Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes

EMAPll/AIMP1 : isolation of putative receptor(s)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Cell-surface Associated p43/Endothelial-monocyte-activating-polypeptide-II in Hepatocellular Carcinoma Cells Induces Apoptosis in T-lymphocytes

The novel, proinflammatory cytokine endothelial-monocyte-activating-polypeptide-II (EMAP-II) was first found in tumour cell supernatants and is closely related or identical to the p43 component of the mammalian multisynthetase complex. In its secreted form, EMAP-II has multiple cytokinelike activities in vitro, including chemotactic, procoagulant and antiangiogenic properties. We recently showed that neoplastic but not normal hepatocytes expresses the 34-kDa molecule on the cell surface in vitro and the cell-surface expression is upregulated by treatment with tumour necrosis factor (TNF)-α/interferon (IFN)-γ and/or hypoxia. We hypothesized an immune-regulatory role of EMAP-II within neo-plastic tissues and investigated its effects on lymphocytes. Methods: To study the role of EMAP-II in tumour cell-induced lymphocyte killing, Jurkat T-cells were co-cultured with a range of hepatocellular carcinoma (HCC) cell monolayers (HuH-7, HepG2 and Alexander cells), which were either untreated or treated with TNF-α/IFN-γ under normoxic and hypoxic conditions over a period of 16-24 hours. Flow cytometric analysis of apoptosis in Jurkat cells was performed using the annexin-V-FITC/propidium iodide technique. Results: rEMAP-II caused a dose-dependent apoptosis in Jurkat T-cells. Co-culture of Jurkat cells with HCC cell monolayers induced significant apoptosis of the Jurkat cells. In general, under normoxic conditions, cytokine-treated HCC cell monolayer caused more apoptosis than untreated cells. This effect was enhanced by hypoxia. Critically, native EMAP-II expressed on the surface of the HCC cells also induced activation of caspase-8 and apoptosis in Jurkat cells, which was partially but significantly blocked by addition of polyclonal antibodies against EMAP-II to the incubation mixture. Conclusion: Our data suggest that membrane-bound EMAP-II is cytotoxic to lymphocytes and, therefore, might constitute a component of a novel, immunosuppressive pathway by which HCC cells may eliminate attacking T-cells and evade the immune system. The mechanism by which it does so is currently under investigation

Transcriptional regulation of the proto-oncogene Zfp521 by SPI1 (PU.1) and HOXC13

The mouse zinc‐finger gene Zfp521 (also known as ecotropic viral insertion site 3; Evi3; and ZNF521 in humans) has been identified as a B‐cell proto‐oncogene, causing leukemia in mice following retroviral insertions in its promoter region that drive Zfp521 over‐expression. Furthermore, ZNF521 is expressed in human hematopoietic cells, and translocations between ZNF521 and PAX5 are associated with pediatric acute lymphoblastic leukemia. However, the regulatory factors that control Zfp521 expression directly have not been characterized. Here we demonstrate that the transcription factors SPI1 (PU.1) and HOXC13 synergistically regulate Zfp521 expression, and identify the regions of the Zfp521 promoter required for this transcriptional activity. We also show that SPI1 and HOXC13 activate Zfp521 in a dose‐dependent manner. Our data support a role for this regulatory mechanism in vivo, as transgenic mice over‐expressing Hoxc13 in the fetal liver show a strong correlation between Hoxc13 expression levels and Zfp521 expression. Overall these experiments provide insights into the regulation of Zfp521 expression in a nononcogenic context. The identification of transcription factors capable of activating Zfp521 provides a foundation for further investigation of the regulatory mechanisms involved in ZFP521‐driven cell differentiation processes and diseases linked to Zfp521 mis‐expression

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

Loss of H1c/H1d/H1e inhibits spontaneous ESC differentiation.

<p>(A) Western blot analysis of OCT4 level in WT and H1 TKO ESCs cultured under indicated conditions for 2 days. (B) Phase images of WT and H1 TKO ESCs cultured either on MEF with LIF (left panel), gelatin coated plate with LIF (middle panel), or gelatin coated plate without LIF (right panel) for 2 days. Scale bar: 100 µm. (C) Growth curves of WT and H1 TKO ESCs cultured on gelatin coated plate with or without LIF. Data are presented as average ± S.D.</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

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