Loss of H1 Impairs Stem Cell Differentiation Figure 5. H1 is necessary for stable repression of Oct4 pluripotency gene during embryogenesis and ESC differentiation. (A) Elevated Oct4 expression and hypomethylation of CpG sites at Oct4 promoters in H1 TKO embryos compared with littermates at E8.5. (i) qRT-PCR analysis of mRNA expression levels of Oct4. Values are means 6 SEM, n = 5 for each genotype. Expression levels were normalized over GAPDH. *: P,0.05. (ii) Bisulfite sequencing analysis of DNA methylation status at Oct4 promoter regions. Results of two wild-type and two knockout E8.5 embryos are shown. The positions of CpG sites analyzed are depicted schematically as vertical ticks on the line. TSS: transcription start site. (iii) Percentage of methylated CpG sites at Oct4 promoter regions in WT and H1 TKO embryos. Statistical analysis was performed using Fisher’s exact test. ***: P,0.001; ****: P,0.0001. (B) Analysis of expression and epigenetic marks at Oct4 pluripotency gene during EB differentiation in rotary suspension culture. Analyses of expression (i), DNA methylation (ii), % of mCpG (iii); and occupancy of H1 and three histone marks (iv) of Oct4 in WT, H1 TKO and RES cells during EB differentiation. Relative expression levels were normalized over GAPDH. Relative fold enrichment is calculated by normalizing the qChIP values (as described in Material and Methods) of ESCs (day 0) or EBs at each time point by that of WT ESCs (WT D0). Values are presented as mean 6 S.D. *: P,0.05; **: P,0.01; ***: P,0.001. (C) Model for H1 in repression of Oct4 during ESC differentiation. ESCs have low H1 content with an relatively ‘‘open’’ chromatin. During differentiation, total H1 content increases, which facilitates local chromatin compaction at Oct4 gene and contributes to establishment and/or maintenance of epigenetic changes necessary for stable silencing of Oct4 pluripotency gene. doi:10.1371/journal.pgen.1002691.g005 indicating that depletion of H1 impairs repression of the expression of pluripotency factors in E8.5 embryos in vivo. DNA methylation of cytosine nucleotide at CpG sites within gene promoter regions contributes to stable gene silencing, and thus is a key determinant in regulating the expression of pluripotency genes [26], so we asked if the DNA methylation status at the Oct4 and Nanog promoters is affected in H1 TKO embryos. Results from bisulfite sequencing analysis demonstrated that the extent of CpG methylation at the Oct4 promoter region was markedly reduced in triple-H1 embryos in comparison with corresponding wild-type littermates (Figure 5Aii), whereas the level of DNA methylation (percent methylation of analyzed CpGs) at Nanog promoter did not display differences between WT and H1 TKO embryos (Figure S4B, S4C). This suggests that H1 participates in establishing and/or maintaining CpG methylation at Oct4 promoter during embryogenesis. To further investigate the mechanisms by which H1 regulates pluripotency genes during ESC differentiation, we analyzed the epigenetic profiles of the Oct4 and Nanog genes during EB differentiation in rotary suspension culture. We demonstrated previously that this method produces a large quantity of homogeneous EBs that progressively differentiate [18], thus the sequential epigenetic events can be readily followed. Expression of Oct4 and Nanog was reduced during continuous suspension culture for WT cultures, but remained high in TKO EB cultures (Figure 5Bi, Figure S6A). DNA methylation analysis by bisulfite sequencing indicated that WT EBs had an increase in the sporadic DNA methylation at specific CpG sites throughout the Oct4 proximal promoter region at day 10 (P = 0.002 and 0.036 for the respective R1 and R2 regions) (Figure 5Bii, iii), whereas TKO EBs remained completely unmethylated at these sites. On the other hand, Nanog promoter region remained unmethylated throughout the differentiation in both WT and H1 TKO cultures (Figure S6B). To further investigate the effect of H1 levels in affecting expression and DNA methylation of pluripotency genes in EB differentiation, we generated ‘‘rescue’’ cell lines (referred to as ‘‘RES’’) by stably overexpressing exogenous H1d in the H1 TKO cells (Figure S5A). RES cells had a H1/nucleosome ratio of 0.31 (Figure S5B), displayed a normal karyotype (Figure S5C), and were able to differentiate into EBs with cystic structures which were observed in WT, but not in TKO, EBs (Figure S5D). RES EBs had elevated expression of differentiation markers, such as AFP and Nkx2.5 (Figure S5E) and reduced expression of Oct4 and Nanog pluripotency genes upon differentiation (Figure 5Bi and Figure S6A), suggesting that the expression of exogenous H1d alleviates the differentiation defects and restores the repression of pluripotency factors in H1 TKO EBs. In addition, the percent of methylated CpG was increased in RES EBs to a level comparable to that of WT EBs at the same time points, suggesting that reintroduction of H1d into the H1 TKO ESCs is able to reestablish DNA methylation and the stable repression of the Oct4 gene in differentiating EBs (Figure 5Bii, iii). We next analyzed the status of H1, H3K4me3, H3K9me3 and H3K27me3 at the promoters of pluripotency genes Oct4 and Nanog by quantitative chromatin immunoprecipitation (qChIP). Whereas H1 occupancy at Oct4 promoter increased in WT and RES cultures during differentiation, it remained unchanged in H1 TKO EBs (Figure 5Biv). It is interesting to note that the occupancy of the replacement subtype, H10, at Oct4 promoter was markedly increased in both WT and RES cultures but only mildly elevated in H1 TKO cultures (Figure S7), suggesting that efficient binding of H10 at Oct4 promoter may be facilitated by sufficient amount of other somatic H1s. Furthermore, wild-type and RES EBs displayed decreasing levels of the active histone mark H3K4me3 accompanied with a significant increase in the levels of the repressive histone mark H3K9me3, at promoter regions of pluripotency genes Oct4 and Nanog upon EB differen- tiation (Figure 5Biv and Figure S6C). In contrast, H1 TKO EBs did not display similar or significant changes in the levels of these histone marks at the same promoter regions (Figure 5Biv and Figure S6C). Levels of H3K27me3, another repressive histone mark, were significantly increased in WT cultures during differentiation at Oct4 promoter, while such increases were not detected at H1 TKO or RES EBs (Figure 5Biv). These analyses suggest that the increase of H1 levels and the changes in histone modifications, such as H3K4me3, H3K9me3 and H3K27me3, precede DNA methylation establishment in mediating Oct4 gene silencing during EB differentiation. Overall, the results indicate that lack of H1c, H1d and H1e impairs the establishment or maintenance of epigenetic changes in DNA methylation and histone modifications that are necessary for stable repression of pluripotent transcription factor Oct4 in differentiated cells (Figure 5C). Discussion Embryonic stem cells, derived from the inner cell mass of the blastocyst stage mammalian embryos [27,28], can self-renew nearly indefinitely in culture and give rise to all cell types of the three germ layers, ectoderm, mesoderm and endoderm, during differentiation. ESCs possess distinctive transcriptional regulatory circuits and chromatin signatures that are critical for maintaining pluripotency and self-renewal [29,30]. Recent studies suggest that ESCs exhibit a relatively ‘‘open’’ chromatin state, and during differentiation, heterochromatin formation increases [2,8,31]. However, whether this ‘‘open’’ chromatin state is necessary for pluripotency and whether the compaction of chromatin is required for ESC differentiation remain to be addressed. Linker histone H1 is the major chromatin architectural protein in mediating higher order chromatin folding. H1 TKO ESCs have PLoS Genetics | www.plosgenetics.org 9 May 2012 | Volume 8 | Issue 5 | e1002691