Primers for qChIP analysis.

<p>Primers for qChIP analysis.</p

qChIP analysis of histone marks at <i>Hox</i> genes in WT and H1 TKO ESCs.

<p>The levels of H3K4me3 (A), H3K36me3 (B), H3K27me3 (C), and H3K9me3 (D) were analyzed by qChIP. Promoter regions of the indicated Hox genes were assayed, except for (B), for which gene body regions were analyzed. Dashed lines denote the highest signal level of control IgG qChIP. *: P<0.05, **: P<0.01, ***: P<0.001.</p

Reduction of <i>Hox</i> gene expression in H1 TKO embryos.

<p>(A) Relative expression of <i>Hox</i> genes with altered mRNA levels in H1 TKO embryos compared with WT. Down-regulated <i>Hox</i> genes are located in <i>HoxA</i> (i), <i>HoxC</i> (ii), and <i>HoxD</i> (iii) clusters. Expression levels of <i>Hox</i> genes were analyzed by qRT-PCR and normalized over <i>GAPDH</i> and represented as a fold change between H1 TKO and WT embryos at E8.5. *: P<0.05, **: P<0.01, ***: P<0.001. Error bars: S.D. (B) The schematic representation of <i>Hox</i> gene clusters with expression patterns in H1 TKO embryos compared with WT.</p

The expression profiles of <i>Hox</i> genes in single-H1 KO ESCs.

<p>Relative expression of <i>Hoxa1, Hoxb8, and Hoxc13</i> in H1c^−/− (A), H1d^−/− (B), and H1e^−/− (C) ESCs were shown. *: P<0.05, **: P<0.01, ***: P<0.001.</p

Primers for qRT-PCR analysis.

<p>Primers for qRT-PCR analysis.</p

Generation and reverse-phase HPLC analysis of single-H1 KO ESCs.

<p>(A) RP-HPLC analysis of total histones from WT and the single-H1 KO ESCs. The identity of the histone subtypes is indicated above each peak. mAU, milli-absorbency at 214 nm. Genotype analyses of single-H1 KO ESCs are shown in insets in respective HPLC profiles. (B) The ratios of individual H1 (left) and total H1 (right) to nucleosome for WT and single-H1 KO ESCs. Ratios were determined from the RP-HPLC and mass spectrometry analyses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038829#s4" target="_blank">methods</a>. ***: P<0.001 (C) The percentage of each H1 subtype among total H1 histones for WT and single-H1 KO ESCs. % total H1 for H1⁰ (marked with arrowhead) is equal to or less than 1%.</p

H1 TKO ESCs fail to undergo neural differentiation.

<p>(A) Neural differentiation scheme for ESCs. (B) Characterization of WT and H1 TKO cultures on day 6+7 under neural differentiation protocol. i). Phase contrast images shows that H1 TKO mutants were unable to adequately form neurites and neural networks. Right panels: zoom-in images of the areas encircled with black rectangles. Scale bar: 100 µm (left panels) and 50 µm (right panels). ii). Left panel: Percentage of neurite-forming EBs. Numbers were averaged from 6 experiments. 80 EBs were counted per experiment. Right panel: Numbers of neurites per neurite-forming EB. Number of neurites was counted from EBs that produced neurites. 58 and 28 neurite-forming EBs from respective WT and TKO were selected and counted for neurite numbers. **: P<0.01; ****: P<0.0001. iii). Immunostaining for expression of TUBB3 and GFAP. Nuclei were stained with Hoechst 33342. Scale bars: 50 µm (left panels) and 20 µm (right panels). Results are representative of three independent experiments. (C) H1 TKO ESCs were unable to adequately repress the pluripotency genes and to efficiently induce the expression of neural genes. Expression levels of pluripotency genes (<i>Oct4</i> and <i>Nanog</i>), neural marker (<i>Nestin</i>), neuronal marker (<i>Tyrosine hydroxylase (TH)</i>), astrocyte marker (<i>GFAP</i>) 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 <i>GAPDH</i> and are presented as average ± S.D.</p

H1 is necessary for stable repression of <i>Oct4</i> pluripotency gene during embryogenesis and ESC differentiation.

<p>(A) Elevated <i>Oct4</i> expression and hypomethylation of CpG sites at <i>Oct4</i> promoters in H1 TKO embryos compared with littermates at E8.5. (i) qRT-PCR analysis of mRNA expression levels of <i>Oct4</i>. Values are means ± SEM, n = 5 for each genotype. Expression levels were normalized over <i>GAPDH</i>. *: P<0.05. (ii) Bisulfite sequencing analysis of DNA methylation status at <i>Oct4</i> 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 <i>Oct4</i> 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 <i>Oct4</i> 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 <i>Oct4</i> in WT, H1 TKO and RES cells during EB differentiation. Relative expression levels were normalized over <i>GAPDH</i>. 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 ± S.D. *: P<0.05; **: P<0.01; ***: P<0.001. (C) Model for H1 in repression of <i>Oct4</i> 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 <i>Oct4</i> gene and contributes to establishment and/or maintenance of epigenetic changes necessary for stable silencing of <i>Oct4</i> pluripotency gene.</p

Expression profiles of linker histones in WT and H1 TKO cultures during EB differentiation.

<p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002691#s4" target="_blank">Materials and Methods</a>. Values are means ± S.D., n = 4. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.</p

H1c/H1d/H1e triple knockout ESCs are impaired in EB differentiation.

<p>(A) Hematoxylin and eosin (H&E) staining of sections of WT EBs (top panels) and H1 TKO EBs (bottom panels) at 7 days, 10 days and 14 days in rotary suspension culture. High magnification images of H&E staining of sections of WT EB (top right) and H1 TKO EBs (bottom right) show that TKO EBs failed to cavitate. WT EBs showed more differentiated morphologies with cysts forming (black arrows). (B) Quantitative RT-PCR analysis of mRNA expression levels of <i>AFP</i> in ESCs (day 0) and EBs throughout 14 days of rotary suspension culture. Data were normalized over the expression level of <i>GAPDH</i> and are presented as average ± S.D. (C) Hierarchical clustering analysis of qRT-PCR SuperArray gene expression profiling of ESCs (day 0) and EBs (day 10) formed from WT and H1 TKO ESCs. Red, green or black represent higher, lower, or no change in relative expression. (D) Scatter Plot analysis of gene expression comparisons of: (i) WT <i>vs.</i> H1 TKO ESCs (day 0); (ii) WT EBs (day 10) <i>vs.</i> WT ESCs (day 0); (iii) H1 TKO EBs (day 10) <i>vs.</i> H1 TKO ESCs (day 0). X- and y- axes are delta CTs using <i>GAPDH</i> to normalize. Genes with more than 2-fold differences lie outside of the blue lines.</p