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

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 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

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

Increased nucleosome repeat length at major satellite repeats in ESCs.

<p>(A) Nucleosome repeat length analyses of bulk chromatin (left), major satellite sequences (middle) and minor satellite sequences (right) in WT ESCs. DNA isolated from ESC nuclei digested with MNase at different time points were analyzed by ethidium bromide (EB) –stained gel (left), transferred to membrane which was sequentially probed with major satellites (middle) and minor satellites (right) using Southern blotting. The positions of di-nucleosomes with 10-minute MNase digestion are marked by *. The dashed line indicates di-nucleosome position of major satellites, which is higher than that of bulk chromatin and minor satellites. (B) The NRLs were calculated from the images presented in (A) by extrapolating the corresponding curves to time “0” as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003417#pgen.1003417-Gilbert2" target="_blank">[72]</a>.</p

Generation of H1d^FLAG knock-in ESCs.

<p>(A) Schematic representation of the H1d^FLAG targeting construct and the knock-in strategy for insertion of the FLAG tag at N-terminus of the endogenous H1d gene. (B) Identification of ESC clones containing the modified FLAG-H1d allele. DNA isolated from Blasticidin resistant ESC clones were analyzed by Southern blotting. <i>Cis vs. trans</i> configurations of the homologous recombination events are schematically illustrated in the diagram above the Southern blotting image. (C) Reverse phase HPLC profiles of histone extracts from ce^het (left panel) and <i>cis</i>-targeted H1d^FLAG ESCs (right panel). mU, milliunits of absorbency at 214 nm. (D) Coomassie staining and Western blotting analysis of individual H1 fractions eluted from HPLC of histone extracts of ce^het (1) and H1d^FLAG (2) ESCs. (E) Calculated ratio of each H1 variant (and total H1) to nucleosome of ce^het and H1d^FLAG ESCs.</p