Loss of H1 Impairs Stem Cell Differentiation morphologies with cysts forming (black arrows). (B) Quantitative RT-PCR analysis of mRNA expression levels of AFP in ESCs (day 0) and EBs throughout 14 days of rotary suspension culture. Data were normalized over the expression level of GAPDH and are presented as average 6 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 vs. H1 TKO ESCs (day 0); (ii) WT EBs (day 10) vs. WT ESCs (day 0); (iii) H1 TKO EBs (day 10) vs. H1 TKO ESCs (day 0). X- and y- axes are delta CTs using GAPDH to normalize. Genes with more than 2-fold differences lie outside of the blue lines. doi:10.1371/journal.pgen.1002691.g002 2Dii, and Figure S2Bii). In contrast, H1 TKO EBs exhibited very similar gene expression signatures to those of ESCs and had less expression changes during differentiation compared with that of WT EBs. (Figure 2C, 2Diii, and Figure S2Biii, S2C), suggesting that the lack of H1c, H1d and H1e leads to diminished changes of transcriptional reprogramming during differentiation. The levels of ectoderm markers, such as Nestin (Nes), mesoderm markers, such as Brachyury (T) and FLT1, and endoderm markers, such as AFP and Gata4, were all markedly less or failed to be expressed in H1 TKO EBs (Figure 2C and Figure S2C), indicating that differen- tiation to all three germ layers was suppressed. H1 is required for neural differentiation of embryonic stem cells To further investigate if and when H1 impacts cell differenti- ation in a specific lineage, we induced differentiation of H1 TKO ESCs under a neural differentiation regimen established using all- trans retinoic acid (RA), which is known to induce neural differentiation in ESCs [19,20]. EBs were prepared using the hanging-drop method, and day 4 EBs were collected and treated with RA for additional two days followed by further differentiation with neural differentiation media on poly-L-ornithine and laminin (PLO+L) coated tissue culture plates (Figure 3A). By day 6+7 of this in vitro neural differentiation scheme, neural cells were clearly established and neurite outgrowth from EBs was seen with neuronal cell proliferation. Neurites are enriched in cylindrical bundles of microtubules, made primarily of b-III tubulin (TUBB3) protein, extending from the body of all neurons, finally differen- tiating into an axon or a dendrite [21]. However, at this time point, neural differentiation of WT and TKO ES cells exhibited several striking differences. While neurite-formation was efficient in WT culture with bundles of neurites cylindrically extending from EB to adjacent EB, H1 TKO EBs had much less neurite outgrowth (Figure 3Bi, 3Bii). Approximately 50% of WT EBs plated for neural differentiation formed neurites compared to only about 10% of H1 TKO EBs forming neurites (Figure 3Bii, left panel). Furthermore, those 10% TKO EBs that were capable of forming neurites only produced on average 8 neurites per EB, whereas each WT EB had on average 18 neurites (Figure 3Bii, right panel). During in vitro neural differentiation, neurons aggregated into mounds of cells forming neuronal clusters (Figure 3Bi; black arrows), connected by bundles of neurites (Figure 3Bi; white arrows), forming a network pattern. While WT cultures showed formation of a neural network with neural clusters inter- connected by bundles of neurites, H1 TKO cultures failed to develop such an extensive intercellular network (Figure 3Bi, ii), evidenced by smaller neuronal clusters with negligible inter- connecting neurites. This was further confirmed with immuno- fluorescence detection of TUBB3 protein expression, and minimal TUBB3 staining was seen in H1 TKO cultures (Figure 3Biii). It appeared that both neurite formation and outgrowth were limited in H1 knock-out mutants, affecting the ability of neurons to form neural networks. We also noted that TKO cultures yielded markedly less glial cells as revealed by much fewer GFAP positive astrocytes in comparison with WT cultures (Figure 3Biii). Since glial cells are essential for the normal growth and development of neurons, the near-lack of glial cells in TKO cultures may contribute to the poor development of TUBB3 positive neuronal cells from TKO EBs. To examine whether the aforementioned defects of the H1 TKO cultures represent a temporary delay or a blockage in neural differentiation, we cultured the cells for an additional 14 days under neural differentiation conditions. As expected, the neural marker (Nestin) and the astrocyte marker (GFAP) were efficiently and progressively induced in WT cell cultures, and the neuronal gene Tyrosine hydroxylase (TH) peaked at day 6+7 when neuronal proliferation occurred (Figure 3C). In contrast, the expression levels of neural genes were significantly curtailed in H1 TKO cultures, suggesting the lack of progression in neural differentiation of H1 TKO culture (Figure 3C). Furthermore, we observed that pluripotency genes Oct4 and Nanog were expressed at higher levels in TKO than WT throughout the differentiation process (Figure 3C). These data suggest that H1 TKO cells are largely blocked in neural differentiation. Levels of H1 increase progressively during differentiation To address the mechanisms by which H1 modulates differen- tiation, we first examined the expression profile of linker histone H1 subtypes during EB formation and differentiation of wild-type ESCs. Histones from wild-type, H1 TKO ESCs and EBs were isolated at various time points during differentiation, and the levels of individual H1 subtype proteins as well as the H1 to nucleosome ratio were quantified from HPLC and mass spectrometry analysis as described previously [15,22,23]. In ESCs (day 0), H10 was nearly undetectable in WT cells but was increased in H1 TKO cultures as we observed previously (Figure 4A and [10]). Upon EB differentiation, the levels of H1c, H1d and H1e and H10 in WT cultures were all progressively increased over time, with the total H1 to nucleosome ratio elevated nearly 40% from 0.45 for ESCs to 0.62 for day 10 EBs (Figure 4B, 4C). Consistent with HPLC analysis, Western blotting showed that levels of total H1 and H10 were increased (Figure S3). The cumulative increase in the protein levels of H1c, H1d and H1e was responsible for 87% of the increase in the total H1 levels during differention (data not shown). Despite less abundant than H1d, H1c and H1e were significantly increased (P,0.001), and H1e levels in differentiated EBs were over 2-fold of that in undifferentiated ESCs (Figure 4C). The protein levels of H1a and H1b remained constant during differentiation, indicating that H1a and H1b were not responsible for the increase of total H1 during ESC differentiation. Albeit higher than that in TKO ESCs (0.25), the ratio of total H1 to nucleosome in day 10 TKO EBs (0.36) remained lower than the ratio in WT ESCs (0.45) (Figure 4B). The increase in the total H1 level in TKO EBs compared with ESCs was largely due to the increase in the level of H10 (Figure 4B, 4C, and Figure S3), indicating H10 being the major H1 subtype upregulated in the face of deficiency of H1c, H1d, and H1e, in both ESCs and EBs. These results show that the levels of H1c, H1d, H1e and H10 are elevated significantly during embryonic stem cell differentiation, PLoS Genetics | www.plosgenetics.org 5 May 2012 | Volume 8 | Issue 5 | e1002691