1 Supplementary Information for Acetylated Histone H3K56 Interacts

Total Page:16

File Type:pdf, Size:1020Kb

1 Supplementary Information for Acetylated Histone H3K56 Interacts Supplementary Information for Acetylated histone H3K56 interacts with Oct4 to promote mouse embryonic stem cell pluripotency Table of contents Supplementary Figures 1-4 and Figure Legends Supplementary Methods Cell culture Plasmid construction and transfection ChIP-Sequencing ChIP-Seq data analysis K-means clustering Co-immunoprecipitation assay In vivo peptide pull-down assay Flag-immunoprecipitation assay In vitro peptide pull-down assay Mononucleosome immunoprecipitation Western blot Quantitative PCR Gel mobility shift assay Supplementary Tables 1-8 Supplementary References 1 Supplementary Figures and Legends 0 1 %&'() %&'() *(+, *(+, !"#$ !"#$ -./01&" -./01&" 023 ()*+ 023 ,'-+ . / %&'() %&'() *(+, *(+, !"#$ !"#$ -./01&" -./01&" 023 !"#$% 023 !$&"' Supplementary Figure 1. The distribution of ChIP-Seq signals for NSO and H3K56ac at Cluster 1 regions. (A-D) Enrichment patterns of Nanog, Sox2 and Oct4 (NSO) and H3K56ac at Oct4 (also known as Pou5f1) (A), Klf4 (B), Nanog (C), and Nodal (D) gene loci are shown by University of California, Santa Cruz (UCSC) genome browser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upplementary Figure 2. H3K56ac positively correlates with the enrichment of Oct4 in mouse ESCs. (A-C) H3K56ac (A), Oct4 (B) and Oct4 binding motifs (MA0142 in JASPAR (1)) (C) signal distribution relative to the center of each cluster of Oct4 regions in Fig. 2A were calculated by CEAS-sitepro (2, 3). The x-axis represents the distance to the center of Oct4 peaks in mouse ESCs. The y-axis represents ChIP-Seq tag count signals normalized by the number of regions in each cluster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upplementary Figure 3. The functional categories for Group I/II/III regions of Cluster 1. (A-B) Functional annotations of Group I (A) and II/III (B) Oct4 ChIP regions in mouse genome were performed using GREAT (4). Mouse Genome Informatics (MGI expression detected) indicates the information on tissue and developmental-stage-specific expression in mouse employed as an ontological category. Group I shows the functional enrichment of regions associated with early embryogenesis and pluripotency. The x-axis values (in logarithmic scale) correspond to the binomial raw p-values. 4 !"#$%&$"'($)*+($,- . / !"# !"# 2"3 2"3 2"4 2"4 $%&'()&*+',-./&*,-*A,B# $%&'()&*+',-./&*,-*0(1,) 2"2 2"2 @4 @# 2 # 4 @4 @# 2 # 4 5&/(6.%&*7.86(19&*6, 5&/(6.%&*7.86(19&*6, :;22*:&(<*9&16&'8*=<>? :;22*:&(<*9&16&'8*=<>? 0 1 4"2 2"C ;"2 2"4 #"2 2"# $%&'()&*+',-./&*,-*D964 $%&'()&*+',-./&*,-*E;FGC(9 !"2 2"2 @4 @# 2 # 4 @4 @# 2 # 4 5&/(6.%&*7.86(19&*6, 5&/(6.%&*7.86(19&*6,* :;22*:&(<*9&16&'8*=<>? :;22*:&(<*9&16&'8*=<>? Supplementary Figure 4. Nanog, Sox2, Oct4 and H3K56ac are enriched at p300 ChIP regions in the mouse genome. (A-D) Average enrichment profiles of Nanog (A), Sox2 (B), Oct4 (C) and H3K56ac (D) relative to p300 peak centers were evaluated by CEAS-sitepro (2, 3). The x-axis represents the distance to the center of selected ChIP regions in mouse ESCs. The y-axis represents average ChIP-Seq signals. 5 Supplementary Methods Cell culture Mouse embryonic stem cell line E14Tg2a (CRL-1820) was obtained from ATCC and cultured in Knockout™ Dulbecco’s Modified Eagle’s Medium (Invitrogen, Cat # 10829- 018) supplemented with 15% ES-qualified FBS (Omega Sci, Cat # FB-05, Lot # 104100), 2mM GlutaMAX™-I Supplement I (Invitrogen, Cat # 35050-061), 0.1 mM MEM Non- Essential Amino Acids Solution (Invitrogen, Cat # 11140-050), 55 nM 2- mercaptoethanol (Invitrogen, Cat # 21985-023) and 1,000 units/ml LIF (Millipore, ESG1107). Mouse ESCs were maintained at 37 °C, 5% carbon dioxide, fed with fresh media daily, and passaged onto new plates at an average ratio of 1:5 after trypsin dissociation. Prior to conducting research with ESCs at UCLA, approval was granted by the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee. Plasmid construction and transfection cDNA of H3.1 was subcloned into the Topo XL vector (Invitrogen) by RT-PCR from murine ES cell RNA with a Flag tag at the C-terminus and sequence verified. Subsequently the H3.1-Flag cassette was transferred into the pcDNA3 vector (Invitrogen) for expression in mouse ESCs. H3.1K56R-Flag, and H3.1K56Q-Flag, H3.1K56A-Flag and H3.1K9A-Flag plasmids were constructed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) following manufacturer’s instruction and confirmed by sequencing. Plasmids were transfected into E14Tg2a cells using Xfect™ transfection reagent (Clontech, Cat # 631320) in accordance with the manufacturer's 6 instructions. shGFP plasmids were obtained from Gerald Crabtree’s lab. shAsf1a plasmids were bought from Open Biosystems. For prolonged decrease of Asf1a, ESCs were selected with puromycin from day 4 to day 9 after transfection. ChIP-Sequencing Chromatin immunoprecipitation (ChIP) was performed using about 2×107 cells with the following protocol described earlier (5). Briefly, E14Tg2a cells were cross linked with formaldehyde for 10 minutes, lysed in 10mM Tris-EDTA pH 8.0 with 1% SDS, and sonicated (Fisher Scientific #550 Sonic Dismembrator) on ice 4 times at 15 second pulses interrupted by 45 second pauses on power 4 followed by 2 times at 20 second pulses interrupted by 40 second pauses on power 2. Clarified sheared chromatin was immunoprecipitated with antibodies to histone H3K56ac (6) overnight at 4°C. Immunoprecipitated DNA was collected with protein A dynabeads for 3 hours, washed twice for 5 minutes at 4°C with wash buffers and eluted with elution buffer containing 1% SDS. Eluates were heated at 65°C over night to reverse crosslinks, treated with RNase and proteinase K, and DNA was purified with the Qiagen PCR purification kit. 10ng DNA was amplified using the ChIP-Seq DNA Sample Prep. Kit (Illumina, P/N # 1003473), and sequenced with Illumina Genome Analyzer Hiseq2000 at the UCLA Broad Stem Cell Research Center high throughput sequencing facility. ChIP-Seq data analysis Raw data for Nanog, Sox2, Oct4, CTCF, pPolII and Smad1 ChIP-Seq in mouse E14Tg2a cells were downloaded from NCBI GEO (GSE11431) (7). H3K56ac ChIP DNA was 7 prepared in our lab and sequenced in the UCLA Broad Stem Cell Research Center High Throughput Sequencing core facility. The raw ChIP-Seq data mapped uniquely to the mouse genome NCBI Build 37 (UCSC, mm9) by Bowtie (8). We then employed the algorithm described in Ferrari’s paper (9) to evaluate the significantly accumulated peaks of these reads in the genome. The distribution of these peaks around other protein’s peak centers was determined by sitepro (2, 3). The Pearson’s correlation was calculated with Cistrome (10). Functional annotation of ChIP regions was obtained with GREAT (4), using the basal plus extension association rules and the mouse genome (mm9) as background. K-means clustering K-means clustering was performed using GENE CLUSTER 3.0 (11, 12) with Euclidean distance measurement and visualized by Java Treeview (13). Co-immunoprecipitation (Co-IP) assay The co-IP assay was performed as described in the Universal Magnetic Co-IP Kit (Active Motif). 1x108 E14Tg2a cells were scraped, and washed with PBS for each co- IP experiment. Nuclear extracts were digested with Enzymatic Shearing Cocktail containing MNase and then were cleared by centrifugation and nutated overnight with 2 µg α-IgG (Millipore, Cat # 12-370), α-Nanog (Santa Cruz, Cat # sc-134218), α- Sox2 (Santa Cruz, Cat # sc-17320) or α-Oct4 (Santa Cruz, Cat # sc-8628) antibody. Protein G beads were added for 3hrs before washing 5 times in co-IP washing buffer. Protein complexes were eluted for western blot assays. IgG (Millipore, Cat # 8 12-370) was used as a control in Fig. 1B. In vivo peptide pull-down assay H3K56 unmodified (47-65) and H3K56ac (47-65) biotinylated peptides were synthesized at the Proteomics Resource Center (Rockerfeller University) and conjugated to streptavidin beads.
Recommended publications
  • Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes the GLC1C Locus
    LABORATORY SCIENCES Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes The GLC1C Locus Frank W. Rozsa, PhD; Kathleen M. Scott, BS; Hemant Pawar, PhD; John R. Samples, MD; Mary K. Wirtz, PhD; Julia E. Richards, PhD Objectives: To develop and apply a model for priori- est because of moderate expression and changes in tization of candidate glaucoma genes. expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expres- Methods: This Affymetrix GeneChip (Affymetrix, Santa sion level, differential expression, and function. Clara, Calif) study of gene expression in primary cul- ture human trabecular meshwork cells uses a positional Conclusions: Only1geneintheGLC1C interval fits our differential expression profile model for prioritization of model for differential expression under multiple glau- candidate genes within the GLC1C genetic inclusion in- coma risk conditions. The use of multiple prioritization terval. models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. Results: Sixteen genes were expressed under all condi- tions within the GLC1C interval. TMEM22 was the only Clinical Relevance: This study identified a small sub- gene within the interval with differential expression in set of genes that are most likely to harbor mutations that the same direction under both conditions tested. Two cause glaucoma linked to GLC1C. genes, ATP1B3 and COPB2, are of interest in the con- text of a protein-misfolding model for candidate selec- tion. SLC25A36, PCCB, and FNDC6 are of lesser inter- Arch Ophthalmol. 2007;125:117-127 IGH PREVALENCE AND PO- identification of additional GLC1C fami- tential for severe out- lies7,18-20 who provide optimal samples for come combine to make screening candidate genes for muta- adult-onset primary tions.7,18,20 The existence of 2 distinct open-angle glaucoma GLC1C haplotypes suggests that muta- (POAG) a significant public health prob- tions will not be limited to rare descen- H1 lem.
    [Show full text]
  • Regulation of Oxidized Base Damage Repair by Chromatin Assembly Factor 1 Subunit a Chunying Yang1,*,†, Shiladitya Sengupta1,2,*,†, Pavana M
    Published online 27 October 2016 Nucleic Acids Research, 2017, Vol. 45, No. 2 739–748 doi: 10.1093/nar/gkw1024 Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A Chunying Yang1,*,†, Shiladitya Sengupta1,2,*,†, Pavana M. Hegde1,JoyMitra1, Shuai Jiang3, Brooke Holey3, Altaf H. Sarker3, Miaw-Sheue Tsai3, Muralidhar L. Hegde1,2,4 and Sankar Mitra1,2,* 1Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA, 2Weill Cornell Medical College, Cornell University, New York, NY 10065, USA, 3Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and 4Houston Methodist Neurological Institute, Houston, TX 77030, USA Received March 23, 2016; Revised October 13, 2016; Editorial Decision October 17, 2016; Accepted October 19, 2016 ABSTRACT INTRODUCTION Reactive oxygen species (ROS), generated both en- ROS, continuously generated in mammalian cells both en- dogenously and in response to exogenous stress, in- dogenously and by environmental genotoxicants, induce duce point mutations by mis-replication of oxidized various genomic lesions, including oxidized bases, abasic bases and other lesions in the genome. Repair of (AP) sites and single-strand breaks (SSBs). If unrepaired or these lesions via base excision repair (BER) pathway mis-repaired, DNA lesions would cause mutations which may lead to cytotoxicity and cell death and also carcino- maintains genomic fidelity. Regulation of the BER genic transformation (1). The base excision repair (BER) pathway for mutagenic oxidized bases, initiated by pathway, responsible for repair of oxidized base lesions NEIL1 and other DNA glycosylases at the chromatin which contribute to drug/radiation sensitivity is highly con- level remains unexplored.
    [Show full text]
  • Differential Requirements for Tousled-Like Kinases 1 and 2 in Mammalian Development
    Cell Death and Differentiation (2017) 24, 1872–1885 & 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1350-9047/17 www.nature.com/cdd Differential requirements for Tousled-like kinases 1 and 2 in mammalian development Sandra Segura-Bayona1,8, Philip A Knobel1,8, Helena González-Burón1,8, Sameh A Youssef2,3, Aida Peña-Blanco1, Étienne Coyaud4,5, Teresa López-Rovira1, Katrin Rein1, Lluís Palenzuela1, Julien Colombelli1, Stephen Forrow1, Brian Raught4,5, Anja Groth6, Alain de Bruin2,7 and Travis H Stracker*,1 The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance.
    [Show full text]
  • TLK2 Antibody (Center) Purified Rabbit Polyclonal Antibody (Pab) Catalog # Ap8102c
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 TLK2 Antibody (Center) Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP8102c Specification TLK2 Antibody (Center) - Product Information Application WB,E Primary Accession Q86UE8 Other Accession Q9UKI7 Reactivity Human, Mouse Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Antigen Region 141-171 TLK2 Antibody (Center) - Additional Information Western blot analysis of anti-TLK2 Antibody Gene ID 11011 (Center) (Cat.#AP8102c) in mouse testis tissue lysates (35ug/lane).TLK2(arrow) was Other Names detected using the purified Pab. Serine/threonine-protein kinase tousled-like 2, HsHPK, PKU-alpha, Tousled-like kinase 2, TLK2 Target/Specificity This TLK2 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 141-171 amino acids from the Central region of human TLK2. Dilution WB~~1:1000 Format Purified polyclonal antibody supplied in PBS TLK2 Antibody (K155) (Cat. #AP8102c) with 0.09% (W/V) sodium azide. This western blot analysis in Y79 cell line lysates antibody is prepared by Saturated (35ug/lane).This demonstrates the TLK2 Ammonium Sulfate (SAS) precipitation antibody detected the TLK2 protein (arrow). followed by dialysis against PBS. Storage TLK2 Antibody (Center) - Background Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw TLK2, a member of the Ser/Thr protein kinase cycles. family, is rapidly and transiently inhibited by phosphorylation following the generation of Precautions DNA double-stranded breaks during S-phase. TLK2 Antibody (Center) is for research use This is cell cycle checkpoint and ATM-pathway only and not for use in diagnostic or dependent and appears to regulate processes therapeutic procedures.
    [Show full text]
  • Induce Latent/Quiescent HSV-1 Genomes
    Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis Camille Cohen, Armelle Corpet, Simon Roubille, Mohamed Maroui, Nolwenn Poccardi, Antoine Rousseau, Constance Kleijwegt, Olivier Binda, Pascale Texier, Nancy Sawtell, et al. To cite this version: Camille Cohen, Armelle Corpet, Simon Roubille, Mohamed Maroui, Nolwenn Poccardi, et al.. Promye- locytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatiniza- tion through a PML NB/Histone H3.3/H3.3 Chaperone Axis. PLoS Pathogens, Public Library of Science, 2018, 14 (9), pp.e1007313. 10.1371/journal.ppat.1007313. inserm-02167220 HAL Id: inserm-02167220 https://www.hal.inserm.fr/inserm-02167220 Submitted on 27 Jun 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. RESEARCH ARTICLE Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis Camille Cohen1, Armelle Corpet1, Simon
    [Show full text]
  • Histone H3.3 and Its Proteolytically Processed Form Drive a Cellular Senescence Programme
    ARTICLE Received 21 Mar 2014 | Accepted 9 Sep 2014 | Published 14 Nov 2014 DOI: 10.1038/ncomms6210 Histone H3.3 and its proteolytically processed form drive a cellular senescence programme Luis F. Duarte1,2,3, Andrew R.J. Young4, Zichen Wang3,5, Hsan-Au Wu1,3, Taniya Panda1,2, Yan Kou3,5, Avnish Kapoor1,2,w, Dan Hasson1,2, Nicholas R. Mills1,2, Avi Ma’ayan5, Masashi Narita4 & Emily Bernstein1,2 The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Here, using models of oncogene-induced and replicative senescence, we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first 21 amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. 1 Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029, USA. 2 Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L.
    [Show full text]
  • Research2007herschkowitzetvolume Al
    Open Access Research2007HerschkowitzetVolume al. 8, Issue 5, Article R76 Identification of conserved gene expression features between comment murine mammary carcinoma models and human breast tumors Jason I Herschkowitz¤*†, Karl Simin¤‡, Victor J Weigman§, Igor Mikaelian¶, Jerry Usary*¥, Zhiyuan Hu*¥, Karen E Rasmussen*¥, Laundette P Jones#, Shahin Assefnia#, Subhashini Chandrasekharan¥, Michael G Backlund†, Yuzhi Yin#, Andrey I Khramtsov**, Roy Bastein††, John Quackenbush††, Robert I Glazer#, Powel H Brown‡‡, Jeffrey E Green§§, Levy Kopelovich, reviews Priscilla A Furth#, Juan P Palazzo, Olufunmilayo I Olopade, Philip S Bernard††, Gary A Churchill¶, Terry Van Dyke*¥ and Charles M Perou*¥ Addresses: *Lineberger Comprehensive Cancer Center. †Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. ‡Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. reports §Department of Biology and Program in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. ¶The Jackson Laboratory, Bar Harbor, ME 04609, USA. ¥Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. #Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA. **Department of Pathology, University of Chicago, Chicago, IL 60637, USA. ††Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132, USA. ‡‡Baylor College of Medicine, Houston, TX 77030, USA. §§Transgenic Oncogenesis Group, Laboratory of Cancer Biology and Genetics. Chemoprevention Agent Development Research Group, National Cancer Institute, Bethesda, MD 20892, USA. Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Section of Hematology/Oncology, Department of Medicine, Committees on Genetics and Cancer Biology, University of Chicago, Chicago, IL 60637, USA.
    [Show full text]
  • Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes the GLC1C Locus
    LABORATORY SCIENCES Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes The GLC1C Locus Frank W. Rozsa, PhD; Kathleen M. Scott, BS; Hemant Pawar, PhD; John R. Samples, MD; Mary K. Wirtz, PhD; Julia E. Richards, PhD Objectives: To develop and apply a model for priori- est because of moderate expression and changes in tization of candidate glaucoma genes. expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expres- Methods: This Affymetrix GeneChip (Affymetrix, Santa sion level, differential expression, and function. Clara, Calif) study of gene expression in primary cul- ture human trabecular meshwork cells uses a positional Conclusions: Only1geneintheGLC1C interval fits our differential expression profile model for prioritization of model for differential expression under multiple glau- candidate genes within the GLC1C genetic inclusion in- coma risk conditions. The use of multiple prioritization terval. models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. Results: Sixteen genes were expressed under all condi- tions within the GLC1C interval. TMEM22 was the only Clinical Relevance: This study identified a small sub- gene within the interval with differential expression in set of genes that are most likely to harbor mutations that the same direction under both conditions tested. Two cause glaucoma linked to GLC1C. genes, ATP1B3 and COPB2, are of interest in the con- text of a protein-misfolding model for candidate selec- tion. SLC25A36, PCCB, and FNDC6 are of lesser inter- Arch Ophthalmol. 2007;125:117-127 IGH PREVALENCE AND PO- identification of additional GLC1C fami- tential for severe out- lies7,18-20 who provide optimal samples for come combine to make screening candidate genes for muta- adult-onset primary tions.7,18,20 The existence of 2 distinct open-angle glaucoma GLC1C haplotypes suggests that muta- (POAG) a significant public health prob- tions will not be limited to rare descen- H1 lem.
    [Show full text]
  • A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family
    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase.
    [Show full text]
  • Supplementary Data
    Supplementary Figure 1 Supplementary Figure 2 CCR-10-3244.R1 Supplementary Figure Legends Supplementary Figure 1. B-Myb is overexpressed in primary AML blasts and B-CLL cells. Baseline B-Myb mRNA levels were determined by quantitative RT-PCR, after normalization to the level of housekeeping gene, in primary B-CLL (n=10) and AML (n=5) patient samples, and in normal CD19+ (n=5) and CD34+ (n=4) cell preparations. Each sample was determined in triplicate. Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Supplementary Figure 2. Cytotoxicity by Nutlin-3 and Chlorambucil used alone or in combination in leukemic cells. The p53wild-type EHEB and SKW6.4 cells lines, and the p53mutated BJAB cell line were exposed to Nutlin-3 or Chlorambucil used either alone or in combination. (Nutl.+Chlor.). In A, upon treatment with Nutlin-3 or Chlorambucil, used either alone (both at 10 μM) or in combination (Nutl.+Chlor.), induction of apoptosis was quantitatively evaluated by Annexin V/PI staining, while E2F1 and pRb protein levels were analyzed by Western blot. Tubulin staining is shown as loading control. The average combination index (CI) values (analyzed by the method of Chou and Talalay) for effects of Chlorambucil+Nutlin-3 on cell viability are shown. ED indicates effect dose. In B, levels of B-Myb and E2F1 mRNA were analyzed by quantitative RT- PCR. Results are expressed as fold of B-Myb and E2F1 modulation in cells treated for 24 hours as indicated, with respect to the control untreated cultures set to 1 (hatched line).
    [Show full text]
  • Molecular Basis of Tousled-Like Kinase 2 Activation
    ARTICLE DOI: 10.1038/s41467-018-04941-y OPEN Molecular basis of Tousled-Like Kinase 2 activation Gulnahar B. Mortuza1, Dario Hermida1, Anna-Kathrine Pedersen2, Sandra Segura-Bayona 3, Blanca López-Méndez4, Pilar Redondo 5, Patrick Rüther 2, Irina Pozdnyakova4, Ana M. Garrote5, Inés G. Muñoz5, Marina Villamor-Payà3, Cristina Jauset3, Jesper V. Olsen 2, Travis H. Stracker3 & Guillermo Montoya 1 Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In 1234567890():,; humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhi- bitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs. 1 Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. 2 Mass Spectrometry for Quantitative Proteomics, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
    [Show full text]
  • Dendritic Cell Maturation Transcription Factor E2F1 Suppresses
    Transcription Factor E2F1 Suppresses Dendritic Cell Maturation Fang Fang, Yan Wang, Rui Li, Ying Zhao, Yang Guo, Ming Jiang, Jie Sun, Yang Ma, Zijia Ren, Zhigang Tian, Feng This information is current as Wei, De Yang and Weihua Xiao of September 29, 2021. J Immunol 2010; 184:6084-6091; Prepublished online 26 April 2010; doi: 10.4049/jimmunol.0902561 http://www.jimmunol.org/content/184/11/6084 Downloaded from References This article cites 40 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/184/11/6084.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Transcription Factor E2F1 Suppresses Dendritic Cell Maturation Fang Fang,*,1 Yan Wang,*,1 Rui Li,* Ying Zhao,* Yang Guo,* Ming Jiang,* Jie Sun,* Yang Ma,* Zijia Ren,* Zhigang Tian,* Feng Wei,† De Yang,†,‡ and Weihua Xiao* Transcription factor E2F1 has been largely studied as a promoter of S-phase transition in the cell cycle and as a regulator of ap- optosis.
    [Show full text]