fourth lysine of histone H3. We now know that the Drosophila and the human homologues of COMPASS are also histone methyltransferases. The posttranslational modifications of histones by methylation have emerged as a key regulatory mechanism for both repression and activation of gene expression. Studies from our laboratory and others during the past few years have brought about a watershed of information defining the molecular machinery and factors involved in the recognition and modification of nucleosomal histones by methylation. I will discuss our recent findings regarding the molecular mechanism and consequences of histone modification by COMPASS and its homolog MLL, in the regulation of gene expression, development and pathogenesis of cancer.

30 Abstracts - Oral

Kevin Struhl

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, CI-204, Boston, MA 02115, U.S.A. Abstract unavailable at time of print

31 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Oliver Rando Genome-scale characterization of chromatin structure

Chih Long Liu, Guo-Cheng Yuan, Tommy Kaplan, Michael Dion, Stuart Schreiber, Steve Altschuler, Nir Friedman and Oliver Rando

Bauer Center, Harvard University, 7 Divinity Ave., Cambridge, MA 02138, U.S.A.

We have designed a tiled oligonucleotide microarray to sequence the primary structure of half a megabase of yeast chromatin. We have found that yeast promoters exhibit a remarkably stereotyped chromatin architecture, with upstream regulatory DNA sequence found in a long nucleosome-free region surrounded by two well-positioned nucleosomes with a characteristic modification pattern. These modifications associated with promoter nucleosomes comprise one of two groups of histone modifications, with the other group consisting of histone modifications that occur over coding regions and whose levels correlate with transcription levels. In yeast, histone modifications occur in few combinations, suggesting that much less information is carried in histone modifications than is theoretically possible.

32 Abstracts - Oral

Rolf Sternglanz Histone mutations that suppress the silencing defects of a yeast sir3 Ala2Gly mutation

Xiaorong Wang, Vinaya Sampath and Rolf Sternglanz.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, U.S.A.

Transcriptional silencing in S. cerevisiae is mediated by the binding of a Sir3-Sir4 complex to the N-terminal regions of histones H4 and H3 after their deacetylation by Sir2. The known histone binding domain of Sir3 occurs in the C-terminal third of the protein. The N- terminus of Sir3 is also important for its silencing function but the binding partner(s) of this region of Sir3 are not known. In order to learn more about the function of the Sir3 N- terminus, we have carried out a screen to identify suppressors of the silencing defect of a sir3 Ala2Gly mutation. One of the suppressors identified was a dominant mutation that changed residue 77 of H3 from Asp to Asn. H3 Asp77 is in a region of the nucleosome where several groups have identified mutations that affect silencing both positively and negatively. It is also very near Lys79, the residue methylated by Dot1. Two recessive suppressor mutations, not in the genes for H3 and H4, were also identified in this screen, and are currently being characterized.

33 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Antonin Morillon Histone variant HT2A-Z promotes promoter-proximal nucleosome dissociation upon transcription activation in S. cerevisiae.

A. Morillon2, E. Kisseleva-Romanova1, N. Karabetsou1, A. Nair1, J. Mellor1

1University of Oxford, Biochemistry department, Oxford, UK, 2CNRS, CGM, Gif/Yvette, France

In addition to transcription factors, chromatin modifications play a crucial role in regulating transcription. It has been shown that chromatin structures can be remodeled by shifting nucleosomes along the DNA, by covalently modifying histones and by changing histone composition of nucleosomes. However, control of histone variants exchange and their functional relationships with others histone modifications remain poorly understood. Here we show that H2A-Z histone variant encoded by HTZ1 in S. cerevisiae is preferentially incorporated onto promoter region during transcriptional repression and dissociated when transcription is activated. We present evidence that Bdf1 and Swr1 are required for the rapid incorporation of H2A-Z during nucleosome re-assembly when GAL1 gene is switched off. During transcription activation H2A-Z dissociation is concomitant with histone H3 depletion on GAL1 and MET16 promoter-proximal regions and is dependent on the histone chaperone Asf1. Finally H2A-Z is required to control the timing of histone H3 depletion suggesting that H2A-Z defines a specialized promoter nucleosome.

34 Abstracts - Oral

Sharon Dent New functions for histone modifying enzymes

Sharon R. Dent, Yvonne A. Evrard, Ping Bu, Wenchu Lin, Ke Zhang and John Latham

Department of Biochemistry and Molecular Biology, U.T. M.D. Anderson Cancer, Houston, Texas 77401, U.S.A.

Our lab uses genetic approaches to define functions of histone modifying enzymes in vivo. We reported previously GCN5 deletion in the mouse leads to embryonic lethality due to increased apoptosis. New data show that this apoptosis is p53 dependent and may be triggered by telomere defects. Telomeres are shortened in cells from GCN5 mutant embryos, and these cells display increased numbers of chromosomal end associations and fusions. In the absence of both p53 and GCN5, an even greater incidence of telomere fusion is observed. These double mutant embryos also display defects in neural tube closure and brain development. Very similar defects in neural development are observed in mice homozygous for GCN5 HAT catalytic site mutations and in mice that carry a hypomorphic allele of GCN5. These data provide the first evidence that GCN5 may be required both for telomere maintenance and proper expression of genes required for neural development in the mouse. In another project, we discovered that deletion of SET1 suppresses defects in chromosome segregation caused by mutations in IPL1 in yeast. Mutations in other components of COMPASS 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, and 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 that Set1 is required for methylation of a kinetochore protein, Dam1. Methylation of Dam1 by Set1 inhibits phosphorylation of flanking serines by Ipl1. 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.

35 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jerry Workman Set2 methylates histone H3 K36 providing transcriptional memory that signals Rpd3S to deacetylate histones in transcribed regions to suppress spurious intragenic transcription Michael J. Carrozza1, Bing Li1, Laurence Florens1, Selene K. Swanson1, Kenneth K. Lee1,Wei-Jong Shia1, Scott Anderson2, John R. Yates2, Michael P. Washburn1 and Jerry L. Workman1

1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, 2The Scripps Research Institute LaJolla, CA 92037

Yeast Rpd3 histone deacetylase plays a surprisingly important role at actively transcribed genes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPIT analysis. Both complexes shared a three subunit core and Rpd3L contains unique subunits consistent with a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific to Rpd3S. Mutants of RCO1 and EAF3 exhibited increased acetylation in the FLO8 and STE11 open reading frames (ORFs) and the appearance of aberrant transcripts initiating within the body of these ORFs. Mutants in the RNA polymerase II-associated SET2 histone methyltransferase also displayed these defects. Set2 functioned upstream of Rpd3S and the Eaf3 methyl-histone binding chromodomain was important for recruitment of Rpd3S and for deacetylation within the STE11 ORF. These data indicate that Pol II-associated Set2 methylates H3 providing a transcriptional memory which signals for deacetylation of ORFs by Rpd3S. This erases transcription elongation-associated acetylation to suppress intragenic transcription initiation.

36 Abstracts - Oral

Nevan Krogan Protein complexes and pathways involved in chromatin function

Nevan J. Krogan1,2, Michael-Christopher Keogh3, Minkyu Kim3, Sean Collins4, Andrew Emili1,2, Jonathan Weissman4, Timothy Hughes1,2, Stephen Buratowski3 and Jack Greenblatt1,2

1Banting and Best Dept. of Medical Research and 2Dept. of Molecular and Medical Genetics, Univ. of Toronto, Toronto, Ontario, Canada M5G 1L6; 3Harvard Medical School, Boston, MA 02115; 4Howard Hughes Medical Institute, Univ. of California, San Francisco, CA 94143, U.S.A.

Proteins are being systematically TAP-tagged in the budding yeast Saccharomyces cerevisiae, then purified and identified by mass spectrometry in order to define the stably associated components of protein complexes and weak interactions among protein complexes. In order to initiate a systems biology approach to chromatin metabolism and the transcription apparatus for RNA polymerase II, including initiation factors, elongation and termination factors, RNA processing proteins, and chromatin modifying proteins, we are combining the clustered protein purification data with synthetic genetic array (SGA) analysis and microarray analysis of gene deletion and Tet-promoter mutants. SGA analysis was used to define proteins that are genetically linked to known components of the RNA polymerase II transcriptional apparatus and chromatin modifiers, and all the resulting proteins were subsequently tagged and purified. Deletion mutants for all the non-essential proteins in this genetic and biochemical network were then assembled into a 384-strain miniarray, and SGA was used to systematically construct double mutants for almost all ~150,000 possible pairwise combinations of deletion mutations. The growth rates of the double mutants were then assessed by an automated vision system, following which the genes, and therefore the protein complexes, were organized into putative pathways according to the similarity of their genetic interaction profiles. Proteins and protein complexes were also organized into putative pathways by clustering the gene expression profiles of a large number of gene deletion and Tet-promoter mutants. Experimental tests of the organization of pathways containing both well-known and newly discovered chromatin modifying complexes and other proteins will be discussed. Protein purification and SGA analysis are also being used to define protein complexes and functional pathways in other yeast biological systems, including protein trafficking and DNA repair.

37 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Bruno Amati Epigenetic determinants of Myc binding to the human genome

Ernesto Guccione, Francesca Martinato, Lucilla Luzi, Giacomo Finocchiaro, Andrea Cocito and Bruno Amati

European Institute of Oncology, IFOM-IEO Campus, Milan, Italy

The determinants of transcription factor (TF) binding to eukaryotic genomes remain largely elusive. Using quantitative chromatin Immunoprecipitation (q-ChIP), we estimated that over 11% of all human loci bear high-affinity Myc-binding sites in their 5’ regulatory regions that conform to the consensus CACGTG (or E-box) [1]. Not all E-box-containing promoters where efficiently bound by Myc, however, suggesting that epigenetic determinants are paramount for selectivity. High-throughput ChIP-on-chip studies suggested that Myc associates with even larger numbers of genomic sites, of which roughly 20% contain an E-box [2, 3]. Starting from the available datasets [1-4], we have used a combination of bioinformatics and large- scale q-ChIP to dissect the correlations between histone marks and Myc binding. Our data show that stretches of H3K4 di- and tri-methylation previously identified by ChIP-on- chip [4] are generally confirmed by q-ChIP and are significantly associated with other marks, including di-methyl H3K79, and acetyl H3K9, H3K18, H4K5 and H4K12. Remarkably, the majority of these H3K4-methylated regions are also associated with CpG islands. We therefore term them “euchromatic islands”. Remarkably, Myc-binding sites are almost exclusively found within euchromatic islands. The presence of tri-methyl H3K4, in particular, shows a very high level of correlation with Myc, consistent with the observation that high-affinity E-boxes are preferentially found within CpG islands [1]. Importantly, our data also show that the E-box is a major specificity determinant, being present in over 60% of all Myc target sites, far above the estimates reached by ChIP- on-chip. In fact, sites previously classified as Myc targets by ChIP-on-chip [2, 3] but lacking tri-methyl H3K4 and E-boxes generally fail to score as targets by q-PCR. Finally, the Myc dimerization partner Max shows a near-perfect correlation with Myc binding, while the E-box binding factors USF1 and USF2 show an overlapping, but distinct pattern. We hypothesize that preferential accessibility of euchromatic islands and/or selective recognition of histone marks are rate-limiting steps for sequence-specific binding of TFs, and determine their selectivity for only a minority of all potential DNA sites in the human genome.

1.Fernandez, P., et al., Genomic targets of the human c-Myc protein. Genes Dev, 2003. 17: p. 1115-1129. 2.Cawley, S., et al., Unbiased Mapping of Transcription Factor Binding Sites along Human Chromosomes 21 and 22 Points to Widespread Regulation of Noncoding RNAs. Cell, 2004. 116(4): p. 499-509. 3.Li, Z., et al., A global transcriptional regulatory role for c-Myc in Burkitt’s lymphoma cells. Proc Natl Acad Sci U S A, 2003. 100(14): p. 8164-8169. 4.Bernstein, B.E., et al., Genomic maps and comparative analysis of histone modifications in human and mouse. Cell, 2005. 120(2): p. 169-181.

38 Abstracts - Oral

Francois Fuks A direct mechanistic link between the Polycomb Group protein EZH2 and DNA methyltransferases

E. Vir, C. Brenner, R. Deplus, L. Di Croce, Y. de Launoit and F. Fuks

Free University of Brussels, GE, CP614, Campus Erasme, 808 route de Lennik, Brussels, Belgium 1070

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. Essential epigenetic systems involved in heritable repression of gene activity are Polycomb Group (PcG) proteins and DNA methylation. Here we show that these two silencing pathways are mechanistically linked. We find that the PcG protein EZH2 interacts with DNA methyltransferases (DNMTs) and associates with DNA methyltransferase activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to the EZH2-repressed MYT1 promoter requires the presence of EZH2 and this correlates with silencing of the MYT1 gene. Furthermore, EZH2 binding to the MYT1 promoter coincides with CpG methylation of MYT1. Our results suggest that EZH2 serves as a molecular beacon for DNA methyltransferases and highlight a previously unrecognised direct connection between two key epigenetic repression systems.

39 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Julie Ahringer Chromatin regulation and sumoylation in the inhibition of Ras induced vulval development in C. elegans

Gino Poulin, Yan Dong, Andrew G. Fraser, Neil A. Hopper and Julie Ahringer

The Gurdon Institute, Tennis Court Road, Cambridge, Cambridgeshire CB2 1QN, U.K.

Regulation of chromatin plays an important role in transcriptional control. Although a large number of chromatin associated proteins have been identified, their cellular functions and associations are not well understood. In C. elegans, a number of synMuv genes encode chromatin associated proteins involved in transcriptional repression, including an ortholog of Rb and components of the NuRD histone deacetylase complex; these are involved in antagonizing Ras signalling in the vulva. Altered regulation of Ras signalling and of these chromatin proteins has been linked with metastasis and/or cancer. To identify other components of this regulatory mechanism, we carried out a genome-wide RNAi screen. After RNAi of 16,757 genes, we found 19 synMuv genes, 9 of which are new. Based on the sequence of these genes and genetic epistasis experiments, we propose that at least four post-translational modifications converge to inhibit Ras-stimulated vulval development: sumoylation, histone tail deacetylation, methylation, and acetylation. We further show that many of the synMuv genes also cooperate in gene regulation outside the vulva, negatively regulating the expression of the Delta homologue lag-2. As most of the genes identified in this screen are conserved in humans, we suggest that similar interactions may be relevant in mammals for control of Ras signalling, cross-talk between these pathways, and cell proliferation.

40 Abstracts - Oral

Peter Becker Site-specific acetylation defines an embryonic form of ISWI that associates with mitotic chromatin

Roger Ferreira, Mariacristina Chioda, Anton Eberharter, Tiziana Bonaldi, Axel Imhof and Peter B. Becker

Adolf-Butenandt-Institut-Molekularbiologie, University of Munchen, 44 Schillerstr, Munchen D-80336, Germany

Nucleosome remodeling enzymes are involved in regulating all aspects of chromatin metabolism, but little is known how their activity is regulated. The nucleosome remodeling ATPase ISWI, which is part of several remodeling complexes in Drosophila, catalyses the sliding of histone octamers on DNA. We now found that ISWI is acetylated at lysine (K) 753 by the acetyltransferase GCN5 in vivo and in vitro. The sequence surrounding K753 resembles the N-terminus of histone H3, where the corresponding lysine, H3K14 is also a prominent substrate of GCN5. This finding provokes the exciting possibility that properties of nucleosomes and remodeling enzymes may be regulated in concert by acetylation. We visualised ISWIK753ac during Drosophila development with a specific antibody. ISWIK753ac is restricted to embryonic development and particularly abundant in the earliest developmental stages. Remarkably, ISWIK753ac was found concentrated on mitotic chromosomes of synchronously dividing preblastoderm nuclei, in contrast to bulk ISWI, which is excluded from condensed metaphase chromatin. Thus, the K753 acetylation marks an ISWI form with novel, unprecedented properties. We hypothesize that the association of this acetylated ISWI form with metaphase chromosomes may contribute to the extreme plasticity of preblastoderm chromatin and to the rapidity of nuclear division cycles during early embryonic development.

41 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Stephen Rea hMOF complex is required for histone H4 lysine 16 acetylation in mammalian cells

Stephen Rea1, Stefan Pfister2, Mikko Taipale1, Peter Lichter2 and Asifa Akhtar1

1Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany, 2Department of Molecular Genetics, German Cancer Research Centre, Heidelberg, Germany

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila MOF, hMOF, is a histone H4 lysine K16 specific acetyltransferase. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 siRNA treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in G2 and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by reduction in hMOF protein levels. Furthermore, hMOF depleted cells show increased number of phospho-ATM and gH2AX foci, and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression. In addition, we have recently purified the hMOF complex and also identified target genes of this complex. These novel findings will also be discussed.

42 Abstracts - Oral

Susana Gonzalo DNA methyltransferases control telomere length in mammalian cells

Susana Gonzalo1, Isabel Jaco1 , Mario F. Fraga2, Taiping Chen3, En Li3, Manel Esteller2 and Mara A. Blasco1

1Telomeres and Telomerase Group. Molecular Oncology Program. Spanish National Cancer Centre (CNIO), Madrid, SPAIN, 2Epigenetics Group. Molecular Pathology Program. Spanish National Cancer Centre (CNIO). Madrid, SPAIN, 3Epigenetics Program. Novartis Institutes for Biomedical Research. Cambridge, Massachussetts, USA

Mammalian telomeres present heterochromatic features, which include trimethylation of H3K9, trimethylation of H4K20, and binding of HP1. Proper assembly of telomeric chromatin is critical for telomere homeostasis. Thus, defects in activities that participate in the assembly of heterochromatin (Suv39h HMTases, Rb family members) result in telomere length deregulation. We demonstrate an unprecedented role for mammalian DNA methyltransferases in telomere length control. Mouse ES cells deficient for Dnmt1, or both Dnmt3a and Dnmt3b show dramatically elongated telomeres compared to wildtype cells. Mammalian telomere repeats (TTAGGG) lack the canonical CpG methylation site, however, we found that mouse subtelomeric regions are highly methylated as indicated by bisulfite sequencing, and that this epigenetic modification is dramatically lost in the absence of the Dnmts. Chromatin immunoprecipitation showed that histone modifications that mark telomeric chromatin, such as H3K9 and H4K20 tri-methylation, remain unaltered at subtelomeric and telomeric domains in the absence of DNA methyltransferase activities. Importantly, lack of Dnmts resulted in increased telomeric recombination as indicated by sister chromatid exchanges involving telomeric sequences. Our results demonstrate that DNA methylation controls the length of TTAGGG repeats at mammalian telomeres independently of histone methylation by repressing recombination between telomeric sequences.

43 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Craig Peterson Chromatin remodeling: Regulation of chromatin higher-order folding and DNA repair

Craig L. Peterson1, Manolis Papamichos-Chronakis1, Michael Shogren- Knaak1,2, Haruhiko Ishii1, Michael J. Pazin3 and Jocelyn Krebs4.

1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605; 2Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011; 3Laboratory of Cellular and Molecular Biology, National Institute of Aging, NIH, Baltimore, MD 21224; 4Department of Biological Sciences, University of Alaska, Anchorage, AK 99508, U.S.A.

Chromosome structure plays a key role in the regulation of all DNA-mediated processes, including transcription and DNA repair. Dynamic changes in chromosome structure are catalyzed by ATP-dependent chromatin remodeling enzymes and by histone modifying enzymes, such as histone acetyltransferases. We will present two stories that illustrate distinct roles for both types of chromatin-based enzymes. First, we will discuss our recent work on Ino80 and Swr1 which are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. We have found that Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA double strand break (DSB). The failure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability to maintain high levels of histone H2AX phosphorylation and an increased incorporation of the Htz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation and checkpoint adaptation. We propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation. The second story will focus on histone H4 lysine 16 acetylation which is a prevalent and reversible post-translational modification in eukaryotic organisms. To characterize the structural and functional role of this mark in the context of chromatin, we have used a native chemical ligation strategy to generate histone H4 homogeneously acetylated at K16. When this histone is incorporated into nucleosomal arrays, we find that acetylation of this single lysine residue inhibits the formation of compact 30 nm-like fibers and impedes the ability of chromatin to form cross-fiber interactions. Histone H4 K16Ac also inhibits the ability of the ACF chromatin remodeling enzyme to mobilize a mononucleosome. This is the first example of a single histone modification that is sufficient to modulate higher-order chromatin structure as well as perturb functional interactions between a nonhistone protein and the chromatin fiber.

44 Abstracts - Oral

Bob Kingston Remodeling chromatin without covalent modification

Jonathan Dennis, Rebecca Dunn, Nicole Francis, Ian King, *Chris Woodcock and Bob Kingston

Massachusetts General Hospital, Boston, MA, U.S.A. and *University of Massachusetts, Amherst, MA, U.S.A.

Master regulatory genes, such as Hox genes, must be maintained in a heritably ‘on’ state in some cell lineages and in a heritably ‘off’ state in other cell lineages. Genetic studies have identified numerous genes that are required for this epigenetic level of regulation to work properly. Many of these genes are classified as Polycomb-Group (PcG) or trithorax-Group (trxG) based on the screen that led to their initial isolation. PcG genes are required to maintain repression. We have identified and characterized a complex called PRC1 that contains four PcG genes. This complex interacts with nucleosomal templates to block transcription and remodeling of those templates. Repressed templates have are differentially sensitive to various enzymatic probes and appear compacted when visualized by microscopy. The Esc/E(z) complex (also called PRC2) has been proposed to target PRC1 action via methylation of lysine 27 of histone H3. We are using recombinant complexes to address functional interactions between the PRC complexes and nucleosomal templates. To further characterize the functions of these complexes, we are developing technologies to analyze chromatin structure in living cells. Our goal is to develop an automatable protocol to allow mapping of chromatin structure across entire Hox clusters in cell lines under different stages of differentiation.

45 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Lianna Johnson DNA and histone methylation in Arabidopsis

Lianna Johnson, Simon Chan, Xiaoyu Zhang, Yana Bernatavichute and Steven Jacobsen

UCLA, Life Sciences, Box 951606, Los Angeles, CA 90095-1606, U.S.A.

Three different classes of DNA methyltransferases are found in Arabidopsis. Each type of methyltransferase is targeted by a different mechanism. siRNAs are involved in targeting DRM2 to various repeated DNAs and also in targeting DNA for de novo methylation. Histone H3 methylation is critical for targeting CMT3 to specific regions of the chromatin. MET1 is specific for methylation of CG residues, and assumed to work like its homologs in mammals (Dnmt1), where hemimethylated DNA resulting from replication is the preferred substrate. Using as a model a SINE element found in euchromatin in Arabidopsis, mutants which block these pathways at various points will be examined for DNA methylation, histone methylation and transcription to arrive at a simple overall model for targeting of DNA and histone methylation.

46 Abstracts - Oral

Eric Miska Short RNAs in C. elegans development and human cancer

Eric Miska

Gurdon Institute, University of Cambridge, The Henry Wellcome Building, Tennis Court Rd., Cambridge CB2 1QN, U.K.

In the last five years microRNAs (miRNAs) have emerged from the obscurity of the C. elegans heterochronic pathway to a new paradigm of gene regulation in plants and animals. Currently, microRNAs represent 2% of all known human genes. Very little is known about their biological function. We have taken a functional genomics approach to study the roles of microRNAs in C. elegans development. We have generated knockout strains corresponding to 96 microRNAs, covering the majority of known microRNA genes. We will present an overview of the classes of mutant phenotypes we have observed. One focus will be the issue of redundancy within families of microRNA genes. This study represents the first comprehensive analysis of microRNA function. We are also interested in the roles of short RNAs in the control of gene expression at the transcriptional level. We will present our work on how these short RNAs work together with a set of argonaute proteins to control germline development in C. elegans.

47 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Ramin Shiekhattar Integrator, a multiprotein mediator of small nuclear RNA processing associates with the C-terminal repeat of RNA polymerase II

David Baillat1, Mohamed-Ali Hakimi2, Anders Näär3, Ali Shilatifard4, Neil Cooch1, and Ramin Shiekhattar1,5

1The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, 2CNRS, UMR5163, Université Joseph Fourier, La Tronche, 38700, France, 3Harvard Medical School and Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, 4Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, Saint Louis, MI 63104

The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is an essential component of transcriptional regulation and RNA processing of protein coding genes. A large body of data also implicates the CTD in the transcription and processing of RNAPII-mediated small nuclear RNA (snRNA) genes. However, the identity of the complex(es) that associate with the CTD and mediate the processing of snRNA genes have remained elusive. Here, we describe an RNA polymerase II complex that contains at least twelve novel subunits, termed the Integrator, in addition to core RNAPII subunits. Two of the Integrator subunits display similarities to the subunits of the cleavage and polyadenylation specificity factor (CPSF) complex. We show that Integrator is recruited to the U1 and U2 snRNA genes and mediates their 3’-end processing. The Integrator complex is evolutionarily conserved in metazoans and directly interacts with the C-terminal domain of the RNA polymerase II large subunit.

48 Abstracts - Oral

Kami Ahmad

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, CI-204, Boston, MA 02115, U.S.A. Abstract unavailable at time of print

49 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jacques Cote Dissection of the NuA4 HAT complex and crosstalk with other chromatin modifications and remodelers

Andranne Auger, Nicolas Lacoste, Marilyn Pray-Grant, Joelle Brodeur, Rhea T. Utley, Yannick Doyon, Olivier Jobin-Robitaille, Stphane Allard, Luc Gaudreau, Patrick Grant and Jacques Cotes

Laval University Cancer Research Center, Hotel-Dieu de Quebec (CHUQ),9 McMahon Street, Quebec City, G1R 2J6 Canada

Acetylation of histone H4 N-termini is a major chromatin modification important for gene activation. The NuA4 histone acetyltransferase complex is the only essential HAT in yeast and is responsible for H4 and H2A hyperacetylation in vivo. Thorough genetic and biochemical analysis of the 13-subunit assembly identified 4 different subcomplexes, each one linked to specific functions in the cell. Protein domains present in the complex regulate its association with chromatin substrates, in part through recognition of specific histone modifications like H4/H2A phosphorylation and H3 methylation. For example, methylation of H3 on lysine 4 and 36 regulates NuA4 action on chromatin. This effect is mediated by the subunit Eaf3 and its chromodomain, which directly associates with MeK4 and MeK36 in vitro. These interactions regulate NuA4 function not only during the process of transcription but also in DNA repair. In addition, findings in yeast, Drosophila and human systems suggests a link between NuA4 activity and the incorporation of histone variant H2AZ in chromatin. We demonstrate new substrate specificity for NuA4 as it efficiently acetylates H2AZ in chromatin substrates. Depletion of NuA4 also affects H2AZ incorporation at specific loci in vivo, supporting the concept of functional cooperation between NuA4 and Swr1 complexes during transcription regulation of specific genes.

50 Abstracts - Oral

Benoit Guillemette H2A.Z is globally localized to the promoters of inactive genes and regulates nucleosome positioning

Benoit Guillemette1, Alain R. Bataille2, Nicolas Gévry1, Maryse Adam1, Mathieu Blanchette3, François Robert2 and Luc Gaudreau1

1Centre de Recherche sur les Mécanismes du Fonctionnement Cellulaire, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada 2Laboratoire de Chromatine et Expression du Génome, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada, 3McGill Center for Bioinformatics, Lyman Duff Medical Building, Montréal, Québec, Canada

H2A.Z is a highly conserved variant of canonical histone H2A that plays many distinct roles. In Saccharomyces cerevisiae, H2A.Z acts as an anti-silencing factor by preventing the spread of heterochromatin. H2A.Z is also functionally redundant with components of SAGA and SWI/SNF, and it is essential for the full expression of certain genes such as PHO5 and GAL1. We have previously shown that H2A.Z co-precipitates with, and is essential for RNAPII recruitment at the GAL1 promoter. Therefore, H2A.Z may possess dual roles in regulation of transcription. In order to better understand the function(s) of H2A.Z in yeast, we used a genome-wide binding assay (ChIP on chip) to map H2A.Z with at ~300bp resolution. We find that H2A.Z is globally located within small regions (1-2 nucleosomes wide) across the genome. These Z- loci are also mostly located in the promoters of yeast genes, and surprisingly, H2A.Z binding is higher in low-, rather than in highly-transcribed genes. A total of 63% of yeast promoters contain a Z-locus. We also show that nucleosomes in genes that contain a Z-locus in their promoter show a regular binding pattern, whereas nucleosomes in non-Z-locus genes are less well organized. Using GAL1 as model gene, we find that deletion of HTZ1 causes a shift in a specific nucleosome positioned over the transcriptional start site. Finally, Z-loci are wider in telomere proximal regions, where the H2A.Z anti-silencing function was shown to be important. These results suggest that H2A.Z contributes gene regulation by poising inactive genes by modifying nucleosome phasing around promoters. They also support a model in which H2A.Z could possess distinct mechanisms in the regulation of telomere-proximal genes than in other genes.

51 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Karolin Luger Structure and function of yeast nucleosome assembly protein 1

Young-Jun Park, Jayanth Chodaparambil and Karolin Luger.

Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA 80523-1870

Nucleosome assembly protein 1 (NAP-1) is a key component in the modulation of chromatin structure. It shuttles histones into the nucleus, assembles nucleosomes, and promotes chromatin fluidity by removal and replacement of histone H2A-H2B dimers, thereby promoting nucleosome sliding. Biochemical and biophysical experiments further suggest additional roles of NAP-1 in ‘scavenging’ misassembled chromatin. Together, these activities are likely to affect transcription of many genes. The 3.0 Å crystal structure of yeast NAP-1 reveals a homodimer with a novel fold. A long a-helix is responsible for dimerization via a novel antiparallel non-coiled coil, and an α/β domain is implicated in protein – protein interactions. The four-stranded anti-parallel β-sheet that characterizes the α/β domain is found in all histone chaperones, despite absence of homology in sequence, structural context, or quarternery structure. This is the first structure of a member of the large NAP family of proteins, and suggests a mechanism by which histones are bound, and by which the shuttling of histones to and from the nucleus is regulated.

Supported by NIH / GM67777

52 Abstracts - Oral

Wolf Reik

Regulation of imprinting and epigenetic reprogramming in mammals

A Lewis, A Murrell, F Santos, H Morgan, W Dean and W Reik

Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, U.K.

Imprinting in mammals has important roles in development, including in fetal growth, postnatal adaptions, and adult behaviour. Imprinted genes occur in clusters, share regulatory elements, and can be epigenetically regulated by imprinting centres. Our recent work reveals how imprinting centres may control long range expression and epigenetic profiles in imprinting clusters. Imprinting centre 1 (the insulator region upstream of H19 on distal chromosome 7 in the mouse) mediates physical contact, on the maternal chromosome, with a silencer sequence in the Igf2 gene, about 100kb away, and the intervening DNA is looped out. On the paternal chromosome, the same region is in contact with an activator sequence in Igf2. This higher order structure results in the maternal Igf2 gene being located in a silent chromosome loop, away from the enhancers, while the paternal Igf2 gene is located outside the silent loop in close proximity to enhancers. Thus the insulator (IC1), presumably through its differential methylation, mediates long distance gene regulation and epigenetic marking in the Igf2-H19 cluster. In the neighbouring IC 2 cluster, a paternally expressed non-coding RNA transcript (Kcnq1ot1) is flanked by several paternally repressed protein coding genes. Most of these genes are specifically imprinted in the placenta, and lack differential DNA methylation. The non-coding transcript, directly or indirectly, mediates the targeting of repressive histone modifications to the flanking genes, and silencing of the flanking genes in the placenta is maintained in the absence of DNA methylation. This mechanism has striking similarities with that of imprinted X chromosome inactivation. We argue that there are at least two different building principles by which different imprinting clusters are constructed.

Murrell et al. (2004) Nature Genet. 36, 889-893, Osborne et al (2004) Nature Genet. 36, 1065-1071, Lewis et al. (2004) Nature Genet. 36, 1291-1295, Constancia et al. (2004) Nature 432, 53-57. Reik and Lewis (2005) Nature Rev. Genet. 6, 403-410.

53 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Aidan Doherty Structure and function of the Jumonji C domain

Andrew Green1, David Lando2, Nigel C. Brissett1, Tony Kouzarides2 and Aidan J. Doherty1

1Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, U.K. 2Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, U.K.

Jumonji domains (JmjC) are present in a wide range of proteins that often contain chromatin binding domains (1). There is significant sequence similarity between JmjC and the FIH-1 family of metalloenzymes. Combined structural and genetic approaches have identified that FIH is a member of the 2-oxoglutarate-dependent dioxygenase superfamily (2,3). The secondary structure of the JmjC domain also predicts enzymatic activity (1,2), and its frequent association with putative chromatin modification activities suggests a role for JmjC in regulating the integrity of chromatin structure (1). This link with chromatin remodelling functions has been supported by the finding that the S. pombe JmjC domain protein, Epe1, modulates heterochromatization in fission yeast (4). Many enzymes regulate chromatin structure via covalent modifications and JmjC represents an excellent candidate for an additional novel modifying activity, possibly a hydroxylase, that regulates the integrity of chromatin structure. As JmJc proteins are similar to members of the FIH family of protein hydroxylases (2), JmjC was assayed for hydroxylase activity. In the presence of histones and co-factors (2- oxoglutarate, oxygen and Fe2+) we observed that JmjC is an active protein hydroxylase and we identified the specific residues modified on the histone substrate. To confirm that this activity is specific to the JmjC protein, active site mutations in the JmjC domain were produced. These mutants were inactive in the hydroxlase assay, confirming that the activity is specific to JmjC. This work establishes, for the first time, that JmjC domains are enzymes (dioxygenases) that can catalyse histone-specific hydroxylation events in vitro. The crystal structure of a Jumonji domain revealed that the enzyme has a beta-barrel ‘jelly- roll’ conformation, similar to FIH-1 (2), that contains an iron co-ordination motif at the catalytic centre with co-substrate 2-oxoglutarate bound in the active site. Thus JmjC appears to represent a new family of histone modifying enzymes.

1. Clissold, P.M. and Ponting, C.P. (2001) Trends Biochem Sci. 1, 7-9. 2. Elkins JM, Hewitson KS, et al. Ratcliffe PJ, Schofield CJ. (2003) J Biol Chem. 278, 1802-6. 3. Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML. (2002) Science 295, 858-861. 4. Ayoub N, Noma K, Isaac S, Kahan T, Grewal SI, Cohen A. (2003) Mol Cell Biol. 23, 4356-70.

54 Abstracts - Oral

Andreas Ladurner Human chromatin as a molecular target of cellular NAD metabolites

Georg Kustatscher, Bjrn Fritz and Andreas G. Ladurner

EMBL, Gene Expression Unit, Meyerhofstrasse 1, Heidelberg 69117, Germany

The role of NAD metabolites in chromatin structure and regulation is receiving increasing biochemical attention. We have recently shown that the SirT1-metabolite O-acetyl-ADP- ribose binds the human macroH2A1.1 histone variant through its C-terminal macro domain. Structural evidence, as well as engineered mutants reveal how the metabolite is selectively recognized. The recognition of nucleotide metabolites by human chromatin suggest the existence of a novel regulatory paradigm in biology. In addition, we find that mutually-exclusive exon use in the MACROH2A1 gene produces a second macroH2A isoforms (known as macroH2A1.2) which cannot bind O-acetyl-ADP- ribose. Alternative splicing may thus regulate the sensitivity of human chromatin toward the potential metabolite regulator. We will discuss our most recent evidence, which suggests distinct tissue distribution between the known macroH2A variants. Further, we will report on our efforts to understand the functional consequences of NAD metabolite binding by chromatin. The histone variant macroH2A is a hallmark of mammalian heterochromatin. Our studies are directed at dissecting the role of these interesting NAD metabolites in mammalian chromatin.

55 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jef Boeke Comprehensive mutagenesis of sites of histone modification

Edel M. Hyland, Michael S. Cosgrove, Henrik Molina, Dongxia Wang, Akhilesh Pandey, Robert J. Cotter and Jef D. Boeke

339 Broadway Research Building,733 North Broadway, Baltimore, MD 21205, U.S.A.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome, and in the DNA repair response. By mutagenizing these residues to mimic their modified and unmodified states we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the rDNA locus and at telomeres, suggesting that post-translational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, R92 and histone H3 K56, K115 lead to hypersensitivity to DNA damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally we allude to the possible molecular mechanisms underlying the effects of these modifications. The project is currently being expanded to histones H2A and H2B.

56 Abstracts - Poster Abstracts - Poster Ted Abel Abstract 1 The role of CBP and histone acetylation in memory storage and synaptic plasticity

T. Abel1, M.P. Kaplan1, C. Vecsey1, K. M. Lattal2, J.M. Stein1, S.A. Fabian1, M.A. Attner1, J.D. Hawk1 and M.A. Wood1

1Biology, University of Pennsylvania, Philadelphia, PA, U.S.A. 2Behavioural Neuroscience, Oregon Health and Science University, Portland, OR, U.S.A.

Transcriptional activation is thought to be a key process in long-lasting forms of memory and synaptic plasticity. This activation is directed by transcription factors and their coactivators, which regulate gene expression via chromatin remodeling, histone modification and interactions with the basal transcription machinery. One type of histone modification associated with transcriptional activation is acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that add or remove acetyl groups from histones, respectively. Recently, we have demonstrated that the transcriptional coactivator CREB-binding protein (CBP), a potent HAT, is involved in specific forms of long- term memory and synaptic plasticity. We have examined mice in which CBP activity in neurons is reduced either by the transgenic expression of an inhibitory form of cbp lacking the HAT domain or by knocking in a mutation of the CREB transcription factor-binding KIX domain of cbp. This genetic approach enabled us to compare the role of CBP-associated HAT activity in memory and synaptic plasticity with the role of the KIX transcription factor- binding domain of CBP. We found that mutant mice expressing an inhibitory form of cbp exhibit impairments in spatial and contextual memory and in long-lasting forms of hippocampal synaptic plasticity. KIX knock-in mice were also observed to have significant impairments in contextual memory. A complementary method to study the role of histone acetylation in synaptic plasticity and memory is to examine the effects of HDAC inhibitors, which increase the level of histone acetylation that correlates with transcriptional activation. We found that increasing histone acetylation using the HDAC inhibitor TSA enhances long- term contextual memory and facilitates synaptic plasticity via the transcription factor CREB. In summary, these results support the idea that histone acetyltransferases and histone acetylation are critical to mechanisms of long-term memory storage. Histone acetylation may provide an epigenetic mechanism for establishing gene-specific modifications that result in the coordinate expression of genes required for long-term memory storage.

57 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Melissa Adkins Abstract 2 Spt6-mediated nucleosome reassembly is required for transcriptional repression of the PHO5 gene

Melissa W. Adkins and Jessica K. Tyler

Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, PO Box 6511, Mail Stop 8101, Aurora, CO 80045 U.S.A.

The packaging of the eukaryotic genome into chromatin is likely to have profound influence on transcription from the underlying genes. We have previously shown that the disassembly of promoter nucleosomes is obligatory for activation of the yeast PHO5 and PHO8 genes. Here we show that the PHO5 promoter nucleosomes are reassembled concomitant with transcriptional repression and displacement of the TATA-binding protein (TBP) and RNA polymerase II. We identify the histone H3-H4 chaperone Spt6 as the factor that mediates replication-independent nucleosome reassembly onto the PHO5 promoter as well as the PHO8 promoter during transcriptional repression. Furthermore, we demonstrate that nucleosome reassembly is essential for transcriptional repression of the PHO5 and PHO8 genes. Finally, we show that in the absence of Spt6-mediated nucleosome reassembly, the activators Pho4 and Pho2 are displaced from the promoter in repressing conditions, yet transcription is sustained. As such, these studies demonstrate that the Pho4 and Pho2 activators are not required for transcription in the absence of competing chromatin reassembly.

58 Abstracts - Poster

Adriana Alejandro-Osorio Abstract 3 The role of the histone deacetylase Rpd3p in coordinating the environmental stress response in yeast.

Adriana L. Alejandro-Osorio1 and Audrey Gasch2

1Department of Biomolecular Chemistry., 2Laboratory of Genetics and Genome Center of Wisconsin, University of Wisconsin-Madison, 3452 Genetics-Biotech Center, 435-g Henry Mall, Madison WI 53706, U.S.A.

Unicellular organisms must be able to withstand frequent and stressful changes in their environment. The budding yeast Saccharomyces cerevisiae initiates a large gene expression program in response to diverse types of environmental stress (Gasch et al. 2000, Causton et al. 2001). This response, called the environmental stress response (ESR), consists of ~300 genes whose expression is induced and ~600 genes whose expression is repressed in response to stressful conditions. A number of transcription factors have been implicated in ESR regulation, however how these expression changes are coordinated is not understood. We have investigated the role of chromatin remodeling in ESR regulation and found that the histone deacetylase, Rpd3p, is required for initiation of the ESR. Cells lacking rpd3 fail to properly regulate expression of the ESR genes and are sensitive to a panel of stresses. This suggests that chromatin remodeling plays an important role in coordinating the expression of this extensive gene expression response.

59 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Erik Andersen Abstract 4 MET-1 and MET-2, two putative histone methyltransferases, are redundantly required for proper vulval cell fate specification

Erik Andersen and Bob Horvitz

MIT Biology 68, Room 417, 77 Massachusetts Ave., Cambridge, MA 02139, U.S.A.

Genetic screens for mutations that affect the vulval cell fate of Caenorhabditis elegans was first to identify many homologs important in the regulation of human cancer. An increase or decrease in the number of cells that adopt vulval cell fates is easily observed as either a Multivulva (Muv) or Vulvaless (Vul) phenotype respectively. These two types of mutants led to the identification of components of the RTK/Ras, Notch, and Wnt pathways. The Muv phenotype of some mutants is due to loss-of-function mutations in two functionally redundant genes, called the synthetic Multivulva (synMuv) genes. The synMuv genes are grouped into three classes, A, B, and C. Animals with mutations in one or more genes within the same class are not Muv, whereas mutations in genes within any two classes are Muv. Several synMuv genes encode counterparts of a transcriptional repression complex, including HDA-1 HDAC, LET-418 Mi2, and LIN-53 RbAp48. Based upon this focus on chromatin remodeling, we wanted to determine if any histone methyltransferases (HMTases) play a role in vulval cell fate specification. We inactivated each of the 33 genes predicted to encode SET domain containing proteins and assayed for vulval defects. Two putative HMTase genes - met-1 and met-2 - conferred a synMuv phenotype in combination with class A mutations. MET-1 is a homolog of yeast SET2p and predicted to methylate H3K36. MET-2 is the homolog of human SETDB1 and predicted to methylate H3K9. Using quantitative western blots of wild-type, met-1, and met- 2 extracts, we found that met-1 and met-2 are required for H3K36 and H3K9 trimethylation, respectively. Interestingly, met-1 and met-2 act redundantly with each other in vulval cell fate specification. Through further genetic studies, we hope to characterize how these putative HMTases function in vivo to specify a developmental cell fate.

60 Abstracts - Poster

Anthony Argentaro Abstract 5 Structure/function studies of the PHD-like domain of the chromatin-remodelling protein, ATRX.

Argentaro A.1, Chapman L.2, Rhodes D.2, Yang J.C.2, Neuhaus D.2, Kowalczyk M.1, Higgs D.1 and Gibbons R.J.1

1MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX39DS, U.K., 2Medical Research Council Laboratories, Cambridge CB22QH, U.K

The chromatin associated protein ATR-X was first identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with a- thalassemia. The ATRX protein contains a highly conserved cysteine-rich N-terminal region, part of which resembles a Plant homeodomain (PHD). NMR studies have shown that PHD domains form a looped structure which binds zinc in a crossed braced topology between anti-parallel beta strands. Such domains are frequently found in chromatin-associated proteins and are thought to mediate protein-protein interactions. The N-terminal region of ATRX is clearly different from the majority PHD domains but similar sequences are found in de novo methyltransferases (DNMT3b) and the related DNMT3L protein, hence this new sequence has been referred to as the ADD domain. At present neither the structure nor function of the ADD domain is known, however since >60% of disease-causing mutations occur within this region of the ATRX protein it is likely to be of considerable functional importance. Based on the previously solved structure of the PHD domain we have made predictions about the fold of the variant PHD domain in ATRX using several structural prediction programmes although preliminary data suggest that the extended PHD-like, ADD domain may form a new fold distinct from that of the PHD domain. Analysis of natural mutations of the ATRX protein may also test structural predictions. Most mutations show significantly reduced levels of ATRX protein despite apparently normal or somewhat increased levels of RNA, suggesting that they de-stabilise the ADD structure. In general, mutations, affecting residues predicted to lie on the surface of the ADD domain have less severely reduced levels of protein and these mutations do not appear to affect the structural integrity of the PHD-like fold. Such mutations may give rise to ATRX syndrome by affecting critical protein interactions involved in the normal nuclear role of ATRX.

61 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Koh Meng Aw Yong Abstract 6 The presence of EBV oriP within a transcription unit inhibits replication as well as transctiption

Koh Meng Aw Yong and Siu Chun Hung

Department of Microbiology, National University of Singapore, Block MD4 Level 5, 5 Science Drive 2, Singapore 119260

The interplay between DNA replication and transcription has never been truly understood. While there has been some work done supporting claims that the two are antagonistic or synergistic to each other, others claim that neither is affected by the other. In our study, we hope to cast more light on this contentious relationship between replication and transcription using the latent origin or replication, oriP of the DNA virus Epstein-Barr virus (EBV). By making use of the fact that EBV initiates replication by recruiting host cellular replication machinery to the oriP in the presence of EBV latent protein EBNA-1, we inserted oriP immediately downstream of a SV40 promoter as part of the transcription unit. We then assayed for DNA replication and transcription using Southern and Northern blots analysis respectively. Our results indicate that DNA replication is inhibited by the presence of the SV40 promoter immediately upstream of the oriP. In addition, transcription was also inhibited such no full length transcripts were produced. This inhibition occurred even in the absence of EBNA-1. Based on the current data observed, our hypothesis is that as the transcriptional elongation complex reads through the transcriptional unit, it is physically stalled at the oriP in a sequence dependent manner. This results in no transcript being generated; and at the same time, it inhibits the initiation of replication by physically preventing the replication machinery complex from assembling on the oriP.

62 Abstracts - Poster

Slobodan Barbaric Abstract 7 Two nucleosomes at the yeast PHO84 promoter demonstrates differential requirement for chromatin remodelling activities

Bojana Sili1, Sabrina Stürzl2, Tim Luckenbach2, Philipp Korber2, Slobodan Barbaric1 and Wolfram Hörz2

1Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia. 2Adolf-Butenandt-Institut Molekularbiologie, Universität München, Schillerstr. 44, 80336 München, Germany.

We have investigated remodeling of the chromatin structure at the yeast PHO84 promoter, which is coregulated with the well studied PHO5 and PHO8 promoters in response to phosphate availability. The PHO84 promoter 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. Upon induction the promoter chromatin structure is altered, so that at least one nucleosome upstream and one downstream from the hypersensitive region are remodeled. This way two additional Pho4 binding sites become accessible. Remodeling leads to histone depletion from the promoter region. We found that the rate of chromatin remodeling and subsequent promoter activation was strongly delayed in mutants deleted for Snf2, Ino80, Gcn5 or the histone chaperone Asf1. Nonetheless, after prolonged induction full remodeling and activation were achieved in the absence of Ino80, Gcn5 or Asf1. However, in the absence of Snf2 remodeling was only partial and resulted in the disruption of the downstream but not of the upstream nucleosome, showing different requirements for remodeling of these two nucleosomes. Therefore, regarding the Snf2 requirement for chromatin remodeling, the PHO84 promoter appears to present a hybrid of the two coregulated promoters: the PHO8 which is fully dependent on Snf2, and the PHO5 where only the rate of remodeling is affected.

63 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Vivian Bardwell Abstract 8 Polycomb group and SCF ubiquitin ligases are found in a BCL6 corepressor complex

Gearhart, M.D., Corcoran, C.M., Wamstad, J.A. and Bardwell, V.J.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, U.S.A.

The corepressor BCOR potentiates transcriptional repression by the oncogene BCL6 and suppresses the transcriptional activity of a common Mixed-Lineage Leukemia (MLL) fusion partner, AF9. Mutations in human BCOR cause X-linked, male lethal Oculofaciocardiodental syndrome. Here we identify a BCOR complex containing several mammalian homologs of Polycomb Group (PcG) proteins, including the chromatin modifier RNF2, an E3 ligase for the mono-ubiquitylation of H2A (Ub-H2A). Thus the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. RNF2 and Ub-H2A are associated with PcG repression of Hox genes in Drosophila and the inactive X chromosome (Xi) in mammals. We find that BCOR complex components and Ub-H2A localize to a BCL6 target in B cells but BCOR does not localize to Xi. The BCOR complex also contains SKP1 and FBXL10, components of a second E3 ubiquitin ligase. In addition its F box and leucine rich repeats, FBXL10 contains chromatin binding motifs and a JmjC domain suggesting the BCOR complex may have enzymatic activities for poly-ubiquitylation and trimethyl histone demethylation. In addition, we are examining the role of BCOR in hematopoietic development using BCOR deficient ES cells and in other aspects of development using a mouse model system.

64 Abstracts - Poster

Ralph Bash Abstract 9 AFM recognition imaging: using antibodies to track proteins during in vitro remodeling

Ralph Bash1,3, Hongda Wang1, Chris Anderson1, Dennis Lohr3 and Stuart Lindsay1,2,3

1Biodesign Institute, 2Department of Physics and Astronomy and 3Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, U.S.A.

We are using recognition imaging to study remodeling of promoter chromatin, a key event in the initiation of transcription. This technique uses antibodies during AFM imaging to recognize specific proteins. Antibodies used were tested against all proteins that might be encountered in the experiments. Some antibodies cross-react badly and others do not. We correlated these results with ELISA assays, finding the two techniques in agreement, so the cross reactions represent limitations of the antibodies and not the recognition technique. We have also begun to establish quantitative criteria for analyzing recognition images, and describe software we have written for this purpose. This technique has been applied to chromatin remodeling reactions with hSwi-Snf, demonstrating removal of histone H2a from nucleosome cores.

65 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Juraj Bies Abstract 10 Pc2, the polycomb group protein recruits the c-Myb into PcG bodies and inhibits its activity.

Marek Sramko, Jan Markus, Linda Wolff and Juraj Bies

Center of Molecular Medicine, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia and Laboratory Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD U.S.A.

Post-translational modifications of c-Myb oncoprotein play an important role in regulation of its activity. We have shown that conjugation of SUMO-1 affects the transactivation capacity and the proteolytic turnover of c-Myb. Importance of this modification is underscored by the fact that both SUMO-1-modified lysines (K499 and 523) are located in the conserved region of the negative regulatory domain, frequently lost during oncogenic activation of c-Myb protein (Bies et al. 2002). c-Myb specific SUMO-ligase PIASy was identified to increase an extent of SUMOylation to the negative regulatory domain of c-Myb (Dahle et al., 2003). However, recent experiments showed, that SUMOylation of c-Myb was unaffected in PIASy deficient mice when compared to wild-type animal (Wong et al., 2004). These results suggest, that in addition to PIASy, there must be another c-Myb-specific SUMO-1 E3 ligase that catalyzes SUMOylation of c-Myb. The polycomb protein Pc2, which has SUMO E3-ligase activity for the corepressors CtBP and CtBP2, interacts with c-Myb and increases conjugation of SUMO-1 to its negative regulatory domain. Pc2 changes subnuclear localization of SUMOylated c-Myb transcription factor. It recruits Myb from PML nuclear bodies into PcG bodies-like structures, where c- Myb colocalizes with another member of the polycomb repressive complex, Bmi. Coexpression of Pc2 strongly decreases transactivation activity of c-Myb. Interestingly, covalent conjugation of SUMO-1 protein to the negative regulatory domain of c-Myb was dispensable for downregulation of c-Myb activity, as similar inhibition of both wild type, and SUMOylation-deficient mutant of c-Myb was detected.

Bies J et al. (2002). J Biol Chem. 277(11): 8999-90 Dahle O et al. (2003) Eur J Biochem.; 270(6):1338-48. Wong et al. (2004). Mol Cell Biol. 24(12):5577-86.

66 Abstracts - Poster

Caroline Bouchard Abstract 11 c-Myc induces localized H2A.Z exchange in the promoters of target genes

Caroline Bouchard, Till Kortüm and Martin Eilers

Institute for Molecular Biology and Tumour Research (IMT), Marburg, Germany

The c-myc proto-oncogene encodes a transcription factor which can both activate and repress gene expression. Post-translational covalent modifications (acetylation, methylation) of the histone tails and replacement of core histones by variant forms play a crucial role in controlling transcription. We have previously found that binding of Myc to its target gene cyclin D2 induces histone H3 and H4 acetylation at a single nucleosome (1). However, the complete and ordered cascade of events leading to the transcription of cyclin D2 is still unclear. The ATPase Domino (the Drosophila melanogaster homolog of the mammalian p400 protein), as part of the Tip60 chromatin remodeling complex, has been shown to catalyze the exchange of the Drosophila histone variant H2Av in its phosphorylated form after acetylation by Tip60 at DNA double strand breaks against unphosphorylated H2Av (2). In mammals, two histone variants (H2A.Z and the DNA-damage-associated H2A.X) have functional overlap with H2Av. The highly conserved H2A.Z is important for the maintenance of constitutive heterochromatin, for setting boundaries between euchromatin and heterochomatin and has also been implicated in both activation and repression of transcription (3). Here, we used chromatin immunoprecipitation (ChIP) to evaluate the Myc-dependent changes in histone methylation and histone replacement at the level of individual Myc target promoters. We now show in different cellular model systems that Myc binding is associated with the local displacement of the histone variant H2A.Z within the promoter region. Our data suggest a universal role of H2A.Z in the regulation by Myc of its target genes. The cofactors recruited by H2A.Z in order to regulate transcription are being identified. Additionally, the molecular mechanism(s) which underlie(s) the displacement of H2A.Z upon Myc binding are currently under investigation.

1. Bouchard C et al., Genes Dev. 2001 Aug 15;15(16):2042-7. 2. Kusch T et al., Science 2004 Dec 17;306(5704):2084-7. 3. Kamakaka RT and Biggins S, Genes Dev. 2005 Feb 1;19(3):295-310.

67 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Ray Camahort Abstract 12 Genome-wide localization of the budding yeast histone variant Cse4

R. Camahort, K. Collins, S. Biggins, C. Seidel and J. Gerton

Stowers Institute for Medical Research, Gerton Lab., 1000 E. 50th St., Kansas City, Jackson 64110 , U.S.A.

The kinetochore is a complex, multi-protein structure required for proper chromosome segregation in all eukaryotes. The Saccharomyces cerevisiae kinetochore consists of over 65 known proteins which work in concert to facilitate equal distribution of the replicated genome. The budding yeast CenH3 histone variant Cse4 is a histone H3-like inner kinetochore protein that is vital to kinetochore function. Cse4 homologs are found in all higher eukaryotes, and include Drosophila melanogaster Cid protein, Caenorhabditis elegans HCP-3, and the functional vertebrate ortholog CENP-A. Cse4 is thought to localize specifically to centromeric nucleosomes, and function exclusively in the recruitment of additional kinetochore proteins and proper chromosome segregation. Using whole genome DNA microarrays, we have looked at genome wide localization of Cse4 in S. cerevisiae. This data indicates that Cse4 may be found at more places along the budding yeast genome than previously thought. Additionally, post-translational modifications and protein- protein interactions have been investigated in the hopes of elucidating the mechanism of Cse4 localization and function.

68 Abstracts - Poster

Kendra Cann Abstract 13 Multiple regions of the DNA-damage response protein TLS/FUS can regulate its relocalization to the nucleolei following transcriptional inhibition.

K. L. Cann, M. Arntfield, L. DeLange and G. G. Hicks

Manitoba Institute of Cell Biology and the University of Manitoba, Winnipeg, MB, Canada

The heterogeneous nuclear ribonucleoprotein TLS (Translocated in LipoSarcoma; FUS) was initially identified as a fusion partner of CHOP in over 90% of human myxoid liposarcomas, and of ERG in human myeloid leukemia. In these fusions, the N-terminus of TLS provides a potent transcriptional activation domain to complement the DNA-binding domain of CHOP or ERG. We have previously shown the TLS-/- mice have widespread genomic instability. We have also found that TLS is required for a proper cellular response to both gamma-irradiation and mitomycin C, which cause DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), respectively. TLS has been implicated in transcription, splicing, and RNA shuttling, but its mechanisms of action in genomic stability and the DNA damage response have yet to be identified. To better understand TLS function, we chose to investigate TLS localization and its known association with the nucleolus, a subnuclear structure involved in rRNA transcription, ribosome assembly and cell cycle regulation. Endogenous TLS and EGFP (enhanced green fluorescent protein)-tagged TLS is restricted to the nucleus, but is excluded from the nucleoli. After transcriptional inhibition, both endogenous TLS and EGFP-TLS relocalize to the nucleoli. Ultraviolet-irradiation and the topoisomerase inhibitor camptothecin can also induce this relocalization. There are two major functional domains in TLS: an N-terminal SYGQQS-repeat region and a C- terminal ribonucleotide-binding domain, containing three RGG repeat regions, a Zn-finger motif, and an RNA-recognition motif (RRM). An EGFP-tagged N-terminal fragment of TLS (amino acids 1-216) still relocalizes to the nucleoli following actinomycin D treatment, identifying this region as containing the necessary response signal. This N-terminal fragment encompasses two regions of SYGQQS repeats, and a deletion that removed the first of these SYGQQS-repeat regions (delta1- 91) retained the ability to relocalize following treatment with actinomycin D. A deletion construct that had both regions of SYGQQS repeat regions removed (delta1-193) did not relocalize to the nucleoli, identifying a critical signalling or interaction domain at amino acids 92-193. An internal deletion of this sequence, the second region of SYGQQS repeats (delta92-193), yielded a modified version of TLS that was still capable of relocalizing following transcriptional inhibition. Because the TLS(delta92-193) construct contained the first region of SYGQQS repeats, these results suggest that there is a redundancy of function between the two SYGQQS repeat regions. Therefore, both SYGQQS repeat regions can function independently to mediate the relocalization of TLS following transcriptional inhibition. This avenue of investigation is identifying and characterizing critical domains of TLS, which will help elucidate its wild-type function in the cellular response to stress. Ultimately, it will also help lead to potential mechanisms of action in genomic stability and the DNA damage response.

69 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Rafael Casellas Abstract 14 Effect of DNA double-stranded break repair on gene expression

Marika Orlov1, Elizabeth Crouch1, Stanley Gorsky2, Andre Nussenzweig3, Tom Misteli2, Robert D. Phair4, Michael Kruhlak3 and Rafael Casellas1

1Genomic Integrity, NIAMS, National Institutes of Health, Bethesda, U.S.A., 2Cell Biology of Genomes, NCI, National Institutes of Health, Bethesda, U.S.A., 3Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, U.S.A., 4Bioinformatics Services, Rockville, U.S.A.

DNA double-stranded breaks (DSBs) result in H2AX phosphorylation and recruitment of DNA repair factors into nuclear micro-domains (foci) at the sites of lesion. Interestingly, both foci formation and damage-induced chromatin remodeling are massive and extend megabases away from the actual DSB. The physiological significant of these higher order chromatin structures is unclear. We have investigated the effects of DSB repair and foci formation on RNA polymerase assembly and elongation. By means of photobleaching techniques and molecular scissors we show that DNA lesions initiate a time dependant inhibition of transcriptional elongation in the vicinity of the DSB. With the use of computational modeling of imaging data, we show that recruitment and incorporation frequencies of polymerase components are affected by DNA damage. Both transcriptional suppression and defects in polymerase assembly are not the direct result of DNA damage per se but they are instead orchestrated by ATM activity. We propose that micro-domains and chromatin remodeling shut down transcriptional activity adjacent to lesion sites to ensure proper repair of DNA ends.

70 Abstracts - Poster

Beverley Chilton Abstract 15 It Takes Two to Tango: autorepression by RUSH-1α/β

Aveline Hewetson and Beverly S. Chilton

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street - MS 6540, Lubbock, TX 79430, U.S.A.

RUSH/SMARCA3 is regulated by a steroid-dependent alternative splicing mechanism. RUSH-1α is the progesterone-dependent isoform. RUSH-1β is the truncated, estrogen- dependent isoform. Although the proteins have identical DNA-binding domains, little is known about their structure-function relationships. Progesterone-dependent transcriptional activation of the RUSH/SMARCA3 gene is mediated by a bipartite PRE half-site/overlapping Y-box combination in the proximal promoter (-162/+90). Estrogen represses the same promoter construct via two Sp3/Sp1 sites. Gel supershift assays confirmed that Sp3/Sp1 proteins bind both sites at a15:1 ratio. ERα/Sp1 interactions were eliminated with gel shift/supershift assays. In transient transfection assays, progesterone (R5020) induced transcriptional activation of the promoter in the presence (p < 0.05) and absence (p < 0.001) of the Sp3/Sp1 sites. Dual ablation of these sites resulted in a dramatic increase (p < 0.001) in promoter activity confirming Sp3 repression of progesterone-dependent transcription. Estrogen-treatment produced a dramatic decrease in the amount of RUSH message with a concomitant shift to RUSH-1β isoform expression. In gel shift assays with nuclear extract from estrous animals, only Sp3 binds the distal site, and neither Sp3 nor Sp1 binds the proximal site.ever, repression also involves an authentic (ChIP) RUSH/SMARCA3 binding site (-616/-611) in the 5’-UTR. Gel shift/supershift assays with nuclear extract from progesterone-treated animals and a-specific antibodies confirmed exclusive, site-specific binding of RUSH-1α when the gene is transcriptionally active. Transient transfection assays with mutant constructs showed that elimination of the binding site increased (p < 0.001) transcriptional activation.m estrous animals and b-specific antibodies confirmed exclusive binding of RUSH-1β to the same site when the promoter is transcriptionally quiescent. alternative splicing and isoform specific autorepression of the RUSH/SMARCA3 gene.

71 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Sung Hee Baek Abstract 16 A chromatin remodeling complex in regulation of a metastasis suppressor gene

Hee June Choi, Jung Hwa Kim, Bogyou Kim, Ji Min Lee, Ik Soo Kim and Sung Hee Baek

Seoul National University 216, 20 Sillim-Dong, Kwanak-Ku, Seoul 151-742, South Korea

Defining the molecular strategies that integrate diverse signaling pathways in expression of specific gene programs critical in homeostasis and disease remains a central issue in biology. In cancer biology, this is particularly pertinent, because down-regulation of tumor metastasis suppressor genes is a common occurrence, and the underlying molecular mechanisms are not well established. Recently, we reported that the down-regulation of a metastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions of beta-catenin, along with a reptin chromatin remodeling complex (Kim et al., Nature 434, 921-926, 2005). This inhibitory function of beta-catenin/reptin chromatin remodeling complex requires both increased levels of beta-catenin expression and recruitment of histone deacetylase activity. The coordinated actions of beta-catenin/reptin components that mediate the repressive state serve to antagonize a Tip60 coactivator complex, required for activation, with the balance of these opposing complexes controlling the expression of KAI1 and metastatic potential. Here we found an intriguing signal recognition code of which signaling factors cause reptin chromatin remodeling complex to confer repressive function to control expression of KAI1 and its metastatic potential. Biochemical purification of a reptin-containing complex revealed that it unexpectedly contains SUMO processing enzymes. Reptin is a direct target of SUMOylation and lack of SUMOylation exerts to antagonize the repressive function of reptin. This work provides a novel insight for linking SUMO modification of chromatin remodeling complex to cancer metastasis. Mutations in the IKBKAP gene, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked with transcriptional elongation and histone acetylation. Here, we used RNA interference (RNAi) to identify Elongator target genes. Strikingly, several of these genes encode proteins implicated in cell motility. Indeed, characterization of IKAP RNAi cells and fibroblasts derived from FD patients uncovered defects in this cellular function. Whereas Elongator is recruited to both target and non-target genes, only target genes display histone H3 hypo-acetylation and progressively lower RNAPII density through the coding region. Decreased target gene transcription in FD fibroblasts can be overcome by treatment with histone deacetylase inhibitor. Taken together, our results indicate that defects in Elongator function result in reduced transcriptional elongation of several genes. The resulting cell motility deficiencies may underlie the neuropathology of FD patients. Our data indicate that FD should be classified as a transcription disease.

72 Abstracts - Poster

Pierre Close Abstract 17 Elongator depletion causes defective transcript elongation of genes that regulate cell motility

Pierre Close1,2, Nicola Hawkes2, Isabelle Cornez1, Catherine Creppe1, Charles A. Lambert3, Bernard Rogister4, Ulrich Siebenlist5, Marie-Paule Merville1, SU.S.A.n A. Slaugenhaupt6, Vincent Bours1, Jesper Q. Svejstrup2 and Alain Chariot1.

1Laboratory of Medical Chemistry and Human Genetics, Center for Biomedical Integrative Genoproteomics, University of Liege, Liege, Belgium., 2Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, United Kingdom., 3Laboratory of Connective Tissues Biology, Center for Biomedical Integrative Genoproteomics, University of Liege, Liege, Belgium., 4Center for Cellular and Molecular Neurobiology, University of Liege, Liege, Belgium., 5Laboratory of Immunoregulation, NIAID, NIH, Bethesda, MD, U.S.A.., 6Center for Human Genetics Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, U.S.A.

Mutations in the IKBKAP gene, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked with transcriptional elongation and histone acetylation. Here, we used RNA interference (RNAi) to identify Elongator target genes. Strikingly, several of these genes encode proteins implicated in cell motility. Indeed, characterization of IKAP RNAi cells and fibroblasts derived from FD patients uncovered defects in this cellular function. Whereas Elongator is recruited to both target and non-target genes, only target genes display histone H3 hypo-acetylation and progressively lower RNAPII density through the coding region. Decreased target gene transcription in FD fibroblasts can be overcome by treatment with histone deacetylase inhibitor. Taken together, our results indicate that defects in Elongator function result in reduced transcriptional elongation of several genes. The resulting cell motility deficiencies may underlie the neuropathology of FD patients. Our data indicate that FD should be classified as a transcription disease.

73 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Megan Cole Abstract 18 Genome-wide map of nucleosome acetylation and methylation in yeast

Dmitry K. Pokholok1, Christopher T. Harbison1, Stuart Levine1, Megan Cole1,2, Nancy M. Hannett1, Tong Ihn Lee1, George W. Bell1, Kimberly Walker1, P. Alex Rolfe3, Elizabeth Herbolsheimer1, Julia Zeitlinger1, Fran Lewitter1, David K. Gifford1,3 and Richard A. Young1,2

1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A., 3MIT Computer Science and Artificial Intelligence, Laboratory,Cambridge, Massachusetts 02139, U.S.A.

Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.

74 Abstracts - Poster

Jean-Francois Couture Abstract 19 Structural insights into lysine multiple methylation by SET domain methyltransferases

Jean-Francois Couture1, Lynnette M. A. Dirk2, Joseph S. Brunzelle3, Robert L. Houtz2 and Raymond C. Trievel1

1University of Michigan, 4412 Medical Science I, 1301 Catherine Street, Ann Arbor, Michigan 48109, U.S.A, 2Department of Horticulture, University of Kentucky, 401D Plant Science Building, Lexington KY 40546-0312, 3Life Sciences CAT, Dept of Mol. Pharm. and Biol. Chem., Feinberg School of Medicine, Northwestern University, Boston MA, U.S.A.

Site-specific protein lysine methylation has emerged as an important covalent modification that regulates numerous genomic functions, including transcription, mitosis, and DNA damage checkpoints in the cell cycle. This modification is catalyzed by a family of protein lysine methyltransferases (PKMTs), the majority of which possess a conserved catalytic SET domain. Sequence and structure variations within the SET domain enable PKMTs to recognize and methylate distinct substrates, including histones, transcription factors, and other nuclear proteins. In addition to their lysine selectivity, these enzymes can catalyze mono-, di-, or trimethylation of the lysine epsilon-amino group. This property is referred to as their product specificity and is biologically important because many methyllysine-binding factors can discriminate among different degrees of methylation. Despite its significance, the mechanism by which SET domain PKMTs catalyze lysine multiple methylation has remained poorly understood. To elucidate this mechanism, we have determined the crystal structures of the Y334F mutant of human SET8 (hSET8), a histone H4 Lys-20-specific methyltransferase, in complex with histone H4 peptides bearing unmodified, monomethylated, and dimethylated Lys-20. The Y334F active site mutation alters the product specificity of hSET8 from a mono- to a dimethylase, but does not otherwise affect histone H4 binding or methylation compared with the native enzyme. Collectively, the structures reveal that the Y334F substitution alters water-mediated hydrogen bonding to the Lys-20 epsilon-amino group, permitting it to adopt alternative conformations that facilitate dimethylation. Moreover, biochemical characterization of the mutant reveals that it binds to the unmodified and monomethylated Lys-20 histone H4 peptides with equivalent affinity, but cannot methylate the monomethylated peptide. These observations suggest that the hSET8 Y334F mutant dimethylates Lys-20 via a processive mechanism in which histone H4 remains bound within the active during the catalytic cycle. Taken together, these studies furnish insights into the structural determinants of lysine multiple methylation by SET domain PKMTs.

75 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Qi Dai Abstract 20 Snail-mediated repression in Drosophila requires Ebi and histone deacetylation

Qi Dai, Mattias Bergman and Mattias Mannervik

Department of Developmental Biology, Wenner-Gren Institute, Arrhenius Laboratories E3, Stockholm University, Stockholm, Sweden

Drosophila Ebi and its mamalian 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. We found that Ebi specifically affects the function of the Snail repressor in Drosophila embryos, since in ebi germline clone mutants 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 the CtBP co-repressor. 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. 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.

76 Abstracts - Poster

Ana D’Alessio Abstract 21 The kinetics of chromatin-driven active DNA demethylation in living cells.

Ana C. D’Alessio, Ian C.G. Weaver and Moshe Szyf.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.

Gene expression profiles of vertebrate cells are regulated through the epigenome, which consists of chromatin, and its modifications, and the pattern of DNA methylation at CpG dinucleotides. Active genes are associated with acetylated histones and hypomethylated DNA, whereas inactive genes are associated with hypoacetylated histones and hypermethylated DNA. However, the relationship between these two components of the epigenome is still unclear. The objective of this study is to define the temporal relationship between chromatin and transcriptional machinery events leading to active DNA demethylation. DNA demethylation of an endogenous gene in a replicating cell could either be an active or a passive replication-dependent process. We took advantage of a previously described transient transfection, replication-independent, active demethylation assay in living cells. In this system, pharmacological induction of histone acetylation by inhibition of histone deacetylases with trichostatin A (TSA) triggers active DNA demethylation. We used ChIP assays and bisulfite mapping to study the temporal and caU.S.A.l relationship between chromatin and active DNA demethylation. We show that histone acetylation is followed by RNA Pol II (Pol II) binding to the methylated promoter. Following interaction of Pol II, the promoter becomes demethylated. Active demethylation of this promoter is dependent on transcription, as demethylation is blocked through the addition of Actinomycin D, an inhibitor of Pol II. DNA demethylation is later followed by appearance of trimethylated lysine 4-H3, which is characteristic of elongating forms of Pol II. In agreement with this model, only demethylated DNA is associated with trimethylated lysine 4-H3. To determine whether the same mechanism applies to endogenous genes, we performed an expression micro-array analysis on TSA treated cells to identify endogenous genes that were induced by histone acetylation. We found that G antigen family (GAGE) could be demethylated in response to TSA. More importantly, the same order of events as found with the exogenous gene, were associated with GAGE. This work provides a mechanism for how genes silenced through histone hypoacetylation and DNA methylation, such as oncogenes, may become activated in tumourigenesis

Supported by grant from the National Institute of Cancer of Canada 2005 to MS.

77 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jan-Hermen Dannenberg Abstract 22 Role of mSin3A in development, regulating transcriptional networks and tumorigenesis.

Jan-Hermen Dannenberg, Gregory David2, Sheng Zhong1, Jaco van der Torre, Wing H. Wong1 and Ronald A. DePinho

Department of Medical Oncology, Dana Farber Cancer Institute; Departments of Medicine and Genetics, Harvard Medical School; Boston, MA, U.S.A. 1Department of Statistics, Stanford University, Stanford, CA, U.S.A., 2Present address: Department of Pharmacology, New York University, School of Medicine, New York, NY, U.S.A.

mSin3A is a core component of a large multi-protein co-repressor complex (mSin3/HDAC) with associated histone deacetylase enzymatic activity. Physical interactions of mSin3A with many sequence-specific transcription factors have implicated a role for the mSin3A co- repressor complex in diverse signaling pathways and linked biological processes. To dissect the complex nature of mSin3A’s actions, we monitored the impact of conditional mSin3A deletion on the developmental, cell biological and transcriptional levels. mSin3A was shown to play an essential role in early embryonic development and in the proliferation and survival of primary, immortalized and transformed cells. Although we established a role for mSin3A/HDAC in p53 deacetylation and activation, genetic deletion of p53 was not sufficient to attenuate the mSin3A null cell lethal phenotype thereby pointing to mSin3A’s broad biological activities beyond regulation of the p53 pathway. Correspondingly, time- course gene expression profiling following mSin3A deletion revealed deregulation of genes involved in cell cycle regulation, DNA replication, DNA repair, apoptosis, chromatin modifications and mitochondrial metabolism. In silico promoter analysis of the mSin3A transcriptome revealed significant enrichment for Myc-Mad, E2F and p53 cis-regulatory elements in promoters of up-regulated genes following mSin3A depletion. Significantly, using bioinformatics combined with ChIP analyses on verified mSin3A target genes we identified specific cis-regulatory elements binding the transcriptional activator Stat and the nucleosome remodeling factor Falz, thereby expanding further the mSin3A network of regulatory factors. Together, these integrated genetic, biochemical and computational studies demonstrate the involvement of mSin3A in the regulation of diverse pathways governing many aspects of normal and neoplastic growth and survival. Studies addressing a role for mSin3A in tumorigenesis showed that mSin3A-heterozygosity results in an increased sensitivity towards DNA-damaging agents and a pronounced resistance to DMBA-induced lymphomagenesis. Mechanisms underlying this novel role for mSin3A in lymphomagenesis will be discussed.

78 Abstracts - Poster

Gregory David Abstract 23 Functional specialization of the mSin3 complex in development and oncogenesis

Gregory David1,2, Natalie Simpson1, Mei Finnerty1, Jan-Hermen Dannenberg2 and Ronald A. DePinho2

1Department of Pharmacology, NYU School of Medicine, New York, 2Departments of Adult Oncology, Dana Farber Cancer Institute, Boston

The highly conserved Sin3-histone deacetylase complex, a prominent co-repressor complex, is characterized in mammalian cells by the presence of the mSin3A protein and the closely related mSin3B protein. Aberrant recruitment of the mSin3 complex alters chromatin structure and is involved in the pathogenesis of several human tumors. We used genetic inactivation in the mouse to elucidate the molecular pathways by which the mSin3 complex affects development and oncogenesis. Specifically, we have generated conditional knock-out mouse strains for mSin3A, mSin3B and the essential component of the mSin3 complex, mSds3, and we have previously shown that an integral mSin3 complex is required for pericentric heterochromatin structure and proper chromosomal segregation. In addition, we have observed that mSin3A and mSin3B are both essential during embryogenesis but their inactivation results in dramatically different developmental and cellular phenotypes. We used biochemical and cellular approaches to dissect the molecular pathways engaged by each component of the mSin3 complex and uncovered specific roles for each of the mSin3 proteins. Although mSin3A engages numerous transcriptional pathways necessary for cell survival and proliferation, mSin3B is dispensable for cell viability. However, organismal and cellular phenotypes resulting from mSin3B inactivation strongly suggest a specific contribution to the E2F repressors pathway. Finally, the role of the mSin3 complex in tumor suppression has been investigated in vivo and will be discussed. Altogether, these studies underline the role of the mSin3 complex in cellular proliferation and differentiation and reveal an unsuspected functional specialization for each one of the mSin3 proteins in higher eukaryotes.

79 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

James Davie Abstract 24 Recruitment of phosphorylated HDAC2 to promoters

Jian-Min Sun, Hou Yu Chen and James R. Davie

University of Manitoba, NA, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba (Province), R3E 0W3, Canada

Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, it is found in the Sin3 and NuRD multi-protein complexes, which are recruited to promoters by DNA binding proteins. In this study, we show that HDAC2 in human breast cancer cells is mostly not phosphorylated. However, a minor population of HDAC2, preferentially cross-linked to DNA by cisplatin and formaldehyde, is mono-, di- or tri-phosphorylated. Moreover, the formation of Sin3 and NuRD complexes, as well as their recruitment to promoters by factors like p53, Rb, YY1, p50, p65, Sp1 and Sp3 are dependent on HDAC2 phosphorylation. The majority of the HDAC2 population, in an unmodified form, is bound to chromatin but is not cross- linked to DNA by formaldehyde and therefore would not be detected in standard chromatin immunoprecipitation (ChIP) assays. This observation raises a cautionary note in the use of the standard ChIP assay that the efficiency of formaldehyde cross-linking of chromosomal proteins to DNA can vary greatly.

80 Abstracts - Poster

Cecile de la Cruz Abstract 25 Developmental regulation of Suz12 localization de la Cruz C.C., Fang J., Plath K., Worringer K.A., Nusinow D.A., Zhang Y. and Panning B.

Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143, U.S.A.

Chromatin modifications are among the epigenetic alterations essential for genetic reprogramming during development. The Polycomb group (PcG) gene family mediates chromatin modifications that contribute to developmentally regulated transcriptional silencing. Trimethylation of histone H3 on lysine 27, mediated by a PcG protein complex consisting of Eed, Ezh2, and Suz12, is integral in differentiation, stem cell self-renewal, and tumorigenesis. Eed and Ezh2 are also implicated in the developmentally regulated silencing of the inactive X chromosome, as they are transiently enriched on the inactive X chromosome when X chromosome silencing is initiated. Here we analyze the dynamic localization of Suz12 during cellular differentiation and X-inactivation. Though Suz12 is a requisite member of the Eed/Ezh2 complexes, we found that Suz12 exhibits a notable difference from Ezh2 and Eed: while Ezh2 and Eed levels decrease during stem cell differentiation, Suz12 levels remain constant. Despite the differential regulation in abundance of Suz12 and Eed/Ezh2, Suz12 is also transiently enriched on the Xi during early stages of X-inactivation, and this accumulation is Xist RNA dependent. These results suggest that Suz12 may have a function that is not mediated by its association with Eed and Ezh2, and that this additional function is not involved in the regulation of X-inactivation.

81 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Stephan Diekmann Abstract 26 Functional complementation of human centromere protein A (CENP-A) by Cse4p

Gerhard Wieland, Sandra Orthaus, Sabine Ohndorf, Stephan Diekmann and Peter Hemmerich

IMB1, Westfluegel, Beutenbergstr 11, Jena, Thueringen 07745, Germany

We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference was used to block the synthesis of proteins CENP-A and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for their ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically recruited to kinetochores and was able to complement the RNAi-induced cell cycle arrest in CENP-A depleted human cells. Thus, Cse4p can structurally and functionally substitute CENP-A cross-species. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized at kinetochores in mitosis but Bik1p did not rescue the CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool to study the function and evolutionary conservation of multiprotein complexes from yeast to man.

82 Abstracts - Poster

Ivana Djuretic Abstract 27 Silencing of a mammalian gene, Interleukin-4 in T helper lymphocytes

Ivana Djuretic1,2, K. Mark Ansel1,2 and Anjana Rao1,2

1Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. , 2CBR Institute for Biomedical Research, Boston, Massachusetts 02115, U.S.A.

T helper cell differentiation has been an excellent model for studying the regulation of mammalian gene expression by changes in chromatin structure. The murine Interleukin- 4/5/13 (T helper 2) locus, in particular, has been a subject of many studies which resulted in identifying various elements that regulate its transcription including enhancers, silencers and a locus control region. In this study we focus on identifying the role of two regulatory elements, HS IV and HSS3, in silencing the Il4 gene in T helper 1 cells. By targeted deletion we determined that HS IV functions as a silencer for Il4 while the function of HSS3 is still unclear. Deletion of both HSS3 and HS IV partially rescues the HS IV-deficient phenotype, suggesting a possible interaction between these two genetic elements. The predominant histone modification at these two sites is H3K27 methylation, indicating that Polycomb proteins might be involved in silencing of Il4. Interestingly, a peak of H3K4 dimethylation in addition to H3K27 trimethylation is also found at HS IV. A combination of these two modifications has not been previously observed. We have also identified two transcription factors, T-bet (T box expressed in T cells) and Runx3, which bind to HS IV. In addition intergenic transcripts can be detected in T helper 2 cells, which express Il4, but not T helper 1 cells, which normally silence it. The final goal of this study will be to determine the relationship between intergenic transcription at the Il4/5/13 locus, histone modifications at HS IV and HSS3, and transcription factors that bind to them, and how they cooperate to regulate the expression of Th2 cytokines.

83 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Tom Donndelinger Abstract 28 Asymmetric eukaryotic cell division and cellular dimorphism

Tom Donndelinger and Elizabeth Oldenkamp

Mercy Medical Center, 1512 12th Ave Rd., Nampa 3686, U.S.A.

Chromatin asymmetry at cell division is strictly conserved in eukaryotic cells and persists in the nuclei of daughter cells. With our greatly improved tissue detail, mitotic asymmetry becomes unavoidably apparent. In over one hundred years of formalin-fixed paraffin embedding tissue processing there have been no significant improvements. Our re- engineered tissue processing method, which preserves structural and dedicated water molecules while cutting at one micron, gives ten to one hundred times better detail than traditional tissue processing methods. Images are referenced to published electron microscopic data at 1000X. Asymmetry in post-mitotic chromatin patterns in daughter cells has been identified and subjected to an extensive phylogenetic trace. The results strongly suggest that all eukaryotic cell division produces some degree of asymmetric structural features of chromatin in the daughter cells. These biologic features of the “0” and “1” cells always occur in cell division point out the possibility of distinct subsetting of overlapping but non-identical proteomics. Cytoplasmic and chromatin differences have been traced in all tissues and all organs of a large number of metazoans and plants with no exceptions observed. The asymmetry continues in cancerous tissues and some virus infected cells. This new detail implies a greater degree of complexity of organisms than was previously understood. Cells as tissue components are not heterogeneous in their morphology; rather, they are dimorphic. Additional studies forcing cells into two-dimensional space from three dimensions, strongly suggests these post-mitotic pairs are preferentially physically entangled. The format of entangled, binucleated protozoans and diatoms has apparently been conserved throughout evolution. We are progressing with proof of concept with high resolution flow cytometry with antibodies to phosphorus biased, strictly conserved histone features in metaphase chromosome sets. Single cell systems biology and communication may be too simple to understand cell life. Basic understandings of cell division, communication, and disease may have to be altered in light of this new data.

84 Abstracts - Poster

Yannick Doyon Abstract 29 ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation

Yannick Doyon1, Christelle Cayrou1, Mukta Ulla2, Anne-Julie Landry1, Valérie Côté1, William S. Lane3, Song Tan4, Xiang-Jiao Yang2 and Jacques Côté1

1Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City Qc, Canada G1R 2J6, 2Molecular Oncology Group, McGill University Health Center, Montreal Qc, Canada H3A 1A1, 3Harvard Microchemistry Facility, Harvard University, Cambridge MA 02138, 4Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park PA 16802, U.S.A.

Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis and DNA repair as important cofactors of p53. In yeast, the ING paralogs are key to the catalysis of chromatin modification by specific HAT or HDAC complexes. In human, ING1 and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes, respectively. We now report the purification of the three remaining members of the human ING family. While ING2 is in a HDAC complex similar to ING1, ING4 associates with the HBO1 acetyltransferase required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs and interacts with the MCM proteins, supporting a role for MYST-ING complexes in DNA replication. As the ING, HBO1 and MOZ/MORF proteins contribute to oncogenic transformation, the multi subunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development.

85 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Katherine Dunn Abstract 30 Independent phosphorylation events on the amino-terminus of histone H3 associated with transcription

Katherine L. Dunn and James R. Davie

Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue Winnipeg, Manitoba R3E 0V9, Canada.

Approximately 30% of human cancers contain a mutation to one of the ras oncogenes. Stimulation of the ras-mitogen activated protein kinase (ras-MAPK) pathway leads to rapid phosphorylation of histone H3 at serines 10 and 28 and expression of immediate-early genes. H3 phosphoacetylated at serine 10 lysine 14 has been found associated with immediate-early genes following stimulation of the Ras-MAPK pathway. Ras-transformed cells contain higher levels of H3 phosphorylated at serine 10 and display a less condensed chromatin structure. We hypothesize that phosphorylation of H3 at serine 28 contributes to chromatin remodeling and immediate early gene activity. Subsequent to ras- MAPK pathway stimulation H3 phosphorylated on serine 10 or 28 was excluded from regions of highly condensed chromatin and was present in increased amounts in ras-transformed cells. H3 phosphorylated at serine 10 or at serine 28 was dynamically acetylated, however H3 tails phosphorylated at serine 28 had a higher steady state level of acetylation. When visualized with indirect immunofluorescence, most foci of H3 phosphorylated at serine 28 do not colocalize with foci of H3 phosphorylated at serine 10 or foci of H3 phosphoacetylated at serine 10 lysine 14, indicating that these two phosphorylation events are targeted separately. Furthermore, foci of H3 phosphorylated at serine 28 associate with foci of phosphorylated RNA polymerase II, as do foci of H3 phosphorylated at serine 10, suggesting that these two modifications promote transcription independently.

This work was supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society, a Canada Research Chair to J.R.D., and a K.M. Hunter/Canadian Institutes of Health Research doctoral research award to K.L.D.

86 Abstracts - Poster

Christine English Abstract 31 ASF1 binds to a heterodimer of histones H3-H4: a two step mechanism for the assembly of H3-H4

Christine M. English1, Jessica K. Tyler1, Brian Tripet1, Nasib K. Maluf2 and Mair E.A. Churchill3

1The Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center at Fitzsimons, Aurora CO 80045, 2Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver CO 80262., 3Department of Pharmacology, University of Colorado Health Sciences Center at Fitzsimons, Aurora CO 80045, U.S.A.

The first step in the formation of the nucleosome is commonly assumed to be the deposition of a histone H3-H4 heterotetramer onto DNA. Anti-silencing function 1 (ASF1) is a major histone H3-H4 chaperone that deposits histones H3 and H4 onto DNA. Towards understanding the mechanism of deposition of histones H3 and H4 onto DNA, we have determined the stoichiometry of the Asf1-H3-H4 complex. We have established that a single molecule of Asf1 binds to a heterodimer of H3-H4 using gel filtration, amino acid, reversed- phase chromatography and analytical ultracentrifugation analyses. We propose that Asf1 blocks H3-H4 heterotetramer formation by occluding the H3-H3 dimerization interface. This work suggests that the formation of the H3-H4 heterotetramer on DNA is likely to occur by the sequential or concerted deposition of two H3-H4 heterodimers.

87 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Alex Erkine Abstract 32 Nucleosome displacement at promoters of yeast heat shock genes is proportional to the degree of transient histone H3 acetylation.

T. Y. Erkina and A. M. Erkine

USD School of Medicine, Lee Medical Bldg., 414 E. Clark Street, Vermillion, SD 57069, U.S.A.

It is often not clear if histones of the promoter nucleosomes are just post-translationally modified or outright removed from promoters during induction of transcription. Using chromatin immuno-precipitation in combination with real-time PCR, we studied the kinetics of chromatin changes at promoters of HSP12, SSA4, HSP82 genes. Our results indicate that the extent of nucleosome displacement differs for individual heat shock gene promoters. Importantly, the HSP12 promoter with a higher degree of nucleosome displacement showed significantly less visible displacement of acetylated H3 than the SSA4 promoter with lower degrees of nucleosome displacement. For HSP12 promoter, the level of acetylated H3 temporarily increased prior to the nucleosome departure. For all promoters tested, the higher level of nucleosome displacement correlated with the transiently increased level of histone H3 acetylation. To test whether this rule is dictated by the promoter elements or by the HSF as a general activator of these genes, we compared individual promoters in strains expressing different truncated versions of HSF. Deletion of either of the two activating regions in HSF led to diminished nucleosome displacement. Deletion of both regions simultaneously severely decreased nucleosome displacement for all the promoters tested. In accordance with the above rule, the histone acetylation level at individual promoters followed the degree of nucleosome displacement showing the dependence of these processes on HSF as a general heat shock gene regulator, and not on individual promoter elements.

88 Abstracts - Poster

Laure Escoubet-Lozach Abstract 33 Application of genome-wide location analysis to study chromatin modifications in a model of signal-dependent gene regulation

Laure Escoubet-Lozach1, Jean Lozach1, Christopher W. Benner1, Roman Sasik1, Ivan Garcia-Bassets2, Rosa Luna1, Donna Reichardt1, Jana Collier1, Colleen Eckhardt3, Kristin Stubben3, Jennifer Lapira3, Gary Hardiman3, B. Ren4, Michael G. Rosenfeld2 and Christopher K. Glass1

1Cellular and Molecular Medicine, School of Medicine, 2Howard Hughes Medical Institute, School of Medicine, 3BioMedical Genomics Microarray Facility, Department of Medicine, 4Howard Hughes Medical Institute, Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A..

Macrophages are phagocytic cells that play essential roles in native and acquired immunity. Upon stimulation with an inflammatory mediator, such as LPS, macrophages regulate several hundred genes that contribute to an anti-microbial response. In this model of signal- dependent gene regulation, we investigate the chromatin modifications occurring in response to LPS and study to which extent these modifications are linked to gene regulation. This study has been performed at a genome-wide level using the ‘ChIP-on-chip’ technology associating Chromatin Immunoprecipitation (ChIP) and promoter microarray (chip). For this purpose, we developed a murine promoter array containing approximately 34,000 features (Mu34K) and representing the proximal promoters of 26,921 genes and tiled regions of 48 genes. Here, we present a comprehensive set of data comparing the H3K9 and H3K18 acetylation marks as well as the H3K4, H4R3, H3R17, H3K9 and H3K27 methylation marks in untreated and LPS-treated macrophages, we analyse their association and their interdependence, their involvement in gene regulation and explore mechanisms by which a signal such as LPS leads to such chromatin modifications.

Grants : P50 HL56989, P30 DK063491, American Heart Association 0325103Y

89 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Yuhong Fan Abstract 34 A new link between linker histone H1 and DNA methylation

Yuhong Fan1, Tatiana Nikitina2, Riddhi Bhattacharyya1, Jie Zhao1, Eric Bouhassira1, Chris Woodcock2 and Arthur Skoultchi1

1Department of Cell Biology, Albert Einstein College of Medicine, 402 Chanin, 1300 Morris Park Ave., Bronx, NY 10461, U.S.A., 2Department of Biology, University of Massachusetts, Amherst, Massachusetts 01003, U.S.A.

H1 linker histones are thought to play a key role in the folding of chromatin into higher order structures. Mice contain eight H1 subtypes that differ in expression during development. Previous studies in our laboratory showed that mice develop normally when any one of six different H1 genes is inactivated homozygously, whereas mice lacking three H1 subtypes, H1c, H1d and H1e, generated by three rounds of gene inactivation in ES cells, die by mid-gestation with a broad range of defects. To further understand the role of H1 in chromatin structure and gene expression, we derived ES cells (embryonic stem cells) from the H1cH1dH1e triple null blastocysts and wild-type littermates. Using these cells, we showed that marked reduction in H1 amount leads to global changes in chromatin structure in vivo, including decreased nucleosome spacing and reduced chromatin compaction. To further study the mechanisms by which H1 affects gene expression, gene expression profiles were examined by Affymetrix microarray analysis. Despite the global changes in chromatin structure in these cells, expression of only a small number of genes is affected. Interestingly, a significant proportion of the affected genes are normally regulated by DNA methylation. Bisulfite sequencing analysis of DNA showed that the level of DNA methylation at certain loci is affected in the triple H1 null ES cells. In contrast, the global DNA methylation status is unchanged. These results suggest that regulation of gene expression by H1 is mediated, at least in part, through epigenetic regulation of DNA methylation.

90 Abstracts - Poster

Barna Fodor Abstract 35 The full complement of Su(var) gene function in Drosophila

Barna D. Fodor, Masato Yonezawa, Amit Sharma, Sandro Lein, Gunter Reuter and Thomas Jenuwein

Research Institute of Molecular Pathology (IMP), 7 Dr. Bohrgasse, Vienna, Landstrasse A- 1030, Austria

The genome of eukaryotes 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. In this study we applied a single nucleotide polymorphism (SNP) mapping approach to identify the previously isolated, unknown Su(var) mutations in Drosophila (~60) in collaboration with the lab of Gunter Reuter. First, Su(var) mutations were mapped between P-elements with molecularly characterized insertion sites by recombination mapping. Later the chromosomes from these recombinants were analyzed by SNP markers. With this approach a genomic region of ~ 50 kb that harbors the Su(var) locus can easily be designated. To date, we conducted linkage analysis on 24 Su(var)s and further mapped 14 by SNP mapping. We were able to identify novel Su(var)s indicating the involvement of the sumoylation pathway in heterochromatin formation. Other mutations have been narrowed down to less then hundred kb segments. In these regions several interesting candidates have been identified. These results prove the utility of our mapping strategy. Once the corresponding genes are identified, study of their mammalian homologs will follow. The project will contribute to the full definition of heterochromatin.

91 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Alexey Fomenkov Abstract 36 Role of p63 in development disorders

Huang Y.P, Kim Y., Fomenkov A. and Ratovitski E.

Johns Hopkins University School of Medicine, Richard Ross Research Building, Room 733 Rutland Avenue, Baltimore, MD 21205, U.S.A.

The stratified epithelia of the skin and oral mucosa are the most protective types of keratinized or cornified epithelia. To maintain this barrier, epithelial tissues undergo constant renewal and repair. A novel gene, p63, was found to be a key regulator of squamous epithelia differentiation/stratification and stem cell renewal during embryonic development. P63 null mice showed crucial defects in skin development, including lack of stratification and structures dependent upon epidermal-mesenchymal interactions, such as hair follicles, teeth, mammary gland, and prostate. However, the molecular mechanisms underlying a critical role for p63 in stratification/development are still poorly understood. By using proteomic, genomic and cellular modeling approaches, our research team has made important contributions into p63 field. p63 physically and functionally associated with members of RNA transcription/splicing machinery (e.g. ABBP1, SRA4, and C-terminal domain of RNA polymerase II). This novel finding supported the idea that p63 modulates specific RNA splicing of the fibroblast growth factor receptor-2 towards epithelial-specific isofom, FGFR2-K-SAM (KGFR) and thus determine epithelial cell fate. p63 function is regulated by a specific proteasome-dependent degradation mechanism through association with RACK1 and stratifin. This process is required phosphorylation and sumoylation of p63 and contributes into a molecular program leading to normal skin and bone differentiation. The environmental stimuli (e.g. ultraviolet, chemical agents, etc.) that induce DNA damage also enhanced p63 protein modifications leading to selective degradation of p63. As a transcriptional factor, p63 plays an important role in regulation of its downstream gene targets by both direct binding to the promoter area and by protein interaction with other components of RNA processing complex. Our results demonstrated that p63 regulates transcription of numerous genes specifically involved in cell-cell adhesion (e.g. K5, K14, plakoglobin, enviplakin, BPAG-1, and ¡V2, integrin alpha3beta1. These proteins were shown to be critical components of cytoskeleton and hemi-desmosomes that define skin stratification and mediate cell-cell junctions. These and other novel findings support the notion why p63 null mice failed to develop proper skin structures. Mutations associated with the p63 DNA-binding domain found in patients with ectodermal dysplasia, facial clefting, split hand/foot malformation, limb-mammary syndrome and acro- dermato-ungual-lacrimal-tooth syndrome and may directly affect the transcription properties of p63. While, a number of mutations associated with AEC syndrome (ankyloblepharon, ectodermal dysplasia and clefting, AEC) and Rapp-Hodgkin syndrome has been mapped inside the p63 sterile ƒÑ-motif. These mutations were suggested to inappropriately modulate the p63 protein-protein interactions mediated by the sterile alpha-motif.

92 Abstracts - Poster

Benjamin Freedman Abstract 37 Linker histone dynamics, structure, and function in interphase and mitotic egg extract chromosomes

Benjamin S. Freedman, Thomas J. Maresca and Rebecca Heald

University of California at Berkeley, Life Science Addition, Room #315, University of California at Berkeley, Berkeley, CA 94720-0001, U.S.A.

Classic electron microscopic and nuclease digestion experiments have established that linker histones bind to nucleosomal arrays and compact them into a thicker chromatin fiber. It is unclear how these experiments, which were assayed at the scale of chromatin under processed conditions, relate to the in vivo organization of entire chromosomes. We recently addressed this issue using physiologically active Xenopus egg extracts and sperm nuclei that progress through the cell cycle. We found that replicated, mitotic chromosomes assembled in extracts depleted of the embryonic linker histone H1M (or B4) had a distorted morphology and were about twofold longer than normal (Maresca T.J. et al., 2005). Thus, H1M helps compact whole chromosomes, which is consistent with the classical role for linker histones at the smaller scale of nucleosomal chromatin. Currently, we are using this roughly physiological, vertebrate system to investigate several aspects of linker histone function: (1) To extend our findings from mitotic to interphase chromosomes, we are using fluorescence microscopy to measure nuclear size, chromosome morphology, and chromosome length (by FISH) in nuclei assembled with or without H1M. (2) H1M must first be loaded onto DNA during interphase in order to compact mitotic chromosomes. To understand the molecular mechanism underlying this loading step, we are performing H1M immunoprecipitations to identify binding partners. We are also comparing the binding dynamics of H1M to chromosomes during interphase versus mitosis. (3) H1M has two major domains, a globular head and a highly basic, unstructured carboxyl tail. We are substituting these domains for the full-length protein to evaluate their separate contributions to chromosome morphology. We are also engineering phosphorylation-state H1M mutants, to investigate the long-standing mystery of why many linker histones are phosphorylated during mitosis.

93 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jennifer Gallagher Abstract 38 Exploring establishment of Sir1-dependent silencing using comparative genomics

Jennifer Gallagher, Lenny Teytelman, Joshua Babiarz and Jasper Rine

University of California, Berkeley, 16 Barker Hall #3202, Berkeley, CA 94720, U.S.A.

Chromosomal regions are silenced by altering the chromatin structure in those regions to form heterochromatin. Silencing in yeast are most evident at the cryptic mating type loci (HM), telomeres, and at eukaryotic genes inserted into rDNA array. Silenced chromatin can be stably inherited through many generations even in the absence of a protein, Sir1p, which functions in its establishment. In the presence of Sir1p, silencing at the HM loci is efficiently established to silence the cryptic mating loci. Recent studies have shown that silenced chromatin can rapidly evolve and the protein components can vary greatly from species to species. Saccharomyces cerevisiae contains one SIR1 gene, while another Saccharomyces species, S. bayanus, contain four orthologs of the Saccharomyces SIR1 gene, all of which are diverged somewhat from each other. We have that least three of the proteins encoded by these orthologs are localized to the HMR-E silencer in Saccharomyces bayanus and all are likely to function in silencing. We hypothesize that multiple Sir1 proteins from S. bayanus may represent a division of labor that is consolidated into one protein in S. cerevisiae. This hypothesis implies that the silencers themselves may have evolved more elaborate structures in S. bayanus. Differences in the HML and HMR silencers may explain why different Sir1 proteins to establish silencing at HML and HMR. In the recently identified Orc1 interaction domain of Sir1, six of eight amino acids essential for the Orc1 interaction are invariant in the closest ortholog of S. bayanus and two are similar (Hou, PNAS 2005). In contrast, the S. bayanus paralogs of the closest Sir1 ortholog all contain from two to four of the eight invariant residues in the Orc interaction domain. Based on the sequence alignments, the Sir1 paralogs could be recruited to the chromatin via a different DNA binding proteins.

94 Abstracts - Poster

Nicolas Gévry Abstract 39 Implication of histone variant H2A.Z in the regulation of the p53/p21CIP1/WAF1 pathway.

Nicolas Gévry and Luc Gaudreau

Departement de Biologie, Faculte des Sciences, Centre de Recherche sur les Mecanismes du Fonctionnement Cellulaire, Universite de Sherbrooke, Sherbrooke, Quebec, Canada.

In addition to post-translational modifications of histone tails and ATP-dependant chromatin remodelling, the incorporation of core histone variants into nucleosome plays a key role in governing gene expression. Histone variant H2A.Z has been shown to be important for the proper induction of several genes in yeast. In spite of the high homology between yeast and human, it remains to be determined if H2A.Z is important for mammalian gene transcription. Here, we show the implication of H2A.Z in the p53/p21CIP1/WAF1 pathway both regulating transcription of p21, and the senescence induction program. High-resolution ChIP analyses using quantitative PCR demonstrates preferential binding of H2A.Z in the region overlapping the p53 binding sites of p21. Moreover, this localization of H2A.Z surrounding the p53 binding sites is not present in p53-deficient cell lines. Down regulation of H2A.Z expression by shRNA technology led to premature senescence of untransformed human fibroblasts, including p21 induction, senescence-associated heterochromatic foci, and beta-gal staining. Collectively, our data suggests that the incorporation of the histone variant H2A.Z into chromatin inhibits p53-p21 transcription and the development of premature senescence.

95 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Anja Groth Abstract 40 The role of human Asf1 in histone metabolism during replicational stress

Groth A., Loyola A., Ray-Gallet D., Quivy J-P., Lukas J., Bartek J. and Almouzni G.

Institute Curie, UMR218, Pavillon Pasteur, 26 Rue d’Ulm, Paris, 75248 France

Maintenance of chromosomal integrity requires tight coordination of histone biosynthesis with DNA replication. We have recently shown that extracts from human cells exposed to replication inhibitors display an increased capacity to support replication-coupled chromatin assembly (Groth et al., 2005). This enhanced activity is dependent on the histone chaperones Asf1a and Asf1b (hAsf1), which can synergize with the chromatin assembly factor CAF-1 in repair- and replication-coupled chromatin assembly (Tyler et al., 1999; Mello et al., 2002). In cytosolic extracts from unperturbed S-phase cells, hAsf1 exists in equilibrium between an active histone-containing multi-chaperone complex and an inactive histone-free form. Interference with DNA replication results in accumulation of soluble S- phase histones and mobilization of the majority of hAsf1 into the active complex. Our data suggest that hAsf1 provides the cells with a buffering system for histone excess generated in response to stalled replication and explains how mammalian cells maintain a critical active histone pool available for deposition during recovery from replicational stresses. Moreover, our findings points to hAsf1 as a candidate sensor of histone availability. We are currently exploring such a potential regulatory function for hAsf1 by searching for novel interactors potentially facilitating integration of histone availability with DNA replication, checkpoint signaling, and histone biosynthesis.

96 Abstracts - Poster

Matthew Guenther Abstract 41 The active and Polycomb-repressed genome in human embryonic stem cells

Matthew G. Guenther1*, Tong Ihn Lee1*, Richard G. Jenner1*, Laurie A. Boyer1*, Brett Chevalier1*, Stuart S. Levine1*, Roshan M. Kumar1, Sarah E. Johnstone1,2, Heather L. Murray1, Jacob P. Zucker3, Bingbing Yuan1, George W. Bell1, Elizabeth Herbolsheimer1, Nancy M. Hannett1, Megan F. Cole1,2, Kaiming Sun1, Duncan T. Odom1, Thomas L. Volkert1, David P. Bartel1,2, Douglas A. Melton3’5, David K. Gifford1,5, Rudolf Jaenisch1,2 and Richard A. Young1,2,5

1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Insitute of Technology, Cambridge, Massachusetts 02139, U.S.A., 3Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, U.S.A., 4MIT CSAIL, 32 Vassar Street, Cambridge, Massachusetts 02139, U.S.A., 5Broad Institute of MIT and Harvard, One Kendall Square, Building 300, Cambridge, Massachusetts 02139, U.S.A. *These authors contributed equivalently to this work

The capacity of embryonic stem (ES) cells to self-renew and to give rise to multiple somatic lineages holds promise for human regenerative medicine. To gain insights into global gene regulation in ES cells, we determined the locations of RNA polymerase II and the Polycomb Repressive Complex-2 (PRC2) subunit Suz12 across the entire non-repeat portion of the human genome. This map of active and repressed portions of the genome identifies active promoters for one-third of human genes and reveals the targeted repression of key developmental regulators by PRC2. Our results produce a high-confidence transcriptome for ES cells and provide insights into how PRC2 contributes to regulation of stem cell pluripotency and self-renewal.

97 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Shweta Hakre Abstract 42 Growth factor mediated gene regulation by TFII-I

Shweta Hakre1,3 and Ananda L. Roy1,2,3,4

Departments of Pathology1 and Biochemistry2 and Programs in Immunology3 and Genetics4 Tufts University School of Medicine, Boston, MA 02111, U.S.A.

TFII-I is a signal induced multi-functional transcription factor that can regulate transcriptional activity of the growth-responsive c-fos gene. Although TFII-I has multiple spliced isoforms, in murine fibroblasts the delta (D) and beta (b) isoforms are predominantly expressed. We show here that these two isoforms have distinct subcellular localization and mutually exclusive transcription functions in the context of growth factor signaling in vivo. While TFII- ID is largely cytoplasmic in the resting state and translocates to the nucleus upon growth factor signaling, TFII-Ib is nuclear in basal state and is exported out of the nucleus upon growth factor stimulation. Employing quantitative chromatin immmunoprecipitation assays and gene silencing experiments with RNAi, we demonstrate that the TFII-Ib is basally recruited to the c-fos promoter in vivo but the recruitment is abolished in the presence of mitogenic stimulation. In contrast, upon growth factor signaling, TFII-ID undergoes induced tyrosine phosphorylation, translocates to the nucleus and is recruited to the same site on the c-fos promoter. Importantly, we also demonstrate that upon growth factor signaling, the TFII-I delta isoform interacts with Erk1/2 (MAPK) kinase in the cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factor signaling. Our results identify a novel growth factor signaling pathway controlled by opposing activities of two TFII-I spliced isoforms.

98 Abstracts - Poster

Melissa Harrison Abstract 43 The MBT repeat-containing protein LIN-61 regulates C. elegans vulval cell-fate specification

Melissa Harrison1, Bob Horvitz1 and Xiaowei Lu2

1Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology,Cambridge, MA 02139., 2Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, U.S.A.

Chromatin remodeling plays an important regulatory role in gene expression, a critical component in controlling cell-fate specification. However, there is limited knowledge of the means by which individual chromatin-remodeling factors cause changes in developmental- fate decisions. Studies of vulval development in the nematode Caenorhabditis elegans have proven to be useful in elucidating mechanisms involved in cell-fate specification. The A dult vulva arises from three of six ectodermal blast cells, all of which are initially competent to adopt a vulval cell fate. Vulval specification is negatively regulated by at least three redundant functions provided by the synthetic Multivulva (synMuv) class A, B, and C genes. Loss-of-function mutations in two or more gene classes result in a Multivulva (Muv) phenotype, characterized by more than the normal three blast cells adopting vulval cell fates. Many of the synMuv genes, including lin-35Rb, dpl-1DP, efl-1E2F, lin-53RbAp48, hda-1HDAC1, let-418Mi-2, trr1TRRAP, mys-1MYST, and epc-1E(Pc), have homologs in other species that function in chromatin remodeling and transcriptional regulation. lin-61, a gene identified in screens for class B synMuv mutants, encodes a protein containing four MBT (malignant brain tumor) repeats. MBT repeats are loosely-defined ~100 amino acid sequences present in several nuclear proteins, such as the Drosophila Polycomb group protein Sex Comb on Midleg. Structural analysis of MBT repeats from other proteins shows that these domains have similarity to Tudor and chromodomains that have been shown to bind modified histones. Antibody staining demonstrates that LIN-61 is a ubiquitously-expressed, DNA-localized nuclear protein. LIN-61 staining patterns are similar in most synMuv mutant backgrounds, suggesting expression is independent of the function of other synMuv proteins. Ten loss-of-function alleles have been identified through genetic screens. Analysis of missense mutants has identified substitutions in highly- conserved residues that may disrupt protein stability, whereas proteins mutant in a non- conserved region of the MBT repeat or outside the MBT repeats are stable, if functionally compromised. Experiments to identify the proteins that interact with LIN-61 in vivo are ongoing.

99 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Michael Hendzel Abstract 44 The regulation of histone H1 binding in vivo

John Th’ng, Ming Ye and Michael J Hendzel

University of Alberta, Rm 3332, Cross Cancer Institute, 11560 University Ave., Edmonton, Alberta, T6G1Z2, Canada

Using fluorescence recovery after photobleaching (FRAP) to quantify the binding properties of histone H1 subtypes in living cells, we have defined the C-terminal domain (CTD) as the primary determinant of binding affinity to chromatin. In this report, I will review experiments that demonstrate that the C-terminal domain of histone H1 becomes structured upon binding to chromatin. Differences in the binding affinity of H1 histone subtypes correlate with the length of the CTD but not with the overall net charge of the domain. Remarkably single amino acid substitutions within the CTD can destabilize binding as much as 90% and identify specific segments of the histone H1 CTD that are particularly important in defining the affinity of binding. By swapping CTDs between individual H1 subtypes, we were also able to define a small contribution mediated by the N-terminal domain. By positioning the fluorescent tag on either the C-terminus or the N-terminus and by using fluorescent tags of substantially different size, we have been able to define the extent that fluorescent tagging disrupts histone H1 binding. These experiments reveal that green fluorescent protein and its variants reduce the binding affinity of H1 histones and raise concerns about conclusions drawn from studies employing histone proteins that use fluorescent protein tags. Using small fluorescent tag of less than 15 amino acids, we find that although H1 subtypes are dynamically bound to chromatin in vivo, they bind more stably than previously appreciated. Histone H1.5, for example, can remain bound for up to 20 minutes before dissociating. Histone H1 subtypes showed different preferences for the regions of chromatin that they bind. Nonetheless, all H1 subtypes were regulated by phosphorylation and this phosphorylation could be inhibited by the cyclin dependent kinase inhibitor, roscovitine. When binding of histone H1 was compared between cancer cell lines and normal cell lines, cancer cell lines were observed to destabilize histone H1 binding to different extents that correlated with differences in steady-state histone H1 phosphorylation status. Interestingly, histone H1 binding affinity also correlated with radiosenstivity. Collectively, our results are beginning to unravel the unique properties of individual histone H1 subtypes, the mechanisms of regulating histone H1 binding, and the existence of tertiary structure in the CTD.

100 Abstracts - Poster

Wilma A.Hofmann Abstract 45 Specific roles for nuclear actin and nuclear myosin I in transcription by RNA Polymerase II

Wilma A.Hofmann1, James A. Goodrich2, Primal de Lanerolle1

1Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, 60612, U.S.A., 2Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO, 80309-0215, U.S.A.

It has been known for many years that actin is abundant in the nucleus. Nevertheless, the physiological functions of actin in the nucleus are not well defined. The discovery of another “cytoplasmic protein” in the nucleus, namely nuclear myosin I, has piqued interest in the functional role of these proteins in the nucleus. We have found that nuclear actin and nuclear myosin I (NMI) are intimately involved in the basic process of transcription. ?-Actin as well as NMI are tightly bound to and co-purify with the RNA polymerase II core enzyme. The functional involvement of actin and NMI in transcription was demonstrated in vivo as well as in vitro. Microinjecting antibodies to actin or NMI inhibited transcription and chromatin immunoprecipitation assays showed that both proteins are present at the promoter region of transcribing genes. In vitro transcription assays using highly purified proteins confirmed that both actin and NMI are required for transcription initiation. However, a combination of pre-initiation complex (PIC) assembly assays and protein-binding studies showed that they are involved in subtly different ways. While actin is needed at the preinitiation stage of transcription and necessary to integrate RNA polymerase II into the developing PIC, NMI is not necessary for PIC-formation per se. In fact, NMI appears to be necessary for the formation of the first phosphodiester bond during transcription initiation. Taken together, our results demonstrate not only a crucial role for nuclear actin and nuclear myosin I in the basic process of transcription but also present mechanistic evidence on how those proteins are involved in transcription.

101 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Sui Huang Abstract 46 Upstream binding factor association induces large scale chromatin decondensation

Danyang Chen, Andrew S. Belmont and Sui Huang

Northwestern University Medical School, Ward 11-240, 303 E. Chicago Ave., Chicago, IL 60611, U.S.A.

The function of upstream binding factor (UBF), an essential component of the RNA polymerase I (pol I) preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats (Capenter, A. E. and Belmont, A. S. 2004, Methods Enzymol 375, 366-381). We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI/SNF and histone acetyltransferases (HATs), and is independent of extensive histone acetylation. However, UBF recruits the pol I specific, TATA box binding protein (TBP) containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA (rDNA) clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating rDNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery.

102 Abstracts - Poster

Dana Huebert Abstract 47 Global analysis of MLL and MLL-fusion mediated histone modifications

Dana J. Huebert1,2, Christina M. Hughes2,3, Matthew Meyerson2,3, Stuart L. Schreiber2,4, Bradley E. Bernstein1,2

1Massachusetts General Hospital, Dept of Pathology, Building 149 13th St, Charlestown, MA, 2Broad Institute of Harvard and MIT, 320 Bent Street, Cambridge, MA, 3Dana-Farber Cancer Institute, Dept of Medical Oncology, 44 Binney St, Boston, MA, 4Howard Hughes Medical Institute, Harvard University, Dept of Chemistry and Chemical Biology, 12 Oxford St., Cambridge, MA, U.S.A.

Rearrangements of MLL (mixed lineage leukemia) are implicated in up to 40% of acute myelogenous leukemias. The MLL protein product methylates lysine 4 of histone 3, a modification generally associated with transcriptional activation. Genomic analyses have shown that lysine 4 methylation is typically punctate and found at gene starts. However, broad, cell-type specific domains span the active portions of the Hox clusters. These domains may keep chromatin in a permissive state, allowing the cell to epigenetically maintain active transcription. MLL has recently been shown to co-localize with RNA polymerase at active genes, an unusual finding given its known role as an oncogene. We investigated the function of an MLL fusion protein using chromatin immunoprecipitation, real-time PCR and microarrays. The data suggest that the fusion protein localizes to regions distinct from those bound by wild-type MLL. We speculate that, while wild-type MLL has an important role in maintaining active chromatin at many genes, the fusion protein functions at a subset of genes, including Hox, which mediate its pathogenesis.

103 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Christina Hughes Abstract 48 Linking Menin mutations to defects in histone methylation and altered expression of target genes

Christina M. Hughes, Laura E. MacConaill, Andrew Goldstein, Orit Rozenblatt-Rosen and Matthew Meyerson

Dana Farber Cancer Institute, Mayer 434, 44 Binney St., Boston, Suffolk 02115, U.S.A.

Some of the mutations that cause cancer directly alter the expression of genes involved in the regulation of cellular growth and differentiation. Gene expression, however, is regulated not only by the sequence of the genes themselves, but by the structure of the chromatin, including modifications of histones. This suggested the possibility that proteins that regulate chromatin structure and histone modification could be involved in the genesis of human cancer. We and others have shown that the tumor suppressor menin, the protein product of the MEN1 gene, which is mutated in familial endocrine neoplasia type 1, associates with a SET1 (COMPASS)-like histone methyltransferase (HMTase) complex. Some, but not all, of the MEN1 mutations associated with familial cancers affected HMTase activity in vitro. To elucidate the role(s) menin plays we are exploring the interaction(s) of menin with the both the MLL and MLL2 proteins, which we believe are key protein components of similar but distinct menin interacting histone methyltransferase complexes. Because we are particularly interested in linking the defects in the HMTase complexes to the ability of the menin mutants to cause cancer, we are analysing the complexes to determine how the patient-derived menin mutations, in particular the mutations that do not have an affect on HMTase activity, alter the complexes. We are also asking how the mutations affect the regulation of menin target genes, for example the homeobox gene Hoxc8, to study the links between the defects gene expression with the formation of the MEN1 tumors. Finally, we have shown that menin is specifically phosphorylated and are in the process of determining whether the phosphorylation of menin affects its association with, or the activity of, the HMTase complexes.

104 Abstracts - Poster

Irena Ivanovska Abstract 49 Histone code in meiosis: histone kinase required for chromosomal architecture in Drosophila oocytes

Irena Ivanovska, Tulasi Khandan and Terry L. Orr-Weaver

Whitehead Institute, 9 Cambridge Center, Cambridge, Massachetts 02142, U.S.A.

Chromosomes undergo dramatic morphological changes that facilitate their functions. During sexual reproduction, meiotic chromosomes assume specialized configurations that ensure accurate segregation of homologous chromosomes and promote faithful propagation of the genetic material. Although histone modifications have well-established functions in chromosomal structural changes, their roles in meiotic chromosomal structures are largely unknown. We discovered a histone code in meiosis characterized by a specific pattern of histone modifications. Phosphorylation of Histone H2A by a recently-identified kinase, NHK- 1, is an important component of this code. Lack of Histone H2A phosphorylation in the Drosophila nhk-1 mutant results in female sterility due to defects in the karyosome (the prophase I arrest state of the chromosomes) and subsequently in the metaphase I arrest. These defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. The absence of Histone H2A phosphorylation in the nhk-1 mutant leads to loss of specific histone acetylations, while other modifications remain unaffected, consistent with the histone code hypothesis. As meiotic progression is easily visualized in Drosophila ovaries, this system has enabled us to decipher the temporal relationships between the relevant histone modifications. Embryos laid by nhk-1 mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis.

105 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Danika Johnston Abstract 50 Role of Drosophila ecdysone receptor and histone methyltransferase TRR in gene regulation

Danika Johnston, Yurii Sedkov, Svetlana Petruk, James B. Jaynes and Alexander Mazo

Thomas Jefferson University, 485 JAH, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.

Type I steroid hormone receptors shuttle between the nucleus and cytoplasm, however most hormone receptors are of the type II category and predominantly nuclear. These nuclear receptors are present at their binding site irrespective of the transcriptional status of their target genes. In the presence or absence of ligand, Type II receptors can recruit coactivator or co-repressor proteins, respectively, to the target gene in order to alter its transcriptional status usually through chromatin structure modifications. The current model for the Drosophila Ecdysone receptor (heterodimer of Usp and EcR) suggests that this receptor belongs to the Type II group. Our results indicate that EcR, and it co-activator, histone methyltransferase (HMTase) Trithorax Related (TRR), can form a protein complex outside DNA and that they are not present in vivo at the promoters of repressed target genes in the absence of ecdysone. Confirming experiments suggest that both EcR and TRR are shuttling between the nucleus and cytoplasm in an ecdysone dependent fashion, but interestingly, these two proteins may be shuttling independently of each other. This data questions the current model of Ecdysone Receptor functioning as a Type II receptor and provoked us to investigate the presence or absence of nuclear receptor components and their co-regulators at the promoters of certain ecdysone induced genes both before and after induction. Preliminary results from chromatin immunoprecipitation experiments indicate that, following induction with ecdysone, EcR and Usp, as well as their co-activator TRR, are recruited to the promoters of both Hsp27 and hedgehog (hh), two ecdysone induced genes. Results also showed an increase in the presence of pCAF, a histone acetyltransferase, and a decrease in SMRTER, a transcriptional co-repressor after ecdysone treatment. These results will aid in understanding the mechanism of nuclear receptors and histone modifying complexes to alter the transcription of developmental genes.

106 Abstracts - Poster

Sarah Johnstone Abstract 51 Embryonic stem cell transcription factors regulate developmental transcription factors

Sarah E.Johnstone1,2, Laurie A. Boyer1,6, Tong Ihn Lee1,6, Megan F. Cole1,2, Stuart S. Levine1, Jacob P. Zucker3, Matthew G. Guenther1, Roshan M. Kumar1, Heather L. Murray1, Richard G. Jenner1, David K. Gifford1,4,5, Douglas A. Melton3,5, Rudolf Jaenisch1,2, and Richard A. Young1,2,5*

1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A., 3Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, U.S.A., 4MIT Computer Science and Artificial Intelligence Laboratory (CSAIL), 32 Vassar Street, Cambridge, Massachusetts 02139, U.S.A ., 5Broad Institute of MIT and Harvard, One Kendall Square, Building 300, Cambridge, Massachusetts 02139, U.S.A.

Embryonic stem (ES) cells have two important properties: the ability to self-renew and the capacity to develop into any cell type. The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated ES cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. Interestingly, target genes for these factors frequently encode transcription factors, many of which are developmentally important homeodomain proteins. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to self-renewal and pluripotency.

107 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Paul Kaufman Abstract 52 Replication-independent histone deposition by the HIR complex and Asf1

Erin M. Green1, Andrew J. Antczak1, Aaron O. Bailey2, Alexa A. Franco1, Kevin J. Wu1, John R. Yates 3rd2 and Paul D. Kaufman3

1Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, U.S.A., 2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., 3Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605-2324, U.S.A.

The orderly deposition of histones onto DNA is mediated by chromatin assembly proteins. In budding yeast, products of four HIR genes (HIR1, HIR2, HIR3 and HPC2) regulate histone gene expression, pericentric chromatin, and heterochromatin, but had been uncharacterized biochemically. Mass spectroscopic analysis of tandem affinity-purified Hir proteins shows that these four proteins comprise the HIR complex. The HIR complex co- purifies with histone deposition protein Asf1 under mild ionic conditions. Each of the four Hir proteins contribute to the stability of the HIR complex and its localization to a histone gene promoter; in contrast, Asf1 is not required for HIR complex localization to a histone gene promoter. The HIR complex and Asf1 together deposit histones onto DNA in a replication-independent manner. However, the HIR complex does not alter the replication-linked histone deposition activity of Chromatin Assembly Factor-1. The HIR and Asf1 proteins thus represent a conserved eukaryotic pathway for histone replacement throughout the cell cycle.

108 Abstracts - Poster

Ik Soo Kim Abstract 53 A chromatin remodeling complex in regulation of crosstalk between the Wnt and the NF-kappaB pathway

Ik Soo Kim, Jung Hwa Kim, Hee June Choi, Bogyou Kim, Ji Min Lee and Sung Hee Baek

Seoul National University 216, 20 Sillim-Dong, Kwanak-Ku, Seoul 151-742, South Korea

Deciphering the molecular strategies, by which specific signal pathways regulate biological processes by switching genes to the appropriate on or off state, remains an important issue in biology. Transcription of most genes in the nucleus is regulated by the coordinate action of cofactors and chromatin remodeling complexes. BAF60c has been reported to be components of ATP-dependent chromatin remodeling complexes. Here we present cDNA microarray data revealing crosstalk between the NF-kappaB and the Wnt/beta-catenin pathway, and found that a subset of NF-kappaB target genes including BAF60c are downregulated by activation of the Wnt/beta-catenin pathway. We will present a molecular mechanism of crosstalk between the NF-kappaB and the Wnt/beta-catenin signaling pathway.

109 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Elena Kisseleva-Romanova Abstract 54 Identification of new mutants that lead to transcription internal entry in S. cerevisiae

Kisseleva-Romanova E., Youdell M., Nair A., Karabetsou N., Morillon A. and Mellor J.

University of Oxford, C8, Dept of Biochemistry, Microbiology Unit, South Parks Road, Oxford, OX1 3QU, U.K.

During transcription, the histone chaperones Spt6 and Spt16 (FACT) assist RNA polymerase II (RNAPII) to disassemble and re-assemble histones on DNA. In S. cerevisiae, it has been shown that in absence of Spt6 or Spt16 activity, nucleosomal histone levels are reduced which result in RNAPII-dependent internal initiation inside FLO8, STE11 or VPS72 genes. To provide a better understanding of this mechanism, we have started to systematically screen for other components involved in internal initiation at STE11 gene. Interestingly, we identified factors interacting with elongating RNAPII and which are related to covalent histones modifications and chromatin remodelling processes. To discriminate between complexes controlling histone expression and histone assembly in these mutants, we compared levels of global histone H3 to the occupancy of histone H3 at several loci including STE11. We will discuss the possibility of a novel aspect of histones chaperone metabolism and its consequence on RNAPII transcription.

110 Abstracts - Poster

Christoph Koch Abstract 55 A microarray based approach to develop high resolution maps of histone modifications in 1% of the human genome

Christoph M. Koch1, Shane C. Dillon1, Robert M. Andrews1, Gayle K. Clelland1, Sarah Wilcox1, Joanna M. Fowler1, Keith D. James1, Alexander W. Bruce1, Phillippe Couttet1, Oliver M. Dovey1, Peter D. Ellis1, Andrew J. Mungall1, Pawandeep Dhami1, Bee Ling Ng1, Heike Fiegler1, Cordelia F. Langford1, Paul Flicek2, Ewan Birney2, Nigel P. Carter1, David Vetrie1, Ian Dunham1

1The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge,CB10 1SA, U.K., 2EMBL – European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK

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 24156 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) and a foetal lung fibroblastoid cell line (HFL-1). Our aim is to investigate whether histone modifications can be used as a robust indicator of functional elements in the genome. Therefore we have developed high resolution maps of histone modifications and correlated these maps 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 trimethylated 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.

111 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Hyockman Kwon Abstract56 BAF53 is essential for maintenance of chromosome territories and higher-order chromatin structure

Ki Won Lee, Mi Jin Kang, Jae Hwan Shim, Su Jin Kwon, Yunhee Kim Kwon and Hyockman Kwon

Department of Bioscience and Biotechnology, 210 Natural Science, 89 Wangsan-ri Mohyeon-myon, Yongin, Kyongki-do, 449-791, Korea

In interphase mammalian cells, chromosomes are compartmentalized into discrete chromosome territories. Chromosome territory has been viewed as a collection of chromosomal subdomains, but the structure of chromosomal subdomain remains largely elusive. Here we show that BAF53 knockdown by siRNA interference causes disintegration of chromosome territories and opens up higher-order chromatin structure. These distinct structural changes of chromosomes were accompanied with a cell cycle defect and the induction of several unexpressed genes such as Prm 2. Relaxation of chromatin fibers from backfolding in chromosomal subdomains explains well these results and can be simulated by dissociation of chromatin loops from loop base spring according to the multi-loop subcompartment model. In agreement with the role of BAF53/beta-actin dimer as a nucleation center for actin oligomerization, we discover that BAF53 knockdown disassembles nuclear actin array. Based on these observations we propose a tentative model for loop base spring.

112 Abstracts - Poster

Eric Lam Abstract 57 Chromatin charting in living plants: a global study.

Eric Lam

Biotech Center, Rutgers University, 216 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901, U.S.A.

In the phenomena known as position effects, integration loci affect the expression levels of transgenes. For instance, a transgene that is highly expressed when integrated into euchromatic regions may be poorly expressed when inserted into heterochromatic regions. Recent studies suggest that chromosomes in interphase nuclei may be organized, and that the 3-D positions of genes in the interphase nuclei may influence their expression. The goals of our Chromatin Charting project are: 1) to produce a set of resources to better quantify position effects in the Arabidopsis genome and 2) to track the three dimensional (3- D) positions and dynamic properties for specific regions of the genome in interphase nuclei of live plants. To these ends, we are presently generating over 5,000 Ds-tagged lines of Arabidopsis. The Ds-tagging cassette contains a CaMV35S::luciferase which allows monitoring of position effects, and a lac operator repeat (array) which allows the tagged loci to be visualized by expressing fusions of fluorescent-proteins with lacI-NLS within the nuclei of live Arabidopsis plants. We are mapping the tagged loci with the goal of selecting 3,000 lines with which tagged loci would occur on average once every 40 kbp. The selected lines will be used to evaluate the position effects on gene activity at a global level. In addition, a visualization line expressing an HcRed-lacI-NLS fusion protein would be crossed to these selected transposants for visual tracking of the inserted loci. Lastly, we are also trying to improve our fluorescent protein tracking system by creating a multicolor system for more quantitative evaluation of the location and dynamics of the tagged loci. These materials should be useful for the characterization of mutations and corresponding genes that influence gene expression through altering chromatin architecture in eukaryotes. Our progress with this project would be presented.

113 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Robert Lane Abstract 58 Does chromatin inaccessibility contribute to mutually exclusive olfactory receptor transcription

Marijo Kambere and Robert P. Lane

Wesleyan University, Hall Atwater Laboratory, Lawn Avenue, Middletown, CT 06457, U.S.A.

The mammalian olfactory system is able to detect and distinguish among tens of thoU.S.A.nds of odorants in the environment. This ability is dependent on an organizing principle in which each sensory neuron in the nose expresses only one olfactory receptor (OR) allele from among >1000 OR genes in the genome. We are interested in understanding the regulatory mechanisms that govern this mutually exclusive expression. Previous genomic and genetic studies suggest that OR genes reside in repressed regions of the genome, and we are exploring the hypothesis that mutually exclusive transcription of only one OR allele is facilitated by limiting transcriptional access by chromatin modifications. We have used chromatin immunoprecipitation (ChIP) assays to generate preliminary results on the histone modifications at an OR locus that we have previously shown becomes active in a differentiated olfactory sensory cell line. For all six modifications tested, histones around the tested OR locus in undifferentiated cells show acetylation and methylation patterns consistent with inactive euchromatin. This result suggests that the ground state for OR loci in premature sensory neurons is “closed”. We are now assaying histone acetylation and methylation patterns at multiple OR loci to test whether all OR genes are similarly “closed” in undifferentiated cells, and investigating how chromatin patterns change during the differentiation process. We anticipate having preliminary results for these studies by the date of this meeting.

114 Abstracts - Poster

Martin Law Abstract 59 Mutations in the ATRX chromatin remodelling protein cause changes in histone H3 lysine 9 methylation.

Martin Law, Richard Gibbons and Douglas Higgs

MRC Molecular Haematology Unit, WIMM, Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 0J1, U.K.

Mutations in the chromatin remodelling protein, ATRX, cause a rare X-linked developmental disorder characterised by severe mental retardation, abnormalities of the face, skeleton and urogenital system with short stature and mild alpha thalassaemia - the ATR-X syndrome. The precise role of the ATRX protein is currently unclear although several lines of evidence suggest that it may influence transcription by altering chromatin structure. In common with members of the SNF2 family of chromatin remodelling proteins ATRX possesses conserved ATPase/helicase motifs and can disrupt histone-DNA contacts in vitro. ATRX also possesses an extended variant PHD domain, which is a cysteine rich zinc finger motif also seen in other chromatin-associated proteins. The PHD-like domain and the ATPase domain are the most common sites of mutations that cause the ATR-X syndrome, underlining their functional importance. ATRX has been shown to interact with several proteins that can influence transcription (DAXX, HP1 and EZH2), although the significance of these interactions is not yet clear. One mechanistic link between ATRX and the alteration of gene expression is that ATR-X patients exhibit diverse changes in DNA methylation patterns. In ATR-X patient cells, ribosomal genes become substantially hypomethylated at the promoter and coding region. In light of the recently described links between chromatin remodelling, DNA methylation and histone methylation, this study sought to further define the role of ATRX in the interconnection of these epigenetic pathways. Here, chromatin immunoprecipitation was used to show that in ATRX patient cell lines, the ribosomal genes show reduced histone H3 lysine 9 trimethylation which parallels the reduction in DNA methylation. These epigenetic changes however produce no detectable alteration in the rate of ribosomal gene transcription. These data support the idea that, in human cells, chromatin remodelling, histone H3 lysine 9 methylation and DNA methylation are linked.

115 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Kenneth Lee Abstract 60 Functional analysis of the ADA histone acetyltransferase complex Kenneth K. Lee, Samantha Pattenden, Daeyoup Lee, Laurence Florens, Selene K. Swanson, Michael Washburn and Jerry L. Workman

Stowers Institute for Medical Research, Kansas City, MO, U.S.A.

The histone acetyltransferase, Gcn5 is present in three macromolecular complexes in Saccharomyces cerevisiae, SAGA, SLIK and ADA. The ADA complex is the least understood of the three and is the only one that does not bind transcriptional activators. Our goal was to characterize the function of the ADA complex through both in vivo and in vivo approaches. Using TAP tag purification in yeast, we purified and separated the three GCN5 containing complexes. MudPIT mass spectrometry of the purified ADA complex revealed two novel subunits, Sgf29 and a previously uncharacterized protein we named Ahc2. We showed that deletion of AHC2 results in the loss of both Ahc2 and Ahc1; however this loss does not affect HAT activity of the remaining complex in vivo. In contrast, deletion of SGF29, which results in the loss of Sgf29 only, does lead to a decrease in HAT activity in vivo. Examination of the DNA and nucleosome binding activity of ADA found that the complex was able to bind both; however binding was reduced in the absence of Ahc1 and Ahc2. Next, we determined the effects of deleting ADA specific components on transcription using expression microarrays. We found a number of genes in the pheromone mating pathway that were downregulated greater than two fold. We are currently examining the role that the ADA complex may play in the regulation of the pheromone pathway. We are also currently examining the binding of the ADA complex throughout the genome using ChIP/chip analysis of both intergenic and open reading frames. Our comprehensive analysis of the ADA complex allows us to understand how it functions independently of SAGA and SLIK.

116 Abstracts - Poster

Young Han Lee Abstract 61 Epigenetic regulation of the tumor suppressor Egr-1 gene by oncogenic Ras Soon Young Shin, Tae Seop Shin and Young Han Lee

Hanyang University, 411, College of Science and Technology, 1271 Sa 1-dong, sangrok- gu/Ansan-si, Gyeonggi-do, 426-791 Korea

The early growth response-1 (Egr-1) gene (also known as NGFI-A, zif268, krox 24, or Tis8) is a transcription factor that contains three zinc-finger motifs in the DNA-binding domain and preferentially binds to GC-rich regulatory elements. Egr-1 is induced rapidly by a variety of growth factors, cytokines, and cellular stresses, suggesting a multifunctional role of Egr-1 in cell growth, survival, differentiation, neuronal plasticity, and development. Egr-1 is downregulated in a variety of human tumors including human breast carcinoma and fibrosarcoma, and decrease of Egr-1 level is strongly associated with the progression of tumorigenesis. Forced expression of the Egr-1 gene into tumor cells inhibits cell growth and tumorigenicity by induction of TGF-beta1, fibronectin, and plasminogen activator inhibitor-1, which are important in the growth control and stabilization of extracellular matrix proteins, implying that functional loss of Egr-1 may contribute to tumorigenic potential. Although a great deal of progress in identifying and characterizing the signaling that positively regulate the Egr-1 gene expression, much less is known about negative regulatory mechanism responsible for loss of Egr-1 expression. Ras-MEK-ERK-Elk-1 is the major signaling pathway which induces the Egr-1 expression through serum response element (SRE)- mediated transcriptional activation in response to mitogenic signals. However, we have found that chronic expression of oncogenic Ras suppresses the Egr-1 transcription which is abrogated by treatment with trichostatin A. In this presentation, well discuss how oncogenic H-Ras paradoxically down-regulates Egr-1 expression in a point of chromatin remodeling.

117 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Lin Li Abstract 62 SP1 and SP3 dynamic association with estrogen regulated promoters Lin Li and James R. Davie

Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9 Canada

Transcription factors Sp1 and Sp3 are involved in the regulation of many genes involved in cellular processes including regulation of estrogen responsive genes. Although Sp1 and Sp3 are structurally similar and bind the same DNA sequence, these factors have several distinguishing features including locating to different subnuclear sites. In this study we applied kinetic chromatin immunoprecipitation assays to investigate the temporal association of Sp1, Sp3 and ER on α-amanitin-synchronized estrogen responsive promoters in MCF-7 cells cultured in the presence or absence of estradiol. The promoters chosen for study have one or more Sp binding sites positioned near an imperfect estrogen response element (ERE) (TFF1), half site ERE (cathepsin D, retinoic acid receptor α, transforming Growth Factor α) or no ERE (cyclin D1, c-myc). Regardless of the promoter context, unliganded and liganded ER cycled on these promoters with different frequencies. Distinct temporal patterns of Sp1 and Sp3 association with these promoters were observed. In the presence of estradiol, Sp3 cycles on these promoters and typically the association of Sp3 lags behind that of liganded ER, suggesting that ER may facilitate the binding of Sp3 to these promoters.

118 Abstracts - Poster

Karen Lower Abstract 63 ATRX acts in a localised manner to down regulate the expression of alpha globin genes in ATR-X syndrome Karen M. Lower, Doug R. Higgs and Richard J. Gibbons

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, John Radcliffe Hospital, Headington, Oxfordshire OX3 9DS, U.K.

ATRX is a member of the SNF2 family of chromatin remodelling proteins, however the exact function of this protein remains unknown. Mutations in the ATRX gene result in the intellectual disability disorder ATR-X syndrome which is associated with a mild form of alpha thalassemia. The latter feature is due to down regulation of the alpha globin genes, which are located at the telomeric end of the short arm of chromosome 16. It is not known how ATRX regulates the expression of the alpha globin genes. One hypothesis is that ATRX acts to remodel the chromatin environment surrounding this gene cluster. If this is the case, it is reasonable to expect that the genes surrounding the alpha globin cluster might also be altered in their expression patterns when ATRX is mutated. To address this hypothesis, we carried out semi-quantitative real time PCR on 20 ATR-X and 20 control lymphoblast cell lines to analyse the expression level of the genes surrounding the alpha globin cluster. Eight genes in the terminal 400 kb of 16p were analysed. Results show that of the genes analysed, expression did not significantly vary between control and ATR-X cell lines. Therefore, this suggests that ATRX is somehow directly targeting the alpha globin locus, and that the down regulation of expression of these genes is not due to a global effect on the chromatin structure of the surrounding region.

119 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Malik Lutzmann Abstract 64 Geminin binding to Cdt1 controls, but does not necessarily block, Cdt1 function during replication origin licensing Malik Lutzmann, Olivier Cuvier, Domenico Maiorano and Marcel Méchali

Institute of Human Genetics, CNRS-UPR1142, 141 rue de la Cardonille, 34396 Montpellier, France

Early in the cell cycle chromosomes become “licensed” to replicate once and only once by formation of pre-replication complexes (pre-RCs) onto DNA replication origins. The last step in this licensing reaction is the essential Cdt1 mediated loading of the MCM2-7 complex onto DNA, the helicase necessary to open up the double stranded DNA during replication initiation. Precise regulation of Cdt1 activity by the multifunctional protein geminin is crucial to prevent re-firing of origins and subsequent re-replication. Geminin was thought to act as a negative regulator of DNA replication by binding Cdt1 and thus blocking its ability to load the MCM2-7 complex onto DNA. Here, we show that a recombinant Cdt1-geminin complex is fully active in MCM2-7 loading and initiation of DNA replication. Moreover, we show that only free Cdt1, but not the licensing active Cdt1-geminin complex leads to re-replication and checkpoint activation if added in excess to Xenopus egg extract. We further demonstrate, that the Cdt1-geminin complex exists in different stoichiometries, of which one is active in loading the MCM2-7 complex and origin licensing, whereas only the other containing more geminin per molecule of Cdt1 renders Cdt1 inactive. Our data give rise to a model in which a complex of Cdt1 and geminin is the normal MCM2-7 loader, which - in contrast to free Cdt1 - can be efficiently regulated. Besides its function in controlling Cdt1, geminin was also shown to possess neuralizing activities and to control embryonic development and transcriptional programs by interacting with several key transcription factors like Hox-and polycomb proteins as well as Brg1, a member of the SWI/SNF chromatin-remodeling complex. Interestingly, origin selection and –activation are in a poorly understood way linked to developmental- and transcriptional programs. Our findings, that geminin-complexed Cdt1 activates replication origins makes it tempting to speculate, that the DNA-replication independent recruitment of geminin to chromatin might also take part in the selection of origins to be activated. This could help to explain, how transcriptional programs and chromatin structure are linked to the spatial and temporal organization of S-phase.

120 Abstracts - Poster

Dale Mackay Abstract 65 Identification of an acetyltransferase of the DNA binding protein Dale T. Mackay, Malcolm White and Garry Taylor

University of St Andrews, BMS Building, North Haugh, St. Andrews, Fife KY16 9ST, U.K.

Members of the crenarchaeal kingdom have a variety of small DNA binding proteins instead of histones. Alba (Sso10b) is an architectural DNA binding protein from Sulfolobus solfataricus, which is controlled by acetylation and deacetylation. Acetylation of a single lysine residue results in weaker binding to DNA, opening the way for transcription, replication and repair. Deacetylation by the enzyme Sir2 leads to transcriptional silencing of chromatin in vitro. The search for a specific acetyltransferase of Alba commenced by searching the Sulfolobus genome for genes encoding a putative acetyltransferase. Two genes sso0082 and sso0209 were identified. From multiple sequence alignments, it appears that Sso0209 has eukaryotic N-acetyltransferase homologues as well as archaeal N-acetyltransferase homologues. This is interesting because the degree of conservation and similarity in most of these cases is high (~70%), indicating that Sso0209 may be a conserved protein from archaea through to humans. Sso0082 shows sequence similarity to acetyltransferase enzymes belonging to other prokaryotes. Both Sso0082 and Sso0209 share at least 25% identity with each other. This poster will describe the initial cloning of the two genes and the biochemical characterisation and crystallisation of Sso0209.

121 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Asoke Mal Abstract 66 Linking HDAC1 and MyoD function in Rhabdomyosarcoma Asoke K. Mal

Lerner Research Institute, Cleveland Clinic Foundation NE-20 Molecular Genetics, 9500 Euclid Avenue, Cleveland, Ohio 44195, U.S.A.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and is a substantial problem in pediatric oncology. RMS thought to arise from myogenic precursor cells and expresses myogenic transcriptional regulator MyoD. The expression of this regulator is necessary for skeletal muscle cell development and differentiation during development, postnatal growth and regeneration in injury and disease. Although MyoD is expressed in RMS cells, it fails to activate differentiation-responsible gene expression in these tumor cells. Our previous work demonstrated that HDAC1 association with MyoD is functionally linked to the inhibition of MyoD-mediated differentiation-responsible gene expression and differentiation in muscle cells that undergo differentiation process (EMBO, 2001; 20: 1739-1753). We investigated whether HDAC1 is responsible for MyoD failure to induce differentiation program in RMS cells. We have observed that HDAC1 is overexpressed in these tumor cells and its expression level remains unchanged when these cells are cultured in condition that is permissible for normal muscle cells to differentiate. In addition, HDAC1 is associated with MyoD in these tumor cells cultured in both growth and differentiation-permissible conditions. These findings are in opposite to the normal muscle cells that undergo differentiation. ChIP and Re-ChIP experiments in RMS cells further shows that MyoD-HDAC1 complex is occupied on the regulatory region of MyoD target muscle genes and histone H3 is in hypoacetylated state. However, RNAi-mediated knockdown of HDAC1 in these tumor cells induces acetylated-H3 on the promoter of muscle genes and restores MyoD-mediated gene transcription. Together, these results suggest that over-expression of HDAC1 inhibits the ability of MyoD to induce differentiation program in RMS cells even under differentiation permissible environment.

122 Abstracts - Poster

Roberto Mantovani Abstract 67 Epigenetic changes to NF-Y controlled genes upon ER induction. Giacomo Donati and Roberto Mantovani

Dipartimento di Scienze Biomolecolari U di Milano, DSBB, Via Celoria 26, Milano, 20133, Italy

The endoplasmic reticulum stress response leads to the induction of several genes. Most, if not all, require NF-Y, a conserved trimer that binds to CCAAT boxes. We evaluated the kinetic induction of several mRNAs, which can be divided in early and late response genes. By Chip experiments, we undertook a systematic analysis of in vivo binding of sequence- specific activators, cofactors and general transcription factors to a number of induced promoters. Furthermore, we evaluated the chromatin changes in terms of specific histone modifications (acetylation and methylations). The picture emerging is one of remarkable complexity, with common themes, as well as peculiar promoters specific differences. Finally, we are evaluating the role of NF-Y in the ER-stress response, by using a dominant negative NF-YA mutant. The results of our analysis will be presented.

123 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jotin Marango Abstract 68 The multiple myeloma SET domain (MMSET) protein is a histone H3 and H4 methyltransferase with properties of a transcriptional co-repressor Jotin Marango1, Manabu Shimoyama1, Boris A Leibovitch1, Ming Ming Zhou2, Yolanda Martinez1, Andres Sirulnik3, Marta Chesi4, P. Leif Bergsagel4 and Jonathan D Licht1.

1Division of Hematology/Oncology, Mount Sinai School of Medicine, New York, NY, U.S.A., 2Structural Biology Program, Mount Sinai School of Medicine, New York, NY, U.S.A., 3Dana Farber Cancer Institute, Boston, MA and 4Department of Hematology/Oncology, Mayo Clinic, Scottsdale, AZ, U.S.A.

Over 40% of cases of multiple myeloma (MM) are associated with translocations of the immunoglobulin heavy (IgH) chain gene gene. The t(4;14) translocation, present in ca. 20% of myeloma cases, results in the overexpression of two potential oncogenes, MMSET and FGFR3, via juxtaposition of their endogenous promoters to regulatory elements of the IgH locus. The presence of t(4;14), and MMSET overexpression, is an adverse prognostic factor in MM irrespective of FGFR3 expression. MMSET contains several conserved motifs found in proteins involved in chromatin function (PWWP, HMG, PHD domains) and in the epigenetic control of transcription (SET domain). Accordingly, we found that the two main isoforms of the MMSET protein exhibit exclusive nuclear localization in both transfected fibroblasts and myeloma cells carrying t(4;14). Towards our goal of defining the ability of MMSET to affect gene regulation and contribute to the disease pathogenesis, we found that the SET domain of MMSET possesses in vitro methyltransferase activity specific for core histones H3 and H4. Using a computational approach and theoretical extrapolation from the solved NMR structure of vSET, we identified residues in the active site of MMSET essential for catalysis, whose mutation drastically reduces enzymatic activity. Reporter assays using Gal4 fusion constructs showed that both the amino terminus of MMSET, containing the PWWP and HMG domains, as well as the SET-containing carboxy terminus act as transcriptional repressors. MMSET interacts physically and functionally with a number of known co- repressor molecules, such as HDAC1, HDAC2, Sin3a, and SIRT1, but not HDAC4 or HDAC6. As such, MMSET co-expression enhances HDAC1 and HDAC2-mediated repression in transcriptional reporter assays, and MMSET repression is partially relieved by the addition of an HDAC inhibitor. A yeast two hybrid screen identified a number of other functional partners of MMSET, including ZNF331/RITA (Rearranged in Thyroid Adenoma), a KRAB domain/zinc finger protein previously implicated in malignancy. MMSET and ZNF331 co-localize in the nuclei of transfected fibroblasts, co- immunoprecipitate, and display cooperative repression in reporter assays. Collectively, these data support the idea that MMSET is a biologically active, bifunctional transcriptional mediator acting as a HMT enzyme in chromatin remodeling and as a complex adaptor in the recruitment of repressor species. Presently we are modeling the biological effects of MMSET through a conditional overexpression system in a B cell line. While low levels of MMSET are ubiquitiously expressed, induction of high levels of MMSET expression in the B cell line is associated with growth suppression and G1 arrest. While paradoxical for a presumed oncoprotein, such actions have been observed for other disease- associated proteins such as Runx1/MTG8. In contrast, a myeloma cell line harboring t(4;14) proliferates in the presence of high level MMSET expression. RNAi-mediated knockdown of MMSET in these cells induces apoptotic cell death. This suggests that MMSET may be critical for growth and survival of myeloma cells. Profiling of gene expression changes in these systems should link the transcriptional and biological activities of MMSET.

124 Abstracts - Poster

Philip Marsden Abstract 69 The cell-specific and hypoxia-regulated expression of endothelial nitric oxide synthase (eNOS) is controlled by chromatin-based mechanisms Fish J.E., Matouk C.C., Yeboah E., Scott J.A. and Marsden P.A.

University of Toronto Room 7358, Medical Sciences Buillding, Room 7358, 1 King’s College Circle, Toronto M5S 1A8, Canada

We have demonstrated that the eNOS gene is regulated in a cell-specific fashion by epigenetic mechanisms, namely proximal promoter DNA methylation (Chan et al., J. Biol. Chem., 2004). Here we present evidence that post-translational modifications of proximal promoter histones also play an important role in the cell-specific and regulated expression of eNOS in endothelial cells. We found that endothelial cells were highly enriched in acetylated histone H3 and H4 and lysine 4 di- and tri-methylation of histone H3 at the eNOS proximal promoter; marks of transcriptionally active chromatin. Endothelial cells were specifically enriched in lysine 9 acetylation of histone H3 and lysine 12 acetylation of histone H4. Treatment of non-endothelial cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased steady-state levels of eNOS mRNA and was accompanied by increased histone H3 and H4 acetylation and lysine 4 methylation of histone H3, suggesting that HDACs repress eNOS transcription in non-endothelial cells. Lysine 4 methylation of histone H3 was found to be necessary for eNOS expression as treatment with methylthioadenosine, a lysine 4 methylation inhibitor, decreased expression of eNOS in endothelial cells and also prevented the upregulation of eNOS in non- endothelial cells treated with TSA. To determine whether chromatin-based mechanisms were also important in the regulated expression of eNOS we assessed chromatin modifications in endothelial cells grown in hypoxic conditions. Hypoxia is known to decrease eNOS mRNA, in part by transcriptional mechanisms. Hypoxia dramatically decreased the amount of RNA Polymerase II present at the eNOS proximal promoter and resulted in decreased histone acetylation by more than 50%, especially on H4. We posit that chromatin-based mechanisms play an essential role in the cell-specific expression of the eNOS gene and may be important in disease processes characterized by a decrease in eNOS expression in endothelial cells, including hypoxia.

125 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Darin McDonald Abstract 70 Polymeric nuclear actin in chromatin modification and double-strand break repair Darin McDonald, Christi Andrin and Michael J Hendzel

Cross Cancer Institute Room 3341, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada

Although it is now well established that beta-actin is an important functional component in transcription and chromatin remodeling, it is largely assumed that this function is mediated by monomeric actin. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that the nuclear actin pool, like the cytoplasmic actin pool, is a dynamic equilibrium between polymeric and monomeric species. We have subsequently identified histone deacetylases and components of the DNA double-strand break repair machinery as nuclear proteins and protein complexes that directly associate with F-actin in vitro. This is consistent with our previously reported experiments where we demonstrated that depolymerizing actin enabled the solubilization of complexes containing histone deacetylases 1, 2, and 3 that were otherwise insoluble in 0.42 M salt solutions. We have also performed experiments to examine the requirement for polymeric actin in DNA double-strand break repair. We find that a brief and reversible treatment of cells with the inhibitor of actin polymerization, latrunculin B, significantly inhibits the phosphorylation of histone H2A.X, a hallmark of sites of DSB repair. We subsequently examined the ability of cells to ligate double-strand breaks following irradiation. By examining the repair of double- strand breaks using the comet assay, which resolves broken DNA from intact DNA by electrophoresis, is completely inhibited when actin is depolymerized by a brief treatment with latrunculin B. In summary, our results provide evidence that actin has important nuclear functions that require polymerization rather than the mere presence of nuclear actin monomers.

126 Abstracts - Poster

Dominik Mojzita Abstract 71 Pdc2, the yeast homologue of CENP-B, is a part of the centromere-kinetochore complex Dominik Mojzita and Stefan Hohmann

Department of Cell and Molecular Biology/Microbiology, Medicinaregatan 9C, S-405 30 Göteborg, Sweden

Pdc2, initially identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast, was characterized as regulatory gene. Pdc2 is necessary for expression of pyruvate decarboxylase structural genes PDC1 and PDC5 and the set of THI genes involved in thiamine synthesis in yeast. Apparently, the production of thiamine and the enzymes using it as a cofactor is coordinated by Pdc2. In a homology search we have found considerable sequence similarity of Pdc2 with centromere binding proteins related to the human CENP-B. In S. pombe there are three homologues, Abp1, Cbh1 and Cbh2. All of these proteins are also involved in formation of centromere. Two-hybrid screen revealed Pdc2 interacting with proteins Mif2 and Spt4. Mif2 is an analogue of human CENP-C and Spt4 is one of the units of functional kinetochore. Interactions found by 2-H screen were confirmed by in vitro assay (co-immunoprecipitation). It was shown that the human homologues CENP-B and CENP-C interact during assembly of centromeres, which makes our observations more relevant. These findings are interesting evidence of Pdc2 involvement in chromosome segregation as a structural protein. In addition to already known facts about Pdc2 function, our results may serve as a good example of a protein acting as transcriptional factor and structural protein at the same time. Pdc2 function is indeed remarkable from an evolutionary point of view. Moreover, some observations of CENP-B localization in the euchromatine, which are unclear, could be possibly clarified by the Pdc2 model. Our results could be an indication for an additional function also for this well studied mammalian protein.

127 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

David Mosser Abstract 72 Chromatin remodeling across the proximal promoter region of the interleukin-10 gene in macrophages Xia Zhang, Shanjin Cao, Justin Edwards and David M. Mosser

Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, College Park, MD 20742, U.S.A.

We previously showed that activation of macrophages in the presence of immune complexes induces the production of high levels of IL-10, through an up-regulation of transcription. We now show that chromatin modifications at the proximal IL-10 promoter region occur following this activation. A rapid phosphorylation at Ser10 of histone 3 (H3) with a peak at 30 min was followed by a modest and slower accumulation of acetylated histones. These modifications were specific for the IL-10 promoter and were not observed as little as 1kB upstream of the transcription start site. The magnitude of the phosphorylation and acetylation of H3 at each of the 12 successive nucleosomes along the proximal promoter region of IL-10 gene was quantitated. Histone phosphorylation directly correlated with sensitivity of chromasomal DNA to micrococcal nuclease and DNase I digestion. Importantly, the remodeling of chromatin permitted the recruitment of two transcription factors, SP-1 and STAT3, to their corresponding DNA binding sites. In summary, we show that the IL-10 gene locus undergoes specific chromatin remodeling in a spatiotemporal manner along the proximal promoter region, which allows the recruitment of transcription factors, SP-1 and STAT3, to their respective binding sites. These events are critical for swift initiation of IL-10 gene transcription and subsequent mRNA accumulation.

128 Abstracts - Poster

Toshinori Nakayama Abstract 73 Long range histone modification of the Type2 cytokine gene loci in developing Th2/Tc2 cells Toshinori Nakayama and Masakatsu Yamashita

Graduate School of Medicine, Chiba University, 4F, Igakubu honkan, 1-8-1 Inohana Chuo- ku, Chiba, 260-8670, Japan

Chromatin remodeling of type 2 cytokine gene loci occurs during differentiation of naive CD4 and CD8 T cells into type 2 helper (Th2) and cytotoxic (Tc2) T cells. IL-4 production and histone hyperacetylation in IL-4-associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells, however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1 and HDAC2, and exhibited repression of IL-4 gene activation. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus.

129 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

James Nicholson Abstract 74 Structural studies of the oxidised histone octamer Christopher M. Wood1, Sirirath Sodngam1, James M. Nicholson2, Stanley J. Lambert1, Alan Brown1, Colin D. Reynolds1 and John P. Baldwin2.

1School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, U.K., 2College of Biology and Medicine, CCLRC Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, U.K.

Oxidizing free radical molecules and their related compounds are present in the human body naturally and may also be produced environmentally by, for example, pollutants and smoking. Oxidized proteins have been implicated in a number of human conditions including: Parkinson’s disease, Alzheimer’s disease and ageing processes, where the amount of oxidized protein increases exponentially with age. The body will seek to maintain a balance between oxidation and reduction by the use of redox reactions. In principle, oxidized proteins should be degraded by proteosomal and lysosomal pathways, but in reality some products either avoid or survive these routes and build up in the cell. To investigate any possible effects of oxidation on the histone proteins, intact octamers purified from chick erythrocyte nuclei were crystallized in the presence of S- nitrosoglutathione (GSNO). GSNO was chosen as the oxidizing agent as it occurs naturally in the human body, and thus any effects seen in the crystallised octamers at 2.1Å resolution can be considered to be a good indicator of that which may occur in vivo. The crystallographic results show that GSNO induces changes in the histone octamer (HO), namely an alteration of the secondary structure by the removal of hydrogen bonds from α- helices. This loss of hydrogen bonds results in a reduction in surface area when compared to the native high-resolution octamer structure [1] and also alterations to the acid-base profile of the octamer’s molecular surface. The changes in the acid-base profile of the oxidized HO may be of fundamental importance to the higher-order chromatin structure, as there are changes in the acid-base patches identified previously [2]. The crystal structure of the oxidised HO has been deposited at the Protein Data Bank (PDB) and has reference number 2ARO. To quantify the degree of secondary structure change and to correlate this with the degree of concentration of oxidizing agent, experiments were carried out using Synchrotron Radiation Circular Dichroism (SRCD). These results show that there is a sudden and pronounced loss of secondary structure within a particular range of concentration of oxidizing agent. Furthermore, the histone octamer secondary structure is restored after dialysis against histone-octamer buffer without the oxidising agent.

[1] Wood, C. M., Nicholson, J. M., Lambert, S. J., Chantalat, L., Reynolds, C. D. and Baldwin, J. P. (2005) Acta Cryst. F61, 541-545. [2] Chantalat, L., Nicholson, J. M., Lambert, S. J., Reid, A. J., Donovan, M. J., Reynolds, C. D., Wood, C. M. and Baldwin, J. P. (2003). Acta Cryst. D59. 1395-1407.

130 Abstracts - Poster

Barbara Nikolajczyk Abstract 75 The IL-1 beta gene is transcribed from a poised promoter architecture in monocytes Michael D. Liang, Yue Zhang, Daniel McDevit, Sylvia Marecki and Barbara S. Nikolajczyk

Boston University School of Medicine, L-516 Department of Microbiology, 715 Albany Street, Boston MA 02118, U.S.A.

IL-1 beta is a key pro-inflammatory cytokine implicated in the generation or exacerbation of multiple diseases. In general, cytokines are transcriptionally regulated by chromatin remodeling occuring concomitantly with mRNA synthesis. However, new data show the IL-1 beta promoter assembles into a poised chromatin structure only in monocytes, as evidenced by accessible chromatin and modification of histones packaging the locus. These parameters do not change upon transcriptional activation. Furthermore, enhancer chromatin structure does not change upon activation. Analysis of transcription factor association with either the promoter or the enhancer fails to uncover significant changes in binding by two key transcriptional activators, PU.1 and C/EBP beta. Hence the IL-1 beta promoter is packaged into a transcriptionally silent poised promoter architecture. Monocyte stimulation, known to activate the protein kinase CK2, results in recruitment of a third factor, IRF-4, to a composite PU.1/IRF site. This recruitment absolutely requires PU.1 phosphorylation, strongly suggesting that IL-1 beta-associated PU.1 is post-translationally modified by CK2 to achieve inducible gene transcription. Overall, the data show that a unique two-step mechanism activates IL-1 beta transcription in the context of cellular chromatin.

131 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Keisuke Nimura Abstract 76 Preferential association of Dnmt3l with Dnmt3a2 on chromatin in ES cells Keisuke Nimura, Yasufumi Kaneda and Kiyoe Ura

Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamadaoka Suita, Osaka, 565-0871 Japan

In mammals, DNA methylation is involved in fundamental processes such as the silencing of gene expression and transposable elements, X-inactivation, genomic imprinting and tumorigenesis. However, control mechanisms of DNA methylation are still largely unknown. Dnmt3l, a Dnmt3 family protein sharing homology with DNA methyltransferase 3a (Dnmt3a) and Dnmt3b but lacking enzymatic activity, has been shown to be required for correct establishment of DNA methylation patterns in germ cells. In this study, we demonstrate a physical link between Dnmt3l and one of Dnmt3 enzymes, Dnmt3a2. Whereas in wild-type ES cells endogenous Dnmt3l is concentrated at heterochromatic foci, it fails to localize to these regions in the absence of only Dnmt3a. In Dnmt3a/Dnmt3b double deficient ES cells, Dnmt3l was defused over the nucleus and cytoplasm and expression of Dnmt3a2, but not Dnmt3a1 or Dnmt3b, relocalized Dnmt3l at hetrochromatic foci. We also show biochemical interaction between Dnmt3l with Dnmt3a2 in ES cell nuclei. Our results demonstrate possible control mechanisms of DNA methylation in which Dnm3a2 recruits Dnmt3l onto specific genomic regions, leading active DNA methylation.

132 Abstracts - Poster

Esperanza Nunez Abstract 77 Repetitive elements, transcriptional control and cell diversity Esperanza Nunez and Victoria Lunyak

University of California at San Diego, CMM-West, Room 345, 9500 Gilman Dr., La Jolla, CA 92093-0648, U.S.A.

The published sequence of the human genome revealed that coding sequences comprise less than 5% of it, whereas repeating sequences account for at least 50% of the genome. Among different types of repeats dominate transposon-derived repeats (~45% of the genome), and, in particular, retroelements including short interspersed nuclear elements, SINEs (~13%), long interspersed nuclear elements, LINEs (~20%), and LTR-containing retroelements (~8%). Provided that regulatory signals entering the nucleus encounter chromatin, not DNA, and the rate-limiting biochemical response that leads to activation of gene expression in most cases involves alterations in chromatin structure; the key issue then becomes that nucleosomes act as a barrier to recognizing binding sites on the DNA (for RNAP or for regulatory proteins). If nucleosomes are positioned so as to avoid the promoter, then clearly nucleosome-mediated repression is avoided. This raises the question of how nucleosomes are positioned, since at first view they would seem to be quite non- specific. Probably some regulatory proteins bind to the DNA before nucleosome assembly, thus phasing the nucleosomes and leaving key parts of the DNA exposed. This implies a competition between regulatory proteins and nucleosomes during formation of new chromatin. The presence of SINEs, especially Alu sequences, in the protein coding regions has been controversial as is their biological meaning and importance. We have preliminary data suggesting the maintained expression of a SINE B2 repeat in the murine pituitary gland as early as E12.5 where it may play a role as a possible boundary determinant. Recent evidence suggests that a main effect of repetitive elements is their ability to influence transcription of neighboring genes. The SINE B2 contains a putative promoter that can initiate transcription of flanking genomic DNA, if not act as RNA regulator to repress transcription. Furthermore, its transcript may be susceptible to epigenetic silencing, which is often stochastic and incomplete, resulting in complex patterns of transcription; which in the case of the pituitary gland this may have acute effects in cell line fate and determination.

133 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Laura O’Neill Abstract 78 Distinctive histone modifications mark specific chromosomes in both human and mouse models Laura P.O’Neill, Matthew ver Milyea, Milan Fernando and Bryan Turner

Birmingham University, IBR, Vincent Drive, Birmingham, West Midlands, B15 2TT, U.K.

The epigenetic code is becoming more complex, with diverse modifications to the N- terminal tails of the core histones correlating with specific aspects of gene regulation. The modification itself, the histone involved and the amino acid residue are all important determinants of these epigenetic marks. Using adult and embryonic stem cells (ES cells) from human and mouse model systems, we have continued to investigate the roles of histone acetylation and methylation in gene silencing and their distribution along chromosome domains. Female ES cells inactivate genes on one of their two X chromosomes as they differentiate in culture. Moreover it has been shown that the active and inactive Xs are differentially methylated at H3 lysines 4, 9 and 27. Using antisera that distinguish lysines with different levels of methylation, we have now been able to show that the X chromosomes carry another mark, irrespective of activity, which distinguishes them from the other chromosomes. Moreover, other chromosomes also carry distinctive combinations of histone marks. In particular chromosome 19, a chromosome rich in genes, shows a distinctive pattern of histone modifications, which are unique to this chromosome. Further development of the N-ChIP procedure has also allowed us to investigate whether the epigenetic marks that we see on specific genes within embryonic stem cells are the same as those isolated from the inner cell mass of a blastocyst from which these cells were originally derived.

134 Abstracts - Poster

Hisanobu Oda Abstract 79 Generation and characterization of Set9 and PR-Set7 conditional gene knockout mice Hisanobu Oda1, Michael M. Shen2 and Danny Reinberg1

1Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, 2Center for Advanced Biotechnology and Medicine and Department of Pediatrics, UMDNJ/RWJMS, RWJMS Research Building, 683 Hoes Lane, Piscataway, Middlesex 08854, U.S.A.

The lysine methyltransferase, Set9, was isolated based on its ability to methylate histone H3 lysine 4; yet Set9 cannot use nucleosomal substrates. We recently found that the tumor suppressor protein p53 is a considerably better substrate for Set9 than is histone H3. Set9 may therefore be involved in various biological processes other than chromatin modulation in vertebrates. Another methyltransferase, PR-Set7 mediates monomethylation of H4-K20. PR-Set7 mutants in Drosophila die at the larval-to-pupal transition and exhibited phenotypes consistent with a failure to complete cell division in their imaginal discs. We have now generated mice carrying a conditional deletion for the gene encoding Set9 or that for PR- Set7. Our goal is to define the function of each of these monomethyltransferases in vivo.

135 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Søren Ottosen Abstract 80 Bromodomain protein 7 (Brd7) promotes nucleolar disruption in response to DNA breaks

Søren Ottosen, Randy Luciano, Shahana Mahajan and Angus Wilson

Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A.

HCF-1 is a chromatin-associated factor required for VP16 to activate herpes simplex virus immediate-early promoters. HCF-1 is also involved in regulation of cellular gene expression, interacting with a number of DNA-binding proteins (LZIP, Sp1, GABP, Krox20), as well as the mSin3/HDAC and Ash2/Set1/MLL methyltransferase complexes. Loss of HCF-1 leads to a G1/G0 cell cycle arrest and defects in chromatin compaction prior to mitosis. The HCF-1 protein is composed of two subunits (HCF-1N and HCF-1C) derived from a single large precursor by internal proteolytic cleavage. We found that bromodomain- containing protein 7 (Brd7) interacts with the C-terminal fibronectin repeats of HCF-1C. Elevated expression of Brd7 leads to an accumulation of HCF-1 in the nucleoli, forming distinctive subnucleolar granules. This coincides with visible changes in nucleolar structure and release of nucleolar proteins into the nucleoplasm. Similar changes occur in response to DNA double-stranded breaks and can be blocked by depletion of either Brd7 or HCF-1. Brd7 expression also results in stabilization of p53 and enhanced activation of p53- responsive promoters. Deletion analysis identified separate domains within Brd7 responsible for HCF-1 subnucleolar granule formation and nucleolar disruption. Brd7 contains a single acetyl-lysine binding bromodomain and treatment of cells with histone deacetylase (HDAC) inhibitors prevents Brd7-mediated activation of p53-responsive genes. This effect is alleviated by mutation of conserved residues within the acetyl-lysine binding pocket. Increased levels of acetylation leads to accumulation of Brd7 in discrete nucleoplasmic speckles and a marked decrease in the overall diffusion rate as measured by fluorescence recovery after photobleaching (FRAP). We propose that in undamaged cells, Brd7 is associated with acetylated chromatin and held in an inactive form. Recognition of double-stranded DNA breaks leads to a change in the acetylation state of these tethers, releasing or relocalizing Brd7 so that it can initiate nucleolar disruption and p53 stabilization.

136 Abstracts - Poster

Mahadeb Pal Abstract 81 The role of TFIIB and the transcription bubble during the earliest stages of transcription by RNA polymerase II Mahadeb Pal and Donal Luse

LRI, Cleveland Clinic Foundation, NE/20, LRI, 9500 Euclid Avenue, Cleveland, Cuyahoga 44195, U.S.A.

We have investigated the role of the transcription bubble in the earliest stages of RNA synthesis using a series of transcription complexes stalled on TATA box promoters with varying TATA to +1 spacing. We found that regardless of the spacing, the upstream edge of the bubble is set at ~20 bp downstream from the TATA box. This edge remains fixed while the bubble expands in the downstream direction, until the bubble grows to more than 17 bases and the transcript length reaches at least 7 nt. At this point the upstream half of the bubble abruptly reanneals (bubble collapse). The bubble collapse transition restores transcription complex stability, confers independence from the TFIIH helicase and greatly diminishes an elongation block (in the region of +7 to +9 nt of RNA synthesis) apparently induced by the B-finger domain of TFIIB. These studies revealed that (1) bubble collapse is a critical determinant of promoter clearance by RNA polymerase II and (2) TFIIB plays an important part in promoter clearance (Pal et al., Mol. Cell 19, 101). Recent structural studies indicate that the B-finger of TFIIB initially occupies the RNA exit channel of pol II and must be displaced by the growing transcript. Our data are consistent with release of TFIIB following the bubble collapse transition. However, when we attempted to directly detect TFIIB release using in vitro transcription with purified transcription factors and bead- bound templates, we found somewhat surprisingly that TFIIB is not released upon bubble collapse or immediately downstream (i.e., +30). Thus far, when using purified components we have been able to demonstrate TFIIB release only when pol II has run off the template. We are currently exploring the possibility that supplementing our reactions with nuclear extract will drive TFIIB release in early elongation complexes.

137 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Maria V. Panchenko Abstract 82 pVHL partner and transcriptional co-activator Jade-1 is a novel substrate for HAT TIP60. Maria V. Panchenko and Herbert T. Cohen.

Boston University School of Medicine, Evans Biomedical Research Center, 650 Albany Street, Boston, MA 02118-2393, U.S.A.

Jade-1 was identified as a protein partner of the von Hippel-Lindau tumor suppressor pVHL. We recently demonstrated that PHD zinc finger protein Jade-1 is localized to the nucleus and possesses intrinsic transcriptional activity. Strikingly, over-expression of the full length Jade-1 in epithelial cells resulted in a global and specific hyperacetylation of endogenous histone H4. PHD zinc fingers appeared indispensable for both transcriptional and HAT- associated Jade-1 functions. Moreover, mutant Jade-1 possesses a dominant negative phenotype, suggesting that Jade-1 may be a part of a HAT complex. In support of this notion, we showed that TIP60 binds and further enhances the HAT-associated function of wild type but not mutated Jade-1. Others have demonstrated that the PHD zinc finger of CBP binds nucleosomal histones in vitro. We hypothesize that PHD fingers provide the association of the Jade-1-TIP60 complex with the nucleosome thereby promoting acetylation of nucleosomal histones by TIP60. We have further investigated outcomes of Jade-1 interactions with TIP60. The endogenous Jade-1 protein band visualized by western blot has diffused appearance characteristic for a post-translationally modified protein. Since Jade-1 polypeptide has numerous lysine residues we investigated whether Jade-1 can be acetylated. Western blot analysis of total cell extracts revealed that the protein band corresponding to endogenous Jade-1 is readily recognized by the Acetyl-lysine antibody, suggesting that Jade-1 can be acetylated. The levels of endogenous Jade-1 acetylation were increased after incubation of cells with Trichostatin A, an inhibitor of HDACs, suggesting that Jade-1 undergoes acetylation/deacetylation mediated by endogenous HATs/HDACs. Because Jade-1 interacts with TIP60, we examined whether Jade-1 can be a substrate for TIP60. Ectopic expression of TIP60 resulted in enhanced levels of acetylated endogenous as well as overexpressed recombinant Jade-1, suggesting that TIP60 can acetylate Jade-1. Surprisingly, Gal4-Jade-1-mediated transcriptional activation of a viral promoter was completely abolished in the presence of wild type TIP60. The potential relationship between TIP60-mediated acetylation of Jade-1 and inhibition of Jade-1 transcriptional function is currently being investigated and will be discussed.

138 Abstracts - Poster

Tej Pandita Abstract 83 Mammalian ortholog of Drosophila MOF is critical for embryogenesis and essential for DNA damage responses A. Gupta1, T. Geraldine Guerin-Peyrou2, M. Agarwal1, G. G. Sharma1, R.K. Pandita1, T. Ludwig2 and T. K. Pandita1

1Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, MO 63108, U.S.A., 2College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A.

Tumor cells and normal cells have significant differences in their chromatin structure. Understanding the role of chromatin modifying factors in cellular responses to DNA damage at the molecular level will provide invaluable insights into questions of both how cancers start and how to cure cancers. Recently, we have identified a chromatin-modifying factor hMOF the human ortholog of Drosophila MOF gene which interacts with ATM. Cellular exposure to IR enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4, independent of ATM function. Inactivation of hMOF results in abrogation of ATM function. Based on the fact that hMOF is involved in ATM function, and tumors show loss of histone H4K16 acetylation, we hypothesize that hMOF is involved in the oncogenic transformation. Since MOF is involved in DNA damage response as well as transcription, its function is indispensable for development, because Mof-deficiency in mouse embryos result in early embryonic lethality. We will discuss the function of mouse Mof (mMof), and the functional links among mMof, histone H4K16 acetylation and spontaneous as well as IR-induced tumor formation.

139 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Biranchi Patra Abstract 84 Genomewide localization of histone modifications during meiosis Biranchi Patra and Animesh Ray

Keck Graduate Institute, 535 Keck, 535 Watson Dr., Claremont CA, 91711 India

Chromatin remodeling by the posttranslational modification of histones has emerged as a major mechanism of eukaryotic gene expression. A diversity of histone modifications such as acetylation, methylation, ubiquitylation, sumoylation are known to dynamically regulate gene activity. Recent studies indicate that histone modification may be important for selection of double strand break sites during meiotic recombination. However, the exact role of histone modification in selecting the DNA break sites is not fully understood. Although much progress has been made on characterization of histone modifying complexes with relation to transcriptional activation and repression in the yeast Saccharomyces cerevisiae and other model systems, much less is known about the chromatin condensation states and transcriptional activity genomewide. In particular, the ubiquitin and SUMO modification of histones are dynamic and reversible. SUMO modification of histone H4 is generally suggested to repress gene expression and ubiquitin modification of histone H2B has generally been associated with gene activation. We report here our progress in genome-wide mapping of sites of H2B ubiquitylation and H4 sumoylation during sporulation of the budding yeast Saccharomyces cerevisiae.

140 Abstracts - Poster

Rushad Pavri Abstract 85 A fully reconstituted chromatin transcription system for the detailed study of epigenetic mechanisms Rushad Pavri, Bing Zhu and Danny Reinberg

University of Medicine and Dentistry of New Jersey , School of Public Health, 683 Hoes Lane, Piscataway, Middlesex 08854, U.S.A.

We have developed a fully reconstituted activator-dependent chromatin transcription system using the endogenous promoter of the RARbeta2 gene and the RSF chromatin assembly system. We previously showed that ligand-dependent transcription from this promoter requires PARP-1 as a novel cofactor. These studies employed crude S190 Drosophila extract to assemble chromatin, limiting our ability to fully characterize the system. We have now fully reconstituted the chromatin assembly reaction using recombinant RSF. In combination with our established reconstituted transcription system comprised of highly purified general transcription factors, RNA polymerase II, Mediator and PARP-1, this fully reconstituted system allows us, for the first time, to study the effects of covalent histone modifications on gene expression in vitro both activation and repression - using RARbeta2 as a paradigm. The high purity of this system enables us to determine the exact contribution of each modification and the minimal set of factors required for gene expression. We find that ligand-dependent transcription is dependent upon histone acetylation and chromatin remodeling as well as upon the histone chaperone FACT. We have also investigated the interplay between histone methylation and ubiquitination on ligand-dependent transcription. Using in vitro ChIP assays, we have begun to correlate RARbeta2 gene expression with the presence or absence of specific histone modifications on the RARbeta2 promoter. Our data demonstrate a link between specific histone modifications and ligand-dependent gene expression with novel mechanistic implications.

141 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Inka Pawlitzky Abstract 86 A novel regulatory region 5’ of the mouse IgH locus Inka Pawlitzky, Christina Angeles, Andrea Siegel, Michelle Stanton, Roy Riblet1 and Peter Brodeur2

Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston MA 02111, U.S.A., 1Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, U.S.A., 2Department of Pathology, Tufts University School of Medicine, Boston MA 02111, U.S.A.

The IgH locus is encoded as gene segments that are assembled during B cell development by a series of recombination events to form continuous heavy chain coding sequences. The targeting of gene segments within Igh is controlled at the level of accessibility to the recombinase machinery. Regulated changes in the chromatin structure that cause a particular region or gene segment to become accessible for recombination ultimately control which gene segment can be targeted for rearrangement. These processes involve cis- acting elements, most notably the E and the 3IgH regulatory region, located near the 3 end the locus. The presence of additional control elements has been postulated to regulate rearrangements of the Vh gene array that extends to the 5 boundary of Igh. To search for novel Igh elements, we physically mapped the most D-distal Vh segments and scanned the 5 flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites approximately 30 kb upstream of Igh. Using cell lines and normal B cell populations we show that the detection of one site, HS-1, is restricted to pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS-1 for PU.1 and Pax5 specifically bind these factors in vitro and these proteins are recruited to HS-1 sequence in a pro-B cell-specific manner. In addition, the Pax5 site is required for repression of transcriptional activity observed in HS-1 containing reporter constructs. Taken together, the position, pro-B cell specificity, and interaction with PU.1 and Pax5 strongly suggest that the identified hypersensitive sites mark a novel 5 IgH regulatory region potentially involved in Vh gene rearrangements.

142 Abstracts - Poster

Laura Perez-Burgos Abstract 87 Domain organization at the chicken β-globin locus Laura Perez-Burgos1, Thomas Jenuwein1, Michael D. Litt2 and Gary Felsenfeld3

1Institute of Molecular Pathology (I.M.P.), Dr. Bohr Gasse, 7, 1030 Vienna, Austria, 2Indiana University School of Medicine - Muncie at Ball State University, 221 N. Celia Ave., MT 201, Muncie, IN 47306, U.S.A., 3N.I.H., Building 5, Room 212, Bethesda, MD 20892-0540. U.S.A.

Histones are subject to a series of post-translational modifications on their N-termini, leading to different phyisological outcomes. For instance, dimethylated lysine 4 of histone H3 (H3-K4) has been found at active loci, whereas trimethylated lysine 9 (H3-K9) is a hallmark of pericentric heterochromatin and trimethylated lysine 27 (H3-K27) is enriched at the inactive X chromosome (Xi). The chicken β-globin locus is an ideal system to study the dynamics of histone modifications within different chromatin domains during development. It encompasses a folate receptor gene, a condensed chromatin region, an insulator (HS4), a series of enhancers (HS3-1) and the globin genes, which are subject to developmental regulation during erythropoeisis. Initial ChIP analysis over this region correlated enhanced H3-K4 dimethylation and H3- and H4 acetylation with expression of the mature β-globin genes, while H3-K9 dimethylation barely changed during development (Litt et al., 2001). We have expanded this study by performing ChIP analysis with our antibodies that are highly specific for mono-, di- and trimethylation at either H3-K9 or H3-K27. In 6C2 cells (an immature progenitor cell line that does not express any globin genes), a coexistence of repressive marks (di- and tri-H3-K9 and di- and tri-H3-K27) was observed over the condensed chromatin region and the silent β-globin genes, while the active folate receptor gene was devoid of these marks. Strinkingly, in red blood cells, which express the mature β- globin chains, H3-K27 di- and tri-methylation were absent from the actively transcribed genes, while persisting over the condensed chromatin region (together with H3-K9 dimethylation). These observations clearly define an anti-correlation between di- and trimethylated H3-K27 and the levels of transcription at the chicken β-globin locus. Moreover, we have deconstructed the β-globin locus into two distinct domains. First, a region of ‘constitutive’ heterochromatin that is enriched in repressive marks (dimethylated H3-K9 and di- and trimethylated H3-K27) at all stages of development. Second, a region of ‘facultative’ heterochromatin, where these repressive marks disappear to allow transcription at the appropriate time during development. Our observations were confirmed with another progenitor cell line (HD24) and with 10-day chick embryo brain cells.

143 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Yuri Postnikov Abstract 88 Modulation of histone modifications by chromatin-binding architectural proteins Yuri Postnikov and Michael Bustin

National Institutes of Health, 37 Convent Dr., Bethesda, MD 20892, U.S.A.

The linker histone H1 and members of the high mobility group (HMG) proteins alter the structure of chromatin and modulate the interaction of regulatory factors with their nucleosomal targets. We find that histone H1 inhibits the ability of PCAF to acetylate H3K14 in chromatin, but not in purified histones. HMGN1, but not HMGN2, inhibits the phosphorylation of H3S10 and enhances the acetylation of H3K14 both in vivo and in vitro. HMGN1- dependent alterations in posttranslational modifications can be detected both genome-wide, and at the single gene level. The results suggest that the binding of HMGN to nucleosomes induces steric changes that alter the ability of histone modifying complexes to modify specific residues in histone tails. FRAP analysis of living cells indicates that H1 and HMG proteins function within a network of proteins that compete for nucleosome binding sites. We suggest that the competitive interactions among members of this network affect the ability of modifying enzymes to access and modify their nucleosomal targets.

144 Abstracts - Poster

Brendan Price Abstract 89 The Tip60 histone acetyltransferase is essential for the acetylation and activation of the ATM protein kinase Brendan D. Price

Dana-Farber Cancer Institute JF222, 44 Binney St., Boston, MA 02115, U.S.A.

The ATM protein kinase is essential for the repair of DNA strand breaks. The repair of DNA strand breaks involves the remodeling of the chromatin structure. Structural changes in chromatin during DNA repair have been linked to post-translational modification of histones by phosphorylation, methylation or acetylation. These histone modifications can create binding sites for DNA repair complexes, including 53BP1, NuA4 and INO80. In addition, activation of the ATM protein kinase can be brought about by changes in chromatin structure. However, the signal transduction pathway linking DNA strand breaks to activation of ATMs kinase activity is not clearly defined. Here, we investigated the role of the Tip60 Histone Acetyltranferase (HAT), a component of the NuA4 complex, in the activation of the ATM protein kinase. ATM is rapidly acetylated in a Tip60-dependent manner following DNA damage. Suppression of Tip60 blocks the activation of ATMs kinase activity and prevents the ATM-dependent phosphorylation of p53 and chk2. Further, inactivation of Tip60 sensitizes cells to DNA damage. ATM and Tip60 exist as a preformed complex in cells; however, interaction between ATM and Tip60 is not regulated in response to DNA damage. Instead, the HAT activity of the ATM-Tip60 complex is specifically activated by DNA damage. This activation of Tip60 requires the chromodomain of Tip60, a domain which can associate with methyl-lysine residues. Finally, the ATM-Tip60 complex is biochemically distinct from the NuA4-Tip60 complex, and functions upstream of NuA4. The results demonstrate that Tip60 is required for the acetylation and activation of the ATM- Tip60 complex following DNA damage. This activation requires the chromodomain of Tip60, suggesting that the ATM-Tip60 complex associates with exposed methyl-lysine residues on histones at sites of DNA damage. These results describe a mechanism to link changes in chromatin structure to the activation of ATM-dependent DNA damage response pathways.

145 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Michael Rehli Abstract 90 Genome-wide profiling of CpG-methylation by methyl-CpG iimmunoprecipitation Claudia Gebhard, Lucia Schwarzfischer, Thu-Hang Pham, Reinhard Andreesen and Michael Rehli

University Hospital, H1 Forschungsbau, F.-J.-Strauss Allee 11, Regensburg, Bayern 93042, Germany

Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor-suppressor genes. We developed a novel and robust technique that allows the detection of CpG-methylation in limited DNA samples, without applying methylation-sensitive restriction endonucleases or bisulfite-treatment. The approach is based on a recombinant, methyl-CpG binding, antibody-like protein that efficiently binds native CpG-methylated DNA depending on its degree of CpG methylation. Its application in methyl-CpG immunoprecipitation (MCIp) facilitates the monitoring of CpG- island methylation either on single candidate gene level (in combination with real-time PCR) or on a genome wide level (in combination with CpG-island promoter microarrays). Genome wide methylation profiling of myeloid leukemia cell lines identified a large number of genes with aberrantly methylated CpG islands. Strikingly, more than half of the identified genes show extremely low or no expression in normal cells, suggesting that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Several individually tested genes were also affected in primary blast cells from AML patients, suggesting that our approach can identify novel potential disease markers.

146 Abstracts - Poster

Kristen Riley Abstract 91 Involvement of the TAC1 complex in transcription elongation Kristen M. Riley1, Sheryl T. Smith2, Svetlana Petruk1, Sususma Hirose3, Hugh W. Brock4, Alexander Mazo1

1Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA, 2Wistar Institute, Philadelphia, PA 19107, U.S.A., 3Department of Developmental Genetics, National Institute of Genetics and Department of Developmental Genetics, Graduate University for Advanced Studies, Mishima, Shizuoka-ken 411-8540, Japan, 4Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada, V6T 1Z4

The Drosophila heat shock genes are rapidly induced upon heat shock, a result of a special, nucleosome-free promoter structure. For elongation to proceed, transcription through nucleosomes downstream of the start site requires the action of a number of elongation factors. A possible role for the TAC1 chromatin-modifying complex in the elongation process has recently been described (Smith et al., 2004). TAC1 localizes to the coding region of hsp70 following heat shock, where it methylates and acetylates histones and actively promotes transcription. We have examined how TAC1 functions in the network of elongation factors by determining how other factors affect its recruitment and activity. We have found that recruitment of TAC1 to hsp70 requires the elongation factor FACT, the kinase Cdk7, which phosphorylates RNA Polymerase II and is required during early elongation steps, and the epigenetic regulator Asx. Interestingly, Asx also requires TAC1 for its recruitment to hsp70, indicating that there is a cooperative mechanism of TAC1 and Asx recruitment. The ability of TAC1 to perform its methylation activity requires the enzyme PARP, which is thought to facilitate loosening of chromatin structure. In addition, examination of TAC1 recruitment in another transcriptional model, the homeotic gene Ubx, shows an almost complete convergence with the heat shock model. The Ubx model also shows that TAC1 is required for recruitment of Cdk7 and FACT, suggesting that there may also be a cooperative recruitment mechanism for these factors with TAC1. Furthermore, staining of polytene chromosomes from larvae lacking Trx shows that TAC1 is required on a broader scale for association of Cdk7 and FACT with their target genes. Our data together suggests that TAC1 is part of the network of interacting factors involved with promoting elongation.

147 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Flavio Rizzolio Abstract 92 Chromatin alteration on POF associated X/A balanced translocation. F. Rizzolio1, S. Alboresi1, S. Gilli1, C. Sala1, S. Bione1,2, T. Pramparo3, M.Goegan1, O .Zuffardi3 and D.Toniolo1.

1DIBIT Ospedale San Raffaele, Milano, 2Istituto di Genetica Molecolare-CNR, Pavia, 3Dipartimento di Biologia Umana ed Ereditaria, sezione di Biologia Generale e Genetica Medica, Universit di Pavia, Pavia, Milan, Lombardia 20132, Italy

Premature ovarian failure (POF) is a heterogeneous disorder characterized by menopause before the age of 40 years. Different chromosome anomalies associated with POF involve the X chromosome. In our laboratory we have mapped 25 X/A balanced translocations interrupting the X chromosome, in the Xq21 critical region. Since only three genes were found interrupted, a position effect on flanking genes was considered. In this work we have analyzed a region of 5 Mb in Xq21 were 13 breakpoints were mapped. RNA in situ hybridization showed that none of the X linked genes is highly or specifically expressed in ovarian follicles, suggesting that the ovary expressed genes responsible for the phenotype may be located on autosomes.We mapped by FISH the autosomal breakpoints of 4 patients, on chromosome 1p35.3, 2q14.2, 3p21 and 5q35. Expression analysis by RNA in situ hybridization demonstrated that many of the genes flanking the autosomal breakpoints were expressed in the ovary and indicated that the phenotype could be due to a position effect of the X chromosome on autosomal genes. To confirm the possible position effect of these breakpoints, we analyzed H3 lysine 9/14 acetylation, H4 lysine 5/8/12/16 acetylation and H3 Lysine 4 methylation on the promoter regions of flanking genes, in lymphoblastoid cells of patients and controls. The preliminary results of this analysis showed that the chromatin modifications were altered in all patients cells compared to controls and tentatively confirmed that the X chromosome critical region may have a chromatin organization or may contain regulatory elements that could alter the expression of autosomal genes involved in the breakpoints.

148 Abstracts - Poster

Orit Rozenblatt-Rosen Abstract 93 Paf1 complex is associated with mRNA processing machinery. Orit Rozenblatt-Rosen1, Suraj J Nannepaga1, Matthew Hoffman2, Kristine Nordick2, Syuzo Kaneko3, Takashi Sugimoto3, Christina M Hughes1, James H. Resau4, Jim Manley3, Judith Jaehning2 and Matthew Meyerson1.

1Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts, U.S.A., 2Department of Biochemistry and Molecular Genetics, Molecular Biology Program, University of Colorado Health Science Center, B121, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A., 3Department of Biological Sciences, Columbia University, New York, NY 10027, U.S.A., 4Analytical, Cellular and Molecular Microscopy Laboratory, Van Andel Institute, Grand Rapids, Michigan, U.S.A.

Human Cdc73 (hCdc73), the product of the HRPT2 tumor suppressor gene is part of the human Paf1 complex. Paf1 complex is known to be important or histone modifications and other co-transcriptional events. Yeast and human Paf1 complexes both contain Cdc73, Ctr9, Paf1 and Leo1protein subunits. The yeast complex contains an additional subunit Rtf1 and the human complex was recently reported to include the Ski8 protein. We now show that hCdc73 is co-purified with mRNA processing factors. Immunoprecipitation and immunodepletion experiments as well as glycerol gradient fractionation further confirm that these complexes interact. Taken together these results suggest that the Paf1 complex serves as a platform between RNA polII and factors modulating mRNA processing.

149 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Ingemar Rundquist Abstract 94 Analyses of linker histone - chromatin interactions in situ in different cell systems Ingemar Rundquist1 and Herbert Lindner2

1Division of Cell Biology, Linkpings universitet, Sweden, 2Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Austria

The linker histones, commonly referred to as H1, are involved in the formation and maintenance of the higher order structure of the chromatin fiber and most likely also in epigenetic modulation of gene expression. In mammals, this family consists of eight subtypes, H1.1-H1.5, H1t, H1 and H1oo. The highly specialized isoforms H5 (in avian species) and H1 accumulate in some terminally differentiated cells. In avian and amphibian erythrocytes, expressing H5 and H1 respectively, these proteins have been linked to cessation of cell proliferation and condensation of chromatin. We have studied the interaction between linker histones and chromatin in a number of cell types, using 46- diamidino-2-phenylindole (DAPI) as an indirect cytochemical probe for linker histone affinity in situ, in combination with high performance capillary electrophoresis and reverse-phase high performance liquid chromatography. Significant differences were detected between some cell types. The results show that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes. This difference may possibly be explained by the high content of arginine-rich H5 in chicken erythrocytes. Our results also indicate that the affinity decreased in differentiating frog erythrocytes, showing the lowest affinity in terminally differentiated cells with highly condensed chromatin. Furthermore, in cultured human fibroblasts the linker histones showed a relatively high affinity for chromatin. However, in highly condensed metaphase chromosomes, the affinity was significantly lower compared to interphase cells. We have also analyzed linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. The results show that the exogenous linker histones were bound with somewhat lower affinity than the native ones and that in vitro phosphorylated linker histones were bound with substantially reduced affinity. Our results also indicate a lack of correlation between linker histone affinity and chromatin condensation.

150 Abstracts - Poster

Michael Scher Abstract 95 SirT3 is A nuclear histone deacetylase that translocates to mitochondria upon stress Michael Scher1,3, Alejandro Vaquero1, Dong-Hoon Lee1,3, Yan Li1,3, Hediye Erdjument-Bromage2, Paul Tempst2 and Danny Reinberg1.

1HHMI-Robert Wood Johnson Medical School. University of Medicine and Dentistry of New Jersey, Piscataway, NJ, 08854, U.S.A.. 2Molecular Biology Program, Memorial Sloan- Kettering Cancer Center, New York, NY 10021. 3UMDNJ-Graduate School of Biomedical Science Piscataway, NJ, 08854, U.S.A..

Sir proteins are essential in the silencing and aging processes in yeast. Sir proteins were discovered in budding yeast as factors required for the establishment of epigenetic repression through specialized heterochromatin formation within portions of the genome, such as telomeric regions, mating type loci, and nucleolar rDNA. Sir2 is involved in the silencing of all three of these regions dependent upon its enzymatic NAD+-dependent deacetylase activity defined by a domain of approximately 250 residues. In humans, there are at least 7 Sir2-like proteins (SirT1-7). The human Sir2 related proteins appeared to target mainly non-histone substrates: SirT1 deacetylates transcription factors, SirT2 localizes to the cytoplasm and deacetylates -tubulin, and SirT3 localizes to the mitochondria. However, we have shown that SirT1 also functions in the formation of facultative heterochromatin through its deacetylation of H3-K9 and H4-K16 and interaction with and deacetylation of histone H1. Contrary to reports that SirT3 localizes to the mitochondria, we have found that SirT3 primarily localizes to the nucleus using highly specific antibodies against the endogenous protein. In addition, certain stimuli trigger SirT3 translocation to the mitochondria. Using microarray analyses together with chromatin immunoprecipitation assays, we also show that SirT3 regulates a specific set of genes in the nucleus. Both the full-length and truncated forms of SirT3 can target H4-K16 for deacetylation in vitro. Overexpression of SirT3 was shown previously to increase the expression of genes involved in mitochondrial function, and promote aging. Our work expands on this and demonstrates a connection between SirT3 and chromatin.

151 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Gunnar Schotta Abstract 96 H4-K20 methylation: a mark important for mouse development? Gunnar Schotta, Stefan Kubicek, Roopsha Sengupta, Michaela Pagani and Thomas Jenuwein

Institute of Molecular Pathology, 7 IMP, Dr. Bohrgasse, Vienna, 1030 Austria

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, the combination of H3-K9 trimethylation and H4-K20 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals.In previous studies we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases, which localize to pericentric heterochromatin in a Suv39h-dependent manner. They act as nucleosome specific H4-K20 trimethylating enzymes suggesting a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. In addition to gene repression, H4-K20 trimethylation has recently been implicated in other biological processes such as DNA damage repair and cancer progression. In order to address these potential functions for mouse development we generated knock-out mice for both Suv4-20h1 and Suv4-20h2. Mice deficient for Suv4-20h1 die perinatally however; Suv4-20h2 null mice do not show obvious defects. The phenotype of Suv4-20h1 is more severe than that of Suv39h double null mice, which may suggest additional functions for this enzyme, independent of pericentric heterochromatin organization.Immunofluorescence and mass-spec analyses of wt vs. Suv4-20h1 and Suv4-20h2 null MEFs revealed that the enzymes could have different in vivo activities. Suv4-20h2 appears to be the major enzyme responsible for pericentric H4- K20 trimethylation, while cells deficient for Suv4-20h1 show a reduction in H4-K20 dimethylation. The different specificities of the enzymes could in part explain the phenotypes observed. However, the identification of H4-K20 di- and trimethylation targets may provide greater insight into the function of these enzymes.

152 Abstracts - Poster

Bernd Schuttengruber Abstract 97 Effect of long-range chromosomal interactions mediated by the Fab-7 element on its target chromatin Bernd Schuttengruber, Fredric Bantignies and Giacomo Cavalli

Institut de Genetique Humeine, 141, rue de la Cardonille, Montpellier 34396, France

Proteins of the Polycomb group (PcG) are key players for the maintenance of cell identity throughout development. In Drosophila, these proteins exert their function through binding to specific DNA elements, known as PcG response elements (PREs). Fab-7 is a well defined chromosomal element that contains a PRE and is involved in the regulation of the homeotic gene Abdominal-B (Abd-B). In addition to an action in cis, homologous PREs can interact over long distances, even if located on different chromosomes. In particular, we show by two-colour DNA FISH that a transgene containing the Fab-7 element located on chromosome 2, can pair with the endogenous element at the Bithorax complex (BX-C) located on chromosome 3, leading to strong and stable repression of a mini-white marker gene which is heritable through mitotic and meiotic cell division. The transgene can be switched to a derepressed state by deletion of the endogenous Fab-7 element that leads to unpairing with the transgene. Here, we investigate what chromatin marks are associated to silencing induced by long-range interactions and how these marks behave during changes in the nuclear localization of the transgene upon unpairing. We could not observe significant differences in the recruitment of PcG proteins to the transgene in the presence or absence of Fab-7 pairing interactions. Similarly, methylation of histone H3 on lysine 27, a hallmark of PcG-mediated silencing, and acetylation levels of core histones are not changed as a function of long-distance interactions. We are currently investigating further chromatin marks as possible candidates to be involved in long-distance chromosomal interactions. Further, by combining DNA FISH with immunostaining of PcG proteins, we test whether the silenced Fab-7 transgene co-localize with PcG foci, and how nuclear compartmentalization to these domains of polycomb action changes upon activation of the transgene.

153 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Alexandra Schulmeister Abstract 98 Histone H3.3 phosphorylation during mitosis and meiosis in the urochordate Oikopleura dioica Alexandra Schulmeister, Philippe J. Ganot and Eric M. Thompson

Sars International Centre, 55 High Technology Centre, Thormohlensgt. 55, Bergen, Hordaland N-5008, Norway

Histone H3.3 phosphorylation during mitosis and meiosis in the urochordate Oikopleura dioicaSars International centre, University of Bergen, Thormhlensgt. 55, N-5008 Bergen, NorwayThe histone variant H3.3 differs from the major histone H3 (H3.1) in five amino acids, where alanine at position 31 is replaced by serine. In contrast to H3.1, the variant H3.3 is expressed throughout the cell cycle and is deposited at transcriptionally active loci independent of S-phase (1, 2). Recent data from mammalian cells suggest that, when phosporylated at Ser-31, H3.3 also plays a role in mitosis (3). Our previous findings, that H3.3 becomes a major H3 variant in the prominent endocycles of the planktonic urochordate Oikopleura dioica, have led us to investigate its state of phosphorylation at different developmental stages. We find that phosphorylation of H3.3 at Ser-31 also occurs in the miotic cells of the planktonic urochordate Oikopleura dioica, suggesting that this histone modification and its function in mitosis is already present at the invertebrate- vertebrate transition. The spatial pattern differs from that of H3.1 phosphorylation at Ser-28. Whereas Ser-28 phosphorylation is enriched in the centromeric region, H3.3 Ser-31 phosphorylation spreads throughout the condensed chromosomes. We further investigated the phosphorylation of H3.3 in meiosis during gametogenesis in Oikopleura dioica. H3.3 phosphorylation occurs after Ser-10 phosphorylation, together with the phosphorylation of Ser-28 in diplotene and persists until oocytes are arrested in metaphase I. We are currently investigating whether the variant H3.3 is phosphorylated via the mitogen-activated protein kinase (MAPK) pathway during meiosis.

1) Schwartz, B. E. and K. Ahmad (2005). Genes Dev 19(7): 804-14. 2) Wirbelauer, C., et al. (2005). Genes Dev 19(15): 1761-6. 3) Hake, S. B., B. A. Garcia, et al. (2005). Proc Natl Acad Sci U S A 102(18): 6344-9.

154 Abstracts - Poster

Jill Schumacher Abstract 99 The Tousled kinase functions in mitosis as a substrate and activator of the Aurora B kinase Zhenbo Han1, Gary M. Riefler1,2, Jennifer R. Saam3, Susan E. Mango3, and Jill M. Schumacher1,2

1Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, 77030, U.S.A., 2Genes and Development Program, Graduate School of Biomedical Sciences, The University of Texas-Houston, Houston, Texas, 77030, U.S.A. 3Huntsman Cancer Institute, University of Utah, Salt Lake City 84112, U.S.A.

The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair. Tousled substrates include the Asf1 chromatin assembly factor and histone H3. We have previously reported that the C. elegans Tousled-like kinase TLK-1 is required for embryonic transcription and viability. Recently, we have found that TLK-1 also has an essential role in mitotic chromosome segregation. Embryos depleted of TLK-1 display abnormalities in metaphase chromosome alignment, anaphase chromosome bridges, and polyploid nuclei. TLK-1 is specifically phosphorylated by the AIR-2 Aurora B kinase during prophase/prometaphase, and this phosphorylation increases TLK-1 kinase activity in vitro. In addition, TLK-1 phosphorylation initiates a feedback loop that increases AIR-2 kinase activity. As predicted for an AIR-2 activator, depletion of TLK-1 enhances the mitotic defects of a hypomorphic AIR-2 allele, revealing that AIR-2 and TLK-1 cooperate to ensure proper mitotic chromosome segregation. These results demonstrate that Tousled kinases have a previously unrecognized role in mitosis and represent a new class of Aurora B activating proteins.

155 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

David Schrump Abstract 100 Gene expression profiling of primary lung cancers exposed to 5 aza 2’ deoxycytidine (DAC), depsipeptide FK228 (DP), or sequential DAC/DP Zhao M.1, Li X.2, Hong J.A.1, Chen G.A.1, Kunst T.F.1, Hancox A.1, Nguyen D.M.1 and Schrump D.S.1

1Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, U.S.A., 2University of Chicago, Chicago, IL, U.S.A.

The emerging relationships between epigenetics and malignant transformation provide impetus for the use of DNA demethylating agents and histone deacetylase (HDAC) inhibitors for cancer therapy. Presently, limited information is available concerning gene expression in primary cancers exposed to these chromatin remodeling agents. During the past several years, more than 70 patients have participated in clinical trials in the Surgery Branch, NCI, examining pharmacokinetics, toxicities, and potential efficacy of DAC, DP, and sequential DAC/DP infusions in thoracic oncology patients. Recently, long-oligo array techniques were used to examine gene expression profiles in RNA amplified from laser-captured tumor cells obtained from fine needle aspirates (FNAs) of 20 individuals receiving DAC, DP, or sequential DAC/DP treatment. Results of pre- and post-treatment arrays from these patients were compared to data derived from analysis of laser-captured tumor cells and normal bronchial epithelia from 15 patients undergoing lung cancer resections at the NCI. A standardized pool of normal lung RNA was used as a reference for all arrays, enabling comprehensive analysis of data within and across treatment groups. Pharmacokinetic analysis confirmed that plasma DAC and DP concentrations in these patients approximated threshold levels for modulation of gene expression in cultured lung cancer cells. Micro-array analysis demonstrated complex, heterogeneous responses to DAC, DP, and sequential DAC/DP in lung cancer cells. Approximately 735 genes were induced or repressed by DAC treatment in a statistically significant manner; 230 genes exhibited significantly altered expression following DP exposure. Interestingly, despite the apparent synergy of DAC and DP in cultured lung cancer cells, only 54 genes were modulated significantly in primary lung cancers following sequential DAC/DP therapy. Sixteen genes were modulated by DAC as well as DP. No genes were modulated by all three treatment regimens; only one gene was altered by DAC and sequential DAC/DP treatment, and no genes were modulated by DP as well as DAC/DP. Thirty- four genes, which exhibited significantly different expression levels in primary lung cancer cells relative to adjacent normal bronchial epithelia were modulated by DAC treatment, whereas 5 genes were modulated by DP. Three genes, which were aberrantly expressed in untreated lung cancer cells, were modulated by DAC as well as DP. Overall, DAC and DP treatment tended to inhibit or induce expression of genes aberrantly over-expressed or silenced, respectively, in primary lung carcinomas. The response to sequential DAC/DP appeared to be less evident, possibly due to pharmacodynamics of DAC and DP in tumor tissues, or effects of stromal elements on gene expression in lung cancer cells. Collectively, these data confirm that DNA demethylating agents and HDAC inhibitors can modulate gene expression in primary lung carcinomas. Despite the complexity of the data, comprehensive analysis of gene expression profiles in laser- captured tumor cells may facilitate rational development of chromatin remodeling agents for lung cancer therapy.

156 Abstracts - Poster

Brian E. Schwartz Abstract 101 Nucleosome assembly pathways discriminate between sites in the H3 and H3.3 histone tails. Brian E. Schwartz and Kami Ahmad.

Harvard Medical School, 204 C1, 240 Longwood Ave., Boston MA 02115, U.S.A.

Nucleosome assembly can occur through at least two known pathways. The bulk of DNA is packaged during S phase via the replication-coupled (RC) pathway, while transcriptionally active chromatin can be rebuilt outside of S phase through the transcription-coupled (TC) pathway. The RC pathway can use both H3 and the conserved variant H3.3 for assembly, while the TC pathway exclusively uses H3.3. H3 differs from H3.3 by only four amino acids, and three of these residues are responsible for targeting the histone to the appropriate assembly pathway. Using Drosophila cultured cells, we show here that lysines in the N terminal tails of H3 also have distinct roles in assembly. In particular, a K4R mutation disables RC assembly but not TC assembly. No other single lysine to arginine mutation affects either pathway. These mutations show that assembly pathways use the N- terminal tail of the H3 subtype histones differently. Additionally, only the RC pathway is disrupted if histone tails are maintained in a constitutively acetylated (neutralized) state with the histone deacetylase inhibitor sodium butyrate. Taken together, our data suggests that the RC deposition machinery binds H3-lysine 4 and requires that N-terminal lysines are transiently de-acetylated during nucleosome assembly. The TC pathway does not require lysine 4, or even the entire N-terminus, as a tail truncation mutant is efficiently incorporated. However, the presence of four point mutations (H3.3-K4,9,14,18R) abrogates TC deposition. We argue that reversible lysine acetylation serves as a mechanism to shuffle histones between chaperones and DNA by temporary disruption of charge interactions.

157 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Kristin Scott Abstract 102 A heterochromatin barrier partitions the fission yeast centromere into discrete chromatin domains Kristin C. Scott, Stephanie L. Merrett and Huntington F. Willard

Duke University Institute for Genome Sciences and 2383 CIEMAS, 101 Science Drive, Durham, 27708, U.S.A.

Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation. Centromeric regions of higher eukaryotes are structurally complex, consisting of various epigenetically modified chromatin types including specialized chromatin at the kinetochore itself, pericentromeric heterochromatin, and flanking euchromatin. Although the features necessary for the establishment and maintenance of discrete chromatin domains remain poorly understood, two models have been proposed based either on the passive convergence of competing activities involved in individual domain formation or, alternatively, on the action of specific genomic sequences and associated proteins to actively block the propagation of one chromatin type into another. Here, functional analysis of centromeric sequences located at the intersection of Schizosaccharomyces pombe central core chromatin and outer repeat heterochromatin identified a chromatin barrier that contains a tRNA alanine gene. Deletion or modification of the barrier sequences result in the propagation of pericentromeric heterochromatin beyond its normal boundary. The tRNA alanine gene is transcriptionally active, and barrier activity requires sequences necessary for RNA polymerase III transcription. Moreover, absence of the barrier results in defective meiotic chromosome segregation. The identification of DNA sequences with chromatin barrier activity at the fission yeast centromere provides a model for establishment of centromeric chromatin domains in higher eukaryotes.

158 Abstracts - Poster

Judith Sharp Abstract 103 XIST functions independently of BRCA1 in X chromosome inactivation Judith Sharp and Barbara Panning

UCSF, Genentech Hall, 600 16th Street, Rm. S374, San Francisco, CA 94158, U.S.A.

In mammals, dosage compensation of the X chromosome is equalized between the sexes by the transcriptional inactivation of one X chromosome in female cells. Precise control of X chromosome dosage is critical for the proper functioning of the embryo: mutant mice unable to enact X chromosome dosage compensation exhibit female specific early embryonic lethality. X inactivation during early female development is mediated by the XIST noncoding RNA. XIST initiates silencing in cis from a region termed the X inactivation center. XIST RNA eventually coats the entire chromosome, resulting in silencing of the majority of genes on the Xi. In terminally differentiated cells, XIST coats the inactive X chromosome but plays a less stringent role in the silencing mechanism. Humans with germline mutations in the BRCA1 gene are predisposed to develop early-onset breast and ovarian cancer. A recent report indicated that BRCA1 was required to maintain XIST localization on the inactive X chromosome in somatic cells and tumor cells, suggesting a correlation between hereditary breast cancer and defects in Xi chromosome structure. Instead, our findings indicate that X chromosome dosage compensation is often misregulated in female cancer cells independently of BRCA1 genotypic status. Further, we found that BRCA1 is dispensable for XIST chromosome-coating activity in somatic cells, suggesting that XIST functions independently of BRCA1 during the maintenance phase of X inactivation.

159 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Shivendra D. Shukla Abstract 104 Histone modifications by ethanol in liver Shivendra D. Shukla, Pil-Hoon Park, Jee-soo Kim and Robert Lim

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri 65212, U.S.A.

Ethanol exposure increases expressions of various genes. Histone acetylation has been shown to induce transcriptional activation. We have therefore investigated the effects of ethanol on histone H3 acetylation in rat liver cells. Ethanol treatment significantly increased H3 acetylation at Lys9 with negligible effect at Lys14, 18 and 23 in primary cultured rat hepatocytes. Treatment of rat hepatic stellate cells with ethanol produced a similar pattern except that acetylation at Lys23 was also increased at high concentration of ethanol. Acute in vivo administration of alcohol in rats for 12 hrs (analogous to binge drinking) increased acetylation of H3-Lys9 in the liver without affecting Lys14, 18 and 23, showing the same results as in vitro with hepatocytes. The in vivo effects of acute ethanol administration occurs in a tissue-specific manner; H3-Lys9 acetylation is seen in lung and spleen, but not the kidney, brain, heart, stomach, colorectum, pancreas and vessels. Inhibitors of ethanol metabolizing enzymes, alcohol dehydrogenase (4-methyl pyrazole) and aldehyde dehydrogenase (cyanamide), diminished ethanol-induced H3 acetylation at Lys9 in hepatocytes, suggesting a role for ethanol metabolism, especially acetate, in histone acetylation. Ethanol exposure increased nuclear histone acetyltransferase (HAT) activity in hepatocytes as monitored by ELISA or immunoblot assays. Chromatin immunoprecipitation (CHIP) assay demonstrated that ethanol increased the association of class I alcohol dehydrogenase (ADH I) gene with acetylated H3-Lys9 domain in the chromatin. Taken together, emerging evidence support the view that post-translational modifications in histones may underlie the mechanism for ethanol induced gene alterations and may constitute an important event in alcoholic liver damage. (Supported by NIH grant #AA14852)

160 Abstracts - Poster

Robert Sims Abstract 105 Regulation of mRNA biogenesis by the coordinator complex, which specifically recognizes methyl H3K4 Robert J. Sims III, Chi-Fu Chen, Scott Millhouse, Patrick Trojer, Subhra S. Mandal, Paul Tempst, Robert P. Perry, Brian A. Lewis, James L. Manley and Danny Reinberg

RWJ-UMDNJ 1, RSB, 683 Hoes Lane, Piscataway, Middlesex 08854, U.S.A.

A contemporary view of mRNA biogenesis depicts an integrated process that incorporates multiple events simultaneously, encompassing transcript elongation and RNA processing, including CAP addition, pre-mRNA splicing, polyadenylation, in addition to mRNA surveillance and export. How chromatin dynamics participate in this integrated process is poorly understood. Here we identify a large protein complex termed the Coordinator, which contains factors that facilitate post-initiation events. This complex was isolated by its ability to recognize the amino terminal tail of histone H3 specifically tri-methylated on lysine 4 (H3K4me3). H3K4 methylation is tightly associated with active loci and corresponds to the 5 region of human genes. The Coordinator contains the chromatin remodelers CHD1 and SNF2h, components of the U2 snRNP, and PAF, a complex implicated in multiple stages of mRNA maturation, as well as chromatin modulation. Human CHD1 directly and selectively binds methylated H3K4, which in contrast to yeast requires two intact chromodomains in tandem. In addition, H3K4me3 recruits factors that assist transcription on chromatinized templates in vitro and methylate H3K4, perhaps identifying a mechanism to propagate this mark. While distinct from the Coordinator, our results also suggest a probable mode of FACT recruitment spatially within an active gene. We hypothesize that the Coordinator integrates multiple processes during mRNA biogenesis, instigated by its recruitment to activated genes through the interaction of CHD1 with H3K4me3.

161 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Jeffrey Smith Abstract 106 Genetic identification of chromatin-related regulators of ribosomal RNA synthesis in Saccharomyces cerevisiae Robert D. Hontz and Jeffrey S. Smith

University of Virginia Health System , Jordan Hall, Box 800733, Charlottesville, VA 22911, U.S.A.

Eukaryotic ribosomal RNA (rRNA) is transcribed from repetitive rDNA genes using RNA polymerase I (Pol I). Despite being responsible for the majority of overall transcription in actively growing cells, the regulation of Pol I, especially at the chromatin level, is poorly understood. To facilitate the identification of chromatin-related factors that regulate this process, we have developed a genetic assay in Saccharomyces cerevisiae that efficiently detects whether a specific gene mutation or growth condition alters the strength of Pol I- mediated rDNA transcription. The assay is based on a modified URA3 reporter gene (mURA3) positioned next to the centromere-proximal (left) end of the rDNA tandem array. The leftmost rDNA repeat has been modified such that Pol I transcription termination does not occur, causing interference with the adjacent mURA3 reporter. Mutations that reduce the level of Pol I transcription therefore relieve the interference with mURA3, resulting in a Ura+ phenotype, whereas mutations that promote Pol I transcription cause more interference with mURA3, resulting in a strong FOA-resistant phenotype. A genetic screen based on these phenotypes has identified approximately 100 different non-essential genes that appear to function in Pol I regulation, including genes encoding components of the RPD3 and HOS2 histone deacetylase complexes, and the SAGA histone acetyltransferase complex. Other chromatin-related factors identified include the SWR1 histone exchange complex, and surprisingly, the Sir1/Sir4 silencing proteins. Development of the new genetic Pol I transcription assay and characerization of the above screen candidates as Pol I regulators will be presented.

162 Abstracts - Poster

Yee Sun Tan Abstract 107 Skp1 regulates transcription via mono-ubiquitylation Yee Sun Tan, Wee Leng Siew, Tze Chin Tan, Debra Morley and Norbert Lehming

Department of Microbiology, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597

The Mediator complex is involved in transcriptional regulation, transducing signals from activators and repressors to RNA polymerase II. The mammalian Mediator complex is comprised of up to 30 subunits, and Med21, previously known as the Suppressor of RNA polymerase B 7 (Srb7), is one of the subunits found in the middle module of the Mediator complex. The amino acids in the N-terminal region of Med21 are highly conserved from budding yeast to humans, and this subunit plays a role both for the activation and the repression of transcription. We have found that Med21 interacts with S-phase kinase associated protein 1A (Skp1A) in Saccharomyces cerevisiae and in human cells as well as in vitro. Skp1 is part of the nuclear SCF complex (Skp1-Cullin-F-box protein) E3 ligase involved in ubiquitylation of nuclear proteins, and it also interacts with proteins like Ctf13 that do not contain F-box motifs. Our work shows that knock-down of Skp1 and point mutations in Skp1 reduced transcriptional activation in human and yeast cells, respectively. Thus, our results indicate that Skp1 plays a role in transcription. In addition, there is increasing evidence of mono-ubiquitylation to act as a regulatory signal in cellular processes like transcription. For instance, mono-ubiquitylation of K123 of H2B leads to methylation of K4 of H3 in S. cerevisiae. As Skp1 is part of the SCF complex, we are investigating the interactions of the various subunits of the SCF complex with numerous transcription factors, as well as the core histones. We will discuss the possibility of mono-ubiquitylation of specific transcription factors by the nuclear SCF complex.

163 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Hannah Tims Abstract 108 Spontaneous and catalyzed nucleosome conformational changes Jonathan Widom and Hannah Tims

Northwestern University, 551 Hinman Ave., Evanston, IL 60202, U.S.A.

DNA wrapped in the nucleosome is sterically occluded, yet must be accessed for vital cellular processes, including transcription, replication, recombination, and DNA repair. We are studying spontaneous and also ATP dependent processes that provide access to buried stretches of nucleosomal DNA. Studies of spontaneous accessibility have focused on characterizing the nucleosome conformational changes that make buried sites accessible, and the frequencies of these conformational changes. We find that the mechanism of spontaneous accessibility is via partial DNA unwrapping starting from one end of the nucleosome and proceeding inward. Spontaneous unwrapping occurs rapidly (>>1 sec-1) and with no loss or exchange of histones. Our studies of the ATP-dependent processes, conducted in collaboration with the Becker group (ABI, Munich), focus on the Drosophila nucleosome remodeling factor ISWI. Studies of the enzymes ATPase activity reveal that at low (nM) ATP concentration, ISWI is self- inhibited, hydrolyzing ATP but failing to release product efficiently. High (mM) ATP concentration greatly stimulates the activity. Together with fluorescent anisotropy studies of ISWI binding to DNA and nucleosomes, these findings suggest that ISWI may be active as a dimer.

164 Abstracts - Poster

Patrick Trojer Abstract 109 A functional interplay between MLL and LSD1 Patrick Trojer1, Rushad Pavri1, Hedjye Erdjument-Bromage2, Paul Tempst2, Ramin Shiekhattar3 and Danny Reinberg1

1University of Medicine and Dentistry New Jersey (UMDNJ), Robert Wood Johnson Medical School (RWJMS), Department of Biochemistry, 683 Hoes Lane West, Piscataway, NJ 08854-0009, U.S.A., 2Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A., 3The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, U.S.A.

Histone H3 lysine 4 tri-methylation (H3-K4m3) is linked with activation of transcription. However, in the mammalian system there is a high redundancy of histone lysine methyltransferases (HKMTs) specific for H3-K4. Knockout models have shown that H3-K4 specific HKMTs are not functionally redundant implying an additional layer of regulation regarding their recruitment and respective role(s). For instance, MLL, a Trithorax group protein with H3-K4-HKMT activity, was shown to be required for the proper regulation of the developmentally important Hox genes. Recently, genome-wide ChIP analysis demonstrated that MLL is present on most active genes suggesting it functions globally in transcriptional activation. Intriguingly, the rise in the H3-K4m3 level upon gene activation is followed by its subsequent decrease after loss of the transcriptional stimuli. This used to be explained by histone exchange. This hypothesis was challenged by the discovery of a histone lysine demethylase (HKDM), LSD1 (BHC110/KIAA0601/PAO), which specifically removes mono- and di-methyl groups from histone H3-K4. Since H3-K4 methylation is an activation mark and LSD1 was found as a component of repressive multiprotein complexes, HKDMs appeared to counteract H3-K4 specific HKMTs analogous to HATs and HDACs. Our data provide evidence that MLL and LSD1 are present in a protein complex of 1.5-2.0 MDa. Each enzyme is active in histone methylation or histone demethylation assays, respectively. However, the complex is only capable of di-methylation, not tri-methylation of H3-K4. The complex acts as a transcriptional repressor in our highly purified reconstituted transcription system. Together with our ChIP data, this suggests that the MLL-LSD1 complex is already present at silenced genes. Upon activation, the LSD-corepressor complex is lost while MLL occupancy is increased concomitant with tri-methylation of H3-K4. Furthermore, based upon the identities of other subunits of this complex it may also play a role in the coordination of transcriptional initiation as well as processes downstream of transcription. The interaction of these two enzymes that modify H3-K4 with opposing catalytic properties represents another critical facet of transcriptional regulation.

165 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Patrick Trojer Abstract 110 L3MBTL1 – the chromatin lock? Patrick Trojer1*, Robert Sims III1*, Guohong Li1*, Alejandro Vaquero1, Piernicola Boccuni2, Nagesh Kalakonda2, Regine Losson3, Hedjye Erdjument-Bromage2, Pierre Chambon3, Stephen Nimer2, Paul Tempst2, Yuh-Hwa Wang1 and Danny Reinberg1 *contributed equally

1University of Medicine and Dentistry New Jersey (UMDNJ), Robert Wood Johnson Medical School (RWJMS), Department of Biochemistry, 683 Hoes Lane West, Piscataway, NJ 08854- 0009, U.S.A., 2Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A., 3Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Parc d´Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, C.U. de Strasbourg, France

Dynamic regulation of chromatin structure is essential for cellular processes like replication, transcription, differentiation, cell-cycle progression and DNA-repair. Posttranslational modifications of histones turned out to be important regulators of these biological processes. However, the histone modifications are not sufficient to carry the biological message. Rather, the histone “marks” serve as recognition modules for proteins which are able to distinguish between different modified residues and can even distinguish between different degrees of modifications on the same residue in the case of histone lysine methylation. The chromodomain of HP1 was shown to bind methylated lysine 9 on histone H3 (H3-K9m) and the tandem-tudor domain of 53BP1 was recently shown to recognize H3- K79m2. It is of great interest to find novel chromatin binding factors to understand how changes of the chromatin structure are regulated. The MBT (malignant brain tumor) domain shows structural similarity with the chromo-, tudor and PWWP-domains and therefore was predicted to bind modified histones. L3MBTL1, the human homolog of the Drosophila tumor suppressor protein D-l(3)mbt, was previously shown to be a repressor of transcription and its mode of action was independent of HDACs We have purified a protein complex using a cell line that stably expresses L3MBTL1. Mass- spectrometry analysis confirmed L3MBTL1-binding to core histones. In addition our data provide evidence that L3MBTL1 binds to the linker histone H1 as well as to HP1 gamma. We show that L3MBTL1 binds to mono- and di-methylated H1-K26 in vitro and in vivo. Using a chromatin compaction assay we demonstrate that recombinant L3MBTL1 is able to compact chromatin in a histone modification specific manner. This, together with the interaction with HP1 suggests that L3MBTL1 functions in the process of chromatin compaction by two distinct mechanisms: (1) Binding of L3MBTL1 to repressive histone marks of different histone tails “locks” histone tails to prevent access of proteins involved in transcriptional activation, (2) Recruitment of factors like HP1 could promote the propagation of chromatin compaction. This work is supported by a fellowship of the Austrian Science Fund (FWF, J2354-B12) to P.T. and NIH(GM-71166) to R.S.

166 Abstracts - Poster

María Isabel Tussié Luna Abstract 111 Pro-proliferative function of the long isoform of PML-RARα involved in acute promyelocytic leukaemia María Isabel Tussié Luna, Liliana Rozo and Ananda L. Roy.

Tufts University School of Medicine. Pathology Department. 150 Harrison Avenue. Boston, MA 02111, U.S.A.

The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML RARα, in which the PML gene fuses to the retinoic acid receptor α (RARα) gene. PML RARα has been shown to promote transcriptional repression of genes involved in myeloid terminal differentiation and to disrupt the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). However, there are several alternatively spliced isoforms of PML RARα. Here we addressed the differences between the short and the long isoforms of PML RARα (L and S) since both are associated with APL. We demonstrate that PML RARαL but not PML RARαS, can directly promote cell growth by transcriptionally activating the pro proliferative gene, c fos, in response to mitogenic stimulation. The activity of the PML RARL is completely sensitive to ATRA. We further show that this activation is not via direct recruitment of the protein to the c fos promoter but indirectly by altering the chromosomal environment of the c fos gene thereby rendering it more accessible to the signal induced transcriptional activators. Our results suggest that in addition to antagonizing the PML tumor suppressor or the PML pro apoptotic activity, PML RARα proteins can also directly promote cell growth by activating c fos.

167 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Wim Vanden Berghe Abstract 112 Differential interleukin-6 gene expression dynamics in benign and metastatic breast cancer cells reflects distinct chromatin signatures at the promoter region Wim Vanden Berghe1, Matladi N. Ndlovu1, Nathalie Dijsselbloem1, Linda Vermeulen1, Carine Van Lint2 and Guy Haegeman1

1Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Molecular Biology, Ghent University, KL Ledeganckstraat 35, B-9000, Gent, Belgium. 2Université Libre de Bruxelles (ULB), Institut de Biologie et de Médecine Moleculaires (IBMM), Service de Chimie Biologique, Laboratoire de Virologie Moleculaire, Rue des Profs Jeener et Brachet, 12, 6041 Gosselies, Belgium.

Previously, we have demonstrated involvement of the mitogen- and stress-activated protein kinase-1 MSK1 as a crucial kinase for NF-kB p65 and histone H3 phosphorylation in IL6 gene expression during inflammatory stress (Vermeulen et al. EMBO J. 2003). As IL6 is also an autocrine growth factor in various cancers, which accelerates tumor progression and promotes metastasis, we have compared NF-kB signaling and chromatin dynamics in a benign and a metastatic breast cancer model. We have therefore analysed the promoter region of the IL6 gene in metastatic MDA-MB-231 cells, producing high levels of IL6, and benign MCF-7 cells, producing background levels of IL6, for the presence of hypersensitive sites (HS) with DNase-I, and for nucleosome positioning with micrococcal nuclease (MNase) and restriction accessibility assays. We detected that elevated IL6 expression levels correlate with increased number of HSs, constitutive NF-kB/DNA binding and elevated MSK1 activity. Of particular interest, soy isoflavones, which are claimed to reduce breast cancer risk, were found to selectively inhibit the MSK1 pathway towards NF-kB and H3 chromatin modifications. These results suggest that MSK1 is a critical trigger for establishing a transcription-competent enhanceosome and a permissive nucleosomal structure of the IL6 promoter, thus restricting NF-kB-driven gene dose responses to a defined physiological frame. Unlimited MSK1 activity may predestine cells towards tumorigenesis or chronic inflammatory disorders at the NF-kB/chromatin interface.

168 Abstracts - Poster

Adam Wood Abstract 113 The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 Adam Wood, Jessica Schneider, Jim Dover, Mark Johnston and Ali Shilatifard

Saint Louis University. Doisy, 1402 South Grand Ave., Saint Louis 63104, U.S.A.

Transcriptional regulation is highly coupled to chromatin structure. Several classes of enzymes have been shown to affect transcription by catalyzing modification of nucleosomes including the methyltransferases COMPASS and Dot1p. By way of our global proteomic screen GPS, we have determined that deletion of BUR2, the gene encoding the cyclin component of the Bur1/Bur2 cyclin-dependent protein kinase, is required for histone H2B monoubiquitination by the Rad6/Bre1 complex. We also demonstrate that the deletion of BUR2 results in a significant decrease in histone H3K4 methylation catalyzed by COMPASS and H3K79 methylation by Dot1. The effect on histone monoubiquitination and methylation are the result of defective Bur1/Bur2-mediated phosphorylation of Rad6 on its serine residue 120 and the recruitment of the Paf1 complex to chromatin. We have demonstrated that serine 120 of Rad6 is required for proper histone H2B monoubiquitination and the regulation of gene expression in vivo. Our results identify in vivo substrates for the Bur1/Bur2 kinase that links its role to transcriptional elongation and demonstrates a potential activation mechanism for histone H2B monoubiquitination by the Rad6/Bre1 complex.

169 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Dag H. Yasui Abstract 114 MeCP2 recruits SNF2h to the 15q11-13 imprinting control region Dag H. Yasui and Janine M. LaSalle

Medical Microbiology and Immunology and Rowe Program in Human Genetics, UC Davis School of Medicine, Medical Microbiology and Immunology, Davis CA 95616, U.S.A.

Rett syndrome is an X-linked neurodevelopmental disorder cause by mutations in MECP2. MECP2 encodes methyl CpG binding protein 2 (MeCP2) that acts as a transcriptional repressor for methylated genes by recruiting factors such as HDAC1, Sin3a, and DNMT1. Recent evidence supports a role for MeCP2 in long-range mechanisms that control gene expression, including chromatin looping and remodeling. We have previously demonstrated binding of MeCP2 to the methylated promoter of SNRPN/Snrpn that acts as an imprinting control region (ICR) for multiple genes on human 15q11-13 and syntenic mouse 7qB5. To identify additional factors specifically binding to the methylated 15q11-13 ICR, DNA affinity chromatography using a methylated or unmethylated SNRPN promoter sequence was performed on nuclear extracts from SH-SY5Y neuroblastoma cells. Proteins isolated from column fractions were identified by mass spectroscopy and confirmed by immunoblot analysis. MeCP2 was isolated specifically from the methylated but not the unmethylated affinity column and co-fractionated with SNF2h, but not Sin3a. SNF2h is a chromatin remodeling ATPase of the SWI/SNF family that requires recruitment by DNA binding proteins for long-range regulation of gene expression. Reciprocal co-immunoprecipitation using anti-MeCP2 and anti-SNF2h confirmed the association in vivo. Immunofluorescence of mouse cortex sections also demonstrated a partial colocalization of MeCP2 and SNF2h in a subset of developing neurons. To determine if MeCP2 binding is required for the recruitment of SNF2h to the methylated ICR, chromatin immunoprecipitation (ChIP) followed by quantitative PCR was performed in SH-SY5Y cells transfected with a methylated oligonucleotide decoy versus a mutant unmethylated control. A decrease in SNRPN signal within both MeCP2 and SNF2h ChIP DNA was observed in SH-SY5Y cells treated with the methylated but not the unmethylated MeCP2-specific decoy, suggesting that MeCP2 binding to methylated CpG sites is required for SNF2h binding to the SNRPN ICR. Experiments to confirm this result in the Mecp2-null mouse model are ongoing. These results are the first demonstration of a chromatin remodeling ATPase associating with an ICR and have direct relevance to understanding the pathogenesis of Rett, Prader-Willi, and Angelman syndromes.

170 Abstracts - Poster Notes

171 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005 Notes

172 Resort Information Resort Information Radisson Hotel telephone numbers Hotel: 242-327- 6000 Hotel General Emergency: 33 (from inside hotel) Concierge: 20 (from inside hotel) Local Services: 0 (from inside hotel) Police: 0 (from inside hotel) Hotel Doctor Service: 242-327-7711 (ask for Medi Center (9-5) not free service.) Hotel Doctor Service (after 5pm): call operator above and a doctor can be arranged at a fee Hotel Security: 242-327-6000 ext.6474

Local telephone numbers Police & Fire Dept: 242-327- 8800 Hospital: 242 322-2861 Walk in clinics: The Hotel recommends the 'Walkin Medical Clinic' (242-328-4014) 24hr pharmacy: 242-326-4629 (regular hours) Taxi: 242- 327-2042 Airport: 242 -377-1759

Organisers Conference organisers: Abcam At the resort from 13th November, +44(0)7968 024628

173 Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005 Radisson Hotel Map To Casino To Beach Golf Course Via Complimentary Shuttle Golf Course Via Escalators to Shops Lucayan Room A Arawak Room Arawak Room B East Wing Rooms 1-43 Elevators Center Stairs to Sports Centre Convention Tour Desks Tour Ballroom Foyer

Pool Convention Centre Elevators Tropical A B C A Bay Street - Eastbound Traffic Bay Street - Eastbound Bay Street - Westbound Traffic Bay Street - Westbound Edgewater Bar Duty Manager Lobby Resort Store Goombay Lounge Rendezvous Lounge Tower Pool Tower Front Desk Stairs to 4th Floor Restaurants Bell Stand Port Cochiere Tour Desk Tour Offices Dolphin Beach Grill Stairs to Beach and Pools

Islands Restaurant Elevators Main Floor Avocados Restaurant Jacuzzi Floor Cascade Pool Fourth The Forge Rum Runner Room Beach West Wing Rooms 51-93 Elevators Amici Restaurant First Floor Water Sports Water Centre Bimini Market Grill

174 Sponsor of 2005 Chromatin Structure & Function Keynote Presentation

Chroma Therapeutics (Oxford, UK) was founded in 2001 and is focused on the exploitation of chromatin biology to discover and develop a new generation of targeted treatments for cancer and inflammatory disorders. Chroma's founding scientists include Tony Kouzarides (University of Cambridge) and David Allis (Rockefeller Institute), leaders in the field of chromatin regulation. In oncology, inhibition of chromatin modifying enzymes (CMEs) such as HDACs, aurora kinases and histone methyltransferases, impacts multiple aspects of tumour progression including proliferation, angiogenesis and invasion while in models of chronic inflammation, CME inhibitors show potent anti-cytokine activity, highlighting their potential as a new class of oral therapies.

Chroma's highly experienced management team aims to build a dominant position in CME inhibitors as treatments for both oncology and inflammatory disorders. The company's resources are focused on the discovery and early stage development of CME inhibitors which, when combined with its access to leading academic institutions working in the CME area, aim to deliver a strong and renewable pipeline of advanced therapeutics. The company is also developing a non-invasive biomarker technology based on the analysis of histone modifications on the tumour- derived chromatin which circulates in the blood of cancer patients.

175 Chromatin and Nuclear Signaling Antibodies, resources and meetings.

Chromatin Abwire Binding proteins, ChIP'ing antibodies, chromosome structure, epigenetics, histones, modifying enzymes, remodeling. www.abcam.com/chromatin Nuclear Signaling Abwire Cardiovascular, cell cycle, DNA/RNA, nuclear signaling pathways, transcription. www.abcam.com/nuclearsignal RNAi Abwire Argonaute, Dicer, RdRP, RISC, RNAi vectors. www.abcam.com/rnai

Abcam “abwires” are gene regulation hotspots! Our resource pages contain exclusive articles, tried and tested protocols, conference information and product guides.

Over 3,500 primary antibodies in Chromatin, Nuclear Signaling and RNAi!