restores the effective repression of expression and dynamic changes in histone modifications and DNA methylation levels during differentiation; 3) the level of H1 is cumulatively increased at the Oct4 promoter during differentiation of WT, but not of H1 TKO, cultures. We suggest that the H1 occupancy at Oct4 promoter in ESCs could be the basal/minimal level for detection by qChIP assay, as H1 has been found to be relatively depleted from active promoters compared with other regions [44,45]. Interestingly, qChIP analysis showed that the association of H10 at Oct4 promoters was significantly higher in RES cells than TKO cells (Figure S7), suggesting that the presence of sufficient H1 proteins may facilitate H10 binding. We surmise that the progressive increase of H1c, H1d and H1e during differentiation and the increased H1 occupancy at Oct4 promoter lead to a transition to a more condensed local chromatin structure necessary for stable silencing of Oct4 during differentiation (Figure 5C). These results together with the observation that OCT4 is present at the promoters of several H1 subtypes in human ESCs [29,35] suggest a potential feedback loop between OCT4 and H1 in stem cell fate determination. Interestingly, we found that CpG methylation of Oct4 promoter in H1 TKO embryos is significantly reduced compared with wild- type littermates. Although less pronounced in EB differentiation, the effects of H1 depletion on DNA methylation at Oct4 promoter are also apparent in day 10 EBs. This observation reinforces the link between H1 and DNA methylation, which was initially discovered at imprinting control regions (ICRs) of H19 and Gtl2 loci [10] and later at regulatory regions of the immunoglobin heavy chain locus and homeobox Rhox gene cluster [46,47]. Future studies on how DNA methylation changes at these regions in H1 TKO ESCs during differentiation will provide additional insights on dynamic profiles of DNA methylation upon differentiation in the face of minimal level of H1 and/or open chromatin structure. H1 TKO EBs do not exhibit the opposite changes in the levels of the active histone mark (H3K4me3) and the repressive histone mark (H3K9me3) at promoters of Oct4 and Nanog that normally occur in wild-type EBs during differentiation. Interestingly, we did observe significant changes in the levels of histone modifications in wild-type EBs at day 7 in rotary culture, before an increase in DNA methylation levels occurred at Oct4 promoter. This result reinforces the notion that DNA methylation is a slower mark to establish compared with histone marks [48]. It is noteworthy that the levels of DNA methylation at the Nanog promoter do not display a difference in WT and H1 TKO embryos at day 8.5 and are not altered during EB differentiation, suggesting that DNA methylation is unlikely to be responsible for gene expression changes of Nanog during this period of time. Our results suggest a role of H1 and chromatin compaction in epigenetic regulation of the pluripotency gene Oct4, likely mediated through DNA methylation and histone modifications. To our knowledge, this represents a novel mechanistic link by which bulk chromatin compaction is directly linked to pluripo- tency, by participating in repression of the pluripotency genes. In ESCs, DNMT3b has been shown to interact with H1 [49]. In vitro studies demonstrated that H1 interacts with HP1 [50,51] which can in turn bind to SUV39H which methylates H3K9. Moreover, H1 has been shown in vitro to stimulate the activity of PRC2 toward methylation of H3K27me3 when H1 is incorporated into nucleosomes [52], and we have also observed interactions between H1 and PRC2 components in ESCs (Cao, Ho, Lasater, and Fan, unpublished observation). Therefore, we envision that during ESC differentiation, H1 levels increase, which may facilitate the recruitment of DNMTs, SUV39H and PRC2 to Oct4 promoter, promoting the establishment and/or maintenance of repressive Loss of H1 Impairs Stem Cell Differentiation epigenetic modifications and silencing the expression of this pluripotency gene (Figure 5C). In summary, we have demonstrated that loss of linker histone subtypes H1c, H1d, and H1e impairs embryonic stem cell differentiation. Furthermore, our results indicate that H1 contrib- utes to silencing of pluripotency factors and participates in mediating changes in DNA methylation and histone marks necessary for silencing of pluripotency genes during differentiation. Thus, modulating the levels of H1 linker histones and chromatin compaction may potentially serve as a new strategy for regulating stem cell pluripotency. Materials and Methods Embryonic stem cell culture ESC lines derived from H1 TKO and wild-type littermates were expanded on mitotically inactivated mouse embryonic fibroblasts feeder layers and cultured feeder-free on tissue culture-treated dishes (Corning) pre-adsorbed with gelatin (Sigma, 0.1% solution in ddH2O) prior to embryoid body differentiation studies. ESC culture media consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin (Mediatech), 2 mM L-glutamine (Mediatech), 16 MEM non-essential amino acids (Mediatech), 0.1 mM b-mercaptoethanol (Fisher Chemical), and 103 U/ml of leukemia inhibitory factor (LIF; ESGRO, Chemicon). Cultures were re-fed with fresh media every other day, and passaged every 2–3 days prior to reaching 70–80% confluence. For spontaneous differentiation studies, 26105 cells were seeded in each well of 6- well plate at day 0 on gelatin coated plate without feeder layer, cultured with media without LIF, and harvested at indicated time points. Cell numbers were determined using a Multisizer 3 Coulter Counter (Beckman). Karyotype analysis Exponentially growing ESCs were cultured in the presence of Karyo-MAX colcemid (Gibco) for 60 minutes, washed with PBS, trypsinized, and collected. ESCs were subsequently treated with hypotonic solution (75 mM KCl) for 6 minutes at 37uC, fixed with fixation solution (3 volumes Methanol, 1 volume Acetic acid), concentrated and dropped onto an angled, humidified microscope slide. The slide was dried and chromosomes were stained with Hoechst dye for 1 h in the dark. Images of metaphase spread were collected at a 606 objective on an Olympus Fluorescence Microscope. Rotary suspension culture and embryoid body differentiation Embryoid bodies were formed by inoculating a single-suspen- sion of ESCs that have been passaged without feeder layers for two generations (referred to as ‘‘day 0’’ culture) at 26105 cells/ml into 100 mm bacteriological grade polystyrene Petri dishes with 10 ml of differentiation media (DMEM, 15% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, 2 mM L- glutamine, 16 MEM non-essential amino acids, 0.1 mM b- mercaptoethanol). The EB cultures were immediately placed on rotary orbital shakers (Lab-Line Lab Rotator, Barnstead Interna- tional) in a humidified incubator (37uC, 5% CO2) and maintained at 40–45 rpm for the entire duration of suspension culture; rotary speed was calibrated daily to ensure accuracy throughout. Rotary orbital culture has been shown previously to significantly enhance the efficiency, yield and homogeneity of EB populations compared to static suspension culture methods [18]. Differentiation media PLoS Genetics | www.plosgenetics.org 11 May 2012 | Volume 8 | Issue 5 | e1002691