were subsequently eluted in 100 ml Elution Buffer (50 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65uC for 15 minutes, and incubated overnight at 65uC. DNA was purified with a Qiagen DNA Isolation column (Qiagen). The amount of each specific DNA fragment in immunoprecipitates was determined by real-time PCR. Triplicate PCR reactions using the iQ SYBR Green Supermix (BioRad) were analyzed in a MyIQ Real-Time PCR Detection System (BioRad). All samples were typically analyzed in triplicate in two independent experiments. The following primers were used: Oct4: forward 59-TGGGCTGAAA- TACTGGGTTC-39, reverse 59- TTGAATGTTCGTGTGCC- AAT-39; Nanog: forward 59-GGCATGGTGGTAGACAAGCC- 39, reverse 59-TTAGTAAGTTGGTCCATGCTTTGG-39. The percentage of input was calculated by dividing the amount of each specific DNA fragment in the immunoprecipitates by the amount of DNA present in the sample before immunoprecipitation (input DNA). The values from ChIP with control antibody (IgG) were typically less than 5% of the ChIP values with the antibodies against histone modifications. Antibodies The following antibodies were used for Western blotting and qChIP: anti-OCT4 (Santa Cruz sc8628), anti-GAPDH (Ambion AM4300), anti-b ACTIN (Sigma-Aldrich A5316), anti-FLAG (Sigma-Aldrich F3165), anti-H1 (Millipore 05-457), anti-H10 (Santa Cruz 56695), anti-H3K4me3 (Millipore 07-473), anti- H3K9me3 (Abcam 8898), anti-H3K27me3 (Millipore 07-449), anti-H3 (Abcam 1791) and IgG (Millipore 12-370). Bisulfite modification, PCR amplification, and sequencing analysis Genomic DNA was prepared from mESCs, EBs, and embryos. 0.1 to 1 mg of DNA was treated with the Bisulfite Conversion Kit (CpG Genome) according to the manufacturer’s manual. 1 ml of treated DNA was used in each PCR reaction as previously described [10]. The primers used to generate PCR products from the bisulfite-converted DNA are specific for the converted DNA sequence of the analyzed regions. The primer sequences were as follows: Oct4 region1: forward 59- GATATGGGTTGAAATAT- TGGGTTTAT-39, reverse 59-AATCCTCTCACCCCTACCT- TAAAT-39; Oct4 region 2: forward 59-AAGGTTGAAAATG- AAGGTTTTTTG-39, reverse 59-TCCAACCATAAAAAAAA- TAAACACC-39; Nanog: forward 59- TTTGTAGGTGGGAT- TAATTGTGAAT-39, reverse 59-AAAAAATTTTAAACAACA- ACCAAAAA-39. The PCR products were subsequently cloned using the TOPO TA Cloning kit (Invitrogen), and clones containing the converted DNA inserts were picked and sequenced. DNA sequences were analyzed with BiQ analyzer [54]. Generation of H1d rescue (RES) cell lines The H1d overexpression plasmid was constructed by cloning a 5 Kb fragment encompassing H1d coding region (with an insertion of FLAG tag at N-terminus) and proximal regulatory sequences into a vector containing a Blasticidin resistant gene. 20 mg of plasmid DNA was transfected into 26107 H1 TKO ESCs as described before [14], and 96 cell clones resistant to blasticidin were picked and analyzed by Western blotting using an anti- FLAG antibody (Sigma-Aldrich). Two cell lines with the highest levels of H1d were selected as RES cell lines for further analysis. Supporting Information Figure S1 (TIF) Chromosome spreads of WT and H1 TKO ESCs. Loss of H1 Impairs Stem Cell Differentiation Figure S2 Gene expression analysis of ESCs and EBs formed in rotary suspension culture. (A) qRT-PCR analysis of expression levels of Nkx2.5 and a-MHC in WT and H1 TKO cells during EB differentiation. Expression levels were normalized over GAPDH. (B) List of genes that displayed more than two-fold differences (P,0.05) in expression shown in Figure 2Di, 2Dii and 2Diii, respectively. (C) Scatter plot analysis comparing the degree of changes in gene expression in WT and H1 TKO cells during EB differentiation. X-axes and y- axes are delta delta CTs. (TIF) Figure S3 Analysis of total H1 and H10 levels during EB differentiation. 2 mg histone proteins were analyzed with immu- noblotting with antibodies indicated. The bottom panel of Western blotting with anti-H3 antibody demonstrates equal loading of proteins in each lane. (TIF) Figure S4 Increased expression of Nanog by H1 depletion in embryos. (A) qRT-PCR analysis of E8.5 embryos indicating the higher levels of Nanog expression in H1 TKO embryos compared with WT. Values are means 6 SEM, n = 5 for each genotype. Expression levels were normalized over GAPDH. *: P,0.05. (B) DNA methylation status of promoter regions of Nanog in E8.5 embryos analyzed by bisulfite sequencing. (C) Percentage of CpG methylation calculated from results in (B). (TIF) Figure S5 Generation and characterization of RES ESC lines. (A) Representative Western blotting analysis of ‘‘rescue’’ clones. Immunoblotting with anti-b-ACTIN antibody indicates equal loading of whole cell lysates. (B) Reverse phase HPLC analysis of a RES cell line with high levels of H1d expression. (C) Chromosome spread of the RES cell shown in B). (D) Hematoxylin and eosin staining of sections of day 10 EBs generated from RES cells in rotary suspension culture. Scale bar: 100 mm. (E) qRT-PCR analysis of differentiation markers in RES cells during EB differentiation. Expression levels were normalized over GAPDH. (TIF) Figure S6 Analysis of expression and epigenetic marks at Nanog promoter. (A) qRT-PCR analysis of Nanog expression in ESCs and day 10 EBs. Expression levels were normalized over GAPDH. (B) DNA methylation status of Nanog promoter in mouse embryonic fibroblasts (MEFs) (left) or in ESCs (day 0) and day 10 EBs (right). (C) qChIP analysis of H1, H3K4me3, H3K9me3 and H3K27me3 levels at Nanog promoters in ESCs (day 0) and day 10 EBs. Data were normalized as described in Figure 5Biv. *: P,0.05; **: P,0.01. (TIF) Figure S7 qChIP Analysis of H10 occupancy at Oct4 promoter during EB differentiation. Data were normalized as described in Figure 5Biv. *: P,0.05; **: P,0.01; ***: P,0.001. (TIF) Acknowledgments We thank colleagues and lab members for critical reading of this manuscript, and the anonymous reviewers for their comments. We thank Samantha Lasater for editorial assistance. Author Contributions Conceived and designed the experiments: YF TCM. Performed the experiments: YZ MC SP KC BK P-YH MM DTB CP YF. Analyzed the data: YZ MC SP KC BK MM TCM YF. Contributed reagents/materials/ analysis tools: YF TCM. Wrote the paper: YF TCM SP KC. PLoS Genetics | www.plosgenetics.org 13 May 2012 | Volume 8 | Issue 5 | e1002691