Author Summary The epigenome and chromatin play critical roles in stem cell fate determination. Linker histone H1 is a major chromatin structural protein that facilitates higher-order chromatin folding. By analyzing the differentiation capac- ity of embryonic stem cells (ESCs) that lack multiple H1 subtypes, we find, for the first time, that H1 and higher- order chromatin compaction are required for proper differentiation and lineage commitment of pluripotent stem cells. Triple-H1 murine ESCs are impaired in both spontaneous differentiation and embryoid body differen- tiation. Furthermore, triple-H1 ESCs are compromised in neural differentiation. Finally, we demonstrate that H1 depletion leads to failure of efficient repression of pluripotency gene expression both in embryos and in ESC differentiation. We present evidence that H1 partici- pates in mediating changes of histone marks and DNA methylation necessary for silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. This finding is important because it provides a mechanistic link by which H1 and chromatin compaction may participate in pluripotent stem cell differentiation through repression of pluripotency gene expression. We have previously derived wild-type (WT) and H1c/H1d/ H1e triple knockout (H1 TKO) embryonic stem cells from the outgrowth of the inner cell masses of blastocysts attained from intercrosses of H1 heterozygous mutants [10]. We have measured that wild-type ESCs have an H1/nucleosome ratio of 0.46 [10], a much lower level compared with a ratio of 0.75,0.83 from various differentiated cell types in mouse tissues [11,15], suggesting that ESCs have a more open chromatin structure compared with differentiated cell types in adult tissues. H1 TKO ESCs have an even lower H1/nucleosome ratio that is close to 0.25, equivalent to 1 H1 per 4 nucleosomes. The compound H1 ES cells display chromatin decondensation in bulk chromatin [10] and an increased nuclear size [16], offering an ideal system to test the necessity of chromatin compaction on ESC pluripotency and differentiation. In the current study, we demonstrate, for the first time, that the differentiation capacity of ESCs that lack multiple H1 subtypes is severely impaired. We find that compound H1 ESCs are more resistant to spontaneous differentiation, impaired in embryoid body differentiation, and largely blocked in neural differentiation. Finally, we present evidence that H1 contributes to efficient repression of the expression of pluripotency factors and partici- pates in establishment and maintenance of epigenetic marks necessary for silencing pluripotency genes during stem cell differentiation and embryogenesis. Results Loss of H1c/H1d/H1e inhibits spontaneous ESC differentiation ESCs exhibit a relatively ‘‘open’’ chromatin structure compared with differentiated cells or lineage committed cells [8]. H1c/H1d/ H1e triple ESCs we derived previously have a significant reduction in total H1 protein levels which leads to further decreased chromatin compaction [10], thus we postulated that loss of H1c, H1d, and H1e may interfere with ESC differentiation. We first compared the spontaneous differentiation tendency of two H1 TKO ESC lines with wild-type littermate ESC lines. Consistent with previous observations [10], H1 TKO ESCs cultured on Loss of H1 Impairs Stem Cell Differentiation mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells with media containing leukemia inhibitory factor (LIF) have comparable growth rate to that of wild-type ESCs (data not shown) and normal karyotypes (Figure S1). In addition, H1 TKO ESCs expressed comparable levels of pluripotency factor OCT4 (POU5F1) (Figure 1A), and displayed a similar ESC colony morphology to that of WT ESCs under culture conditions which promote ESC self-renewal (Figure 1B, left panel). However, when cultured in a feeder-free manner on gelatin-coated plates without MEFs, the H1 TKO cells displayed higher levels of OCT4, a more homogeneous, undifferentiated colony morphology, and a higher growth rate than WT ESCs under the same condition (Figure 1A, 1B middle panel, and 1C). Furthermore, upon removal of LIF, the majority of H1 TKO ESCs continued to retain high expression levels of OCT4 (Figure 1A) as well as a tightly packed colony morphology typical of undifferentiated ESCs (Figure 1B, right panel) for a week. In contrast, wild-type ESCs differentiated readily, with approximate 90% of the cells appearing to differentiate by 2 days after LIF removal in feeder free culture, as judged by diminishing OCT4 expression and the loss of a compact colony morphology (Figure 1A, 1B right panel). Removal of LIF reduced the growth of both WT and H1 TKO ESCs (Figure 1C), consistent with LIF’s known role in promoting self- renewal and proliferation of ESCs [17]. Collectively, these results suggest that ESCs lacking H1c, H1d, and H1e are more refractory to spontaneous ESC differentiation in vitro. Loss of H1c, H1d, and H1e impairs EB differentiation To assess whether loss of H1c, H1d and H1e impairs cellular differentiation of any of the three germ layers, we examined the ability of H1 TKO ESCs to form embryoid bodies (EB) using a rotary orbital suspension culture system to induce differentiation in vitro. We have previously shown that the rotary suspension culture method offers improved efficiency and homogeneity of embryoid body production compared with the common practice of forming EB aggregates in static suspension culture [18]. During EB culture in serum-containing media, ESCs form aggregates and differen- tiate into cell types of all three primitive germ layers: endoderm, mesoderm and ectoderm, offering a temporal window to investigate specific defects in lineage differentiation. After 10 days of culture in rotary suspension, the wild-type EBs had a distinct outer endoderm-layer surrounding differentiated cell morpholo- gies representing the three germ layers, including different epithelial cell types and mesenchymal cell populations (Figure 2A). In contrast, although H1 TKO ESCs were able to form putative EBs, most H1 TKO EBs appeared blocked in the differentiation process in rotary suspension culture, forming undifferentiated masses of stem cells that lacked cavity formation and other types of differentiated structures even after prolonged culture in rotary suspension (up to 14 days) (Figure 2A). Quantitative RT-PCR analyses also indicated that the expression of differentiation markers, such as the endoderm marker, alpha- fetoprotein (AFP), was drastically increased in WT EBs, but significantly curbed in H1 TKO EBs (Figure 2B). The mRNA levels of other lineage specific markers, including mesoderm markers, such as the cardiac transcription factor Nkx2.5, and the sarcomeric muscle marker, alpha myosin heavy chain (aMHC), also progressively increased over time in WT EBs, but were not detected at similar levels in H1 TKO EBs (Figure S2A). To gain a more comprehensive view of the scope of genes affected by linker histone H1 depletion during differentiation, we performed quantitative PCR SuperArray analysis of wild-type and H1 TKO cells at the start (day 0) and the end point (day 10) of rotary suspension culture. The genes analyzed included pluripo- PLoS Genetics | www.plosgenetics.org 2 May 2012 | Volume 8 | Issue 5 | e1002691