SUPPLEMENTAL INFORMATION SetDB1 Contributes to Repression of Genes Encoding Developmental Regulators and Maintenance of ES Cell State Steve Bilodeau, Michael Kagey, Garrett M. Frampton, Peter B. Rahl, Richard A. Young CONTENTS Supplemental Tables Supplemental Figures Supplemental Data Files Supplemental Experimental Procedures Growth Conditions for Embryonic Stem Cells High-Throughput shRNA Screening Library Design and Lentiviral Production Lentiviral Infections Immunofluorescence Image Acquisition and Analysis Validation of shRNAs Lentiviral Production and Infection Immunofluorescence Chromatin Immunoprecipitation ChIP-Seq Sample Preparation and Analysis Sample Preparation Polony Generation and Sequencing ChIP-Seq Data Analysis ChIP-Seq Density Heatmaps Heatmap display of similarity between genomic occupancy of multiple factors Gene Ontology Analysis Comparison to Previous Results Comparison of H3K9me3 ChIP-Seq datasets H3K9me3 Antibody Specificity SetDB1 Antibody Specificity PCR primers (mm8) RNA Extraction, cDNA, and TaqMan Expression Analysis Microarray Expression Analysis Supplemental Discussion 1 H3K9 Associated Validation SetDB1 Validation Criteria for Identifying Chromatin Regulators Screening Results Comparison Comments on Design and Saturation of Screen Comments on Chromatin Regulators Identified in the Screen H3K9 Methylation Associated HDAC Associated Polycomb Repressive Complex Cohesin Complex Members Uncharacterized Methyltransferases H3K4 Methyltransferases Histone Chaperones SWI/SNF Arginine Methylation H3K36 Methylation Associated Supplemental References Supplemental Tables Supplemental Table 1 – Results of chromatin regulator shRNA screen Supplemental Table 2 – Z-scores of shRNAs used in the screen Supplemental Table 3 – Genes bound by SetDB1, H3K9me3, H3K4me3, H3K27me3, H3K36me3, H3K79me2, Pol2, Oct4, Sox2, Nanog and Tcf3 in mES cells Supplemental Table 4 – H3K9me3 at DNA repeats Supplemental Table 5 – SetDB1 at DNA repeats Supplemental Table 6 – Summary of ChIP-Seq data used Supplemental Table 7 – Genomic regions bound by H3K9me3 (3 worksheets) in mES cells Supplemental Table 8 – Genomic regions bound by SetDB1 in mES cells Supplemental Table 9 – Summary of gene ontology analysis results for Fig. 2B Supplemental Table 10 – Complete ES cell gene expression data Supplemental Table 11 – Gene expression data for Fig. 3G Supplemental Table 12 – RSA screen analysis Supplemental Table 13 – Comparison of the different published screen Supplemental Table 14 – Normalized ChIP-Seq data for H3K9me3 in shRNA GFP and shRNA SetDB1 mES cells Supplemental Figures Supplemental Figure 1 – Validation of H3K9 Associated shRNAs. (A) The two best shRNAs targeting SetDB1, Suv39h2, Ehmt1 and Ube2i/Ubc9 identified in the screen result in efficient knockdown and also a decrease in Oct4 expression. 2 Murine ES cells were split off of MEF feeder cells, infected with the indicated shRNA knockdown lentivirus or a shRNA GFP control lentivirus. Expression levels were determined by real-time qPCR. RNAi Consortium Oligo ID (TRC) numbers are shown for each shRNA. shRNA sequences are available from Open Biosystems. Supplemental Figure 2 – Validation of SetDB1 shRNAs and loss of ES cell state. (A) SetDB1 knockdown with shRNA #1 and #2 results in morphological changes and decreased Oct4 staining intensity. Murine ES cells were split off of MEF feeder cells, infected with the indicated SetDB1 knockdown lentivirus or a shRNA GFP control lentivirus. Cells were crosslinked and stained with Hoechst and for Oct4. Z-scores from the screening results and RNAi Consortium Oligo ID (TRC) numbers are shown for each shRNA. shRNA sequences are available from Open Biosystems. (B) SetDB1 knockdown results in decreased expression of Oct4. Real-time qPCR indicates a loss of Oct4 expression following infection of mES cells with shRNAs #1 and #2 targeting SetDB1. (C) SetDB1 knockdown in 129/cast mES cells results in loss of Oct4. Supplemental Figure 3 – Specificity of SetDB1 antibodies. Western blot of (A) SetDB1 SC-66884 (H-300, Santa Cruz) and (B) SetDB1 11231-AP (Proteintech Group) using nuclear extract of mES cells infected with a GFP or SetDB1 shRNA. (C) ChIP PCR using both SetDB1 antibodies at Polrmt, Nnat and Mybl2. (D) ChIP PCR with SetDB1 H-300 antibody in mES cells infected with a GFP or SetDB1 shRNA. Supplemental Figure 4 – Reproducibility and specificity of ChIP-Seq experiments with H3K9me3 antibodies. (A) Venn diagram representation of genes occupied by histone H3K9me3 in mES cells profiled with two antibodies (Ab8898 and Up07-442) and in previously published data (Mikkelsen et al., 2007). (B, C, D) Examples of H3K9me3 histone modification profiles for the three datasets. (E, F, G) Peptide competition assays to validate antibody specificity. Each antibody binding was competed with unmodified H3, H3K9me3, H3K4me3 and H3K27me3. (H, I, J) Comparison of H3K9me3 and H3K4me3. (K, L, M) Comparison of H3K9me3 and H3K27me3. Supplemental Figure 5 – SetDB1 and H3K9me3 co-occupy a common set of genes. Venn diagram representation of genes called ± 5kb from the TSS. Supplemental Figure 6 – SetDB1 dependent H3K9me3. (A) Analysis of normalized H3K9me3 ChIP-Seq in mES cells infected with GFP or SetDB1 shRNAs. Genes that are bound by SetDB1 and H3K9me3 were classified based on their relative level of H3K9me3 following SetDB1 knockdown. The percentage indicates the fraction of SetDB1 and H3K9me3 occupied genes. (B, C) SetDB1 and H3K9me3 profiles showing a loss of H3K9me3 or (D) no change. 3 Supplemental Figure 7 – Supplemental gene tracks. SetDB1 and histone H3K4me3, H3K27me3 and H3K9me3 occupancy profiles for (A) Olig2, (B) Sumo3, (C) Jmjd1a and (D) Pramel1. Supplemental Figure 8 – Occupancy of H3K4me3, H3K27me3 and H3K9me3 at nucleosomes adjacent to the transcription start site. (A to C) H3K4me3, H3K27me3 and H3K9me3 occupancy profiles for Pax7, Lmx1b, Dlx1. Boxes represent nucleosomes with evidence of all three marks. Supplemental Data Files The following files contain data formatted (.WIG) for upload into the UCSC genome browser (Kent et al., 2002). To upload the files, first copy the files onto a computer with internet access. Then use a web browser to go to http://genome.ucsc.edu/cgi-bin/hgCustom?hgsid=105256378 for mouse. In the “Paste URLs or Data” section, select “Browse…” on the right of the screen. Use the pop-up window to select the copied files, then select “Submit”. The upload process may take some time. MM8_mES_H3K9me3_AB8898.WIG.gz MM8_mES_H3K9me3_UP7442.WIG.gz MM8_mES_SetDB1_H300.WIG.gz MM8_mES_WT_shRNAGFP_shRNASetDB1_normalized _H3K9me3.WIG.gz These files present ChIP-Seq data for H3K9me3 with two different antibodies and SetDB1 in mES cells. They also include normalized ChIP-Seq data for H3K9me3 for cells infected with GFP and SetDB1 shRNAs. The first track for each data set contains the ChIP-Seq density across the genome in 25bp bins. The minimum ChIP-Seq density shown in these files is 1.5 reads per million total reads. Subsequent tracks identify genomic regions identified as enriched. Supplemental Experimental Procedures Growth Conditions for Embryonic Stem Cells 4 V6.5 and 129/Cast hybrid murine embryonic stem (mES) cells were grown on irradiated murine embryonic fibroblasts (MEFs) unless otherwise stated. Cells were grown under standard mES cell conditions as described previously (Boyer et al., 2005). Briefly, cells were grown on 0.2% gelatinized (Sigma, G1890) tissue culture plates in ESC media; DMEM-KO (Invitrogen, 10829-018) supplemented with 15% fetal bovine serum (Hyclone, characterized SH3007103), 1000 U/mL LIF (ESGRO, ESG1106), 100 μM nonessential amino acids (Invitrogen, 11140-050), 2 mM L-glutamine (Invitrogen, 25030-081), 100 U/mL penicillin, 100 μg/mL streptomycin (Invitrogen, 15140-122), and 8 nL/mL of 2-mercaptoethanol (Sigma, M7522). High-Throughput shRNA Screening Library Design and Lentiviral Production Small hairpins targeting 197 chromatin regulators were designed and cloned into pLKO.1 lentiviral vectors as previously described (Moffat et al., 2006). On average 5 different shRNAs targeting each chromatin regulator were used. Lentiviral supernatants were arrayed in 384-well plates with negative control lentivirus (shRNAs targeting GFP, RFP and LacZ) (Moffat et al., 2006). Lentiviral Infections Murine ES cells were split off MEFs and placed in a tissue culture dish for 45 minutes to selectively remove the MEFs. Murine ES cells were counted with a Coulter Counter (Beckman) and seeded using a μFill (Bioteck) at a density of 1500 cells/well in 384-well plates (Costar 3712) treated with 0.2% gelatin (Sigma, G1890). An initial cell plating density of 1500 cells/well was established so that an adequate amount of cells would survive puromycin selection for analysis. However, the initial cell plating density was kept low enough to avoid wells reaching confluency during the timeframe of the assay. One day following cell plating the media was removed, replaced with ESC media containing 8 μg/ml of polybrene (Sigma, H9268-10G) and cells were infected with 2 µl of shRNA lentiviral supernatant from the chromatin regulator set. Infections were performed in quadruplicate on separate plates. Control wells on each plate were mock infected and designated as “Empty”. Positive control wells on each plate were infected with 3 µl of validated control shRNA lentiviral supernatant targeting Oct4 (TRCN0000009613), Tcf3 (TRCN0000095454) and Stat3 (TRCN0000071454) that was generated independently of the chromatin regulator set. Plates were spun for