Loss of H1 Impairs Stem Cell Differentiation hydroxylase (TH)), astrocyte marker (GFAP) from WT and H1 TKO cultures at indicated days in differentiation cultures were determined by qRT-PCR. Data were normalized over the expression level of GAPDH and are presented as average 6 S.D. doi:10.1371/journal.pgen.1002691.g003 and that the H1 TKO EB has a total H1 level lower than the WT ESC. H1c/H1d/H1e is necessary for efficient transcriptional repression of pluripotency genes Oct4 and Nanog during embryogenesis and ESC differentiation The results from the aforementioned experiments suggest that H1c/H1d/H1e triple ESCs are less effective than WT ESCs in repressing the expression of pluripotency genes, such as Oct4 and Nanog, during spontaneous differentiation, rotary suspension differentiation, and neural differentiation in vitro (Figure 1A, Figure 2C, and Figure 3C). Therefore, we next investigated if H1 contributes to stable repression of pluripotency gene expression in vivo during embryogenesis. Oct4 is expressed in undifferentiated cells in the preimplantation embryo, and is progressively down- regulated in differentiating embryonal cells during gastrulation, becoming restricted to germ cell precursors after E8.5 [24], whereas Nanog expression is largely downregulated after E4.5 [25]. We analyzed expression of Oct4 and Nanog from E8.5 embryos, when many of the surviving TKO embryos appeared comparable to WT littermates. E8.5 embryos were harvested from intercrosses of H1c/H1d/H1e triple heterozygotes and the expression levels of Oct4 and Nanog in TKO and WT embryos were analyzed from three litters using quantitative RT-PCR. On average, expression levels of Oct4 and Nanog in TKO embryos were more than 4-fold of that from WT littermate controls (Figure 5Ai, Figure S4A), Figure 4. Expression profiles of linker histones in WT and H1 TKO cultures during EB differentiation. (A) Reverse-phase HPLC and Mass Spectrometry (inset) analysis of histones from WT and H1 TKO ESCs. X axis: elution time; Y axis: absorbency at A214. mAU, milli-absorbency units. Inset shows the relative signal intensity of H1d and H1e mass spectral peaks in the H1d/H1e fraction collected from HPLC eluates of WT histones. (B,C) H1/ nucleosome ratio of the total H1 (B) and individual H1 subtype (C) during EB formation and differentiation. Day 0, day 7 and day 10 of EB cultures were collected and HPLC analyses as shown in (A) were performed. The ratio of total H1 (or individual H1 subtype) to nucleosome was calculated as described in Materials and Methods. Values are means 6 S.D., n = 4. *: P,0.05; **: P,0.01; ***: P,0.001; ****: P,0.0001. doi:10.1371/journal.pgen.1002691.g004 PLoS Genetics | www.plosgenetics.org 7 May 2012 | Volume 8 | Issue 5 | e1002691