1 Supplementary Information for Acetylated Histone H3K56 Interacts
Total Page:16
File Type:pdf, Size:1020Kb
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.