Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development

Suv4-20h Histone Methyltransferases Promote Neuroectodermal Differentiation by Silencing the Pluripotency-Associated Oct-25 Gene

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Brg1 chromatin remodeling ATPase balances germ layer patterning by amplifying the transcriptional burst at midblastula transition.

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning

<i>In vitro</i> induction of xSuv4-20h double-morphant animal cap explants.

<p>(A) Noggin-dependent neuralisation. XK81, Nrp1 and Xbra expression is monitored in uninjected control caps and double-morphant caps with or without Noggin mRNA. Note that explants coinjected with xSuv4-20h MOs together with Noggin mRNA show reduced Nrp1 expression, but normal downregulation of XK81 mRNA. (B) Muscle induction by Activin A in uninjected, ctrl-MO injected, and xSuv4-20h double morphant animal caps. Top row demonstrates comparable expression of myosin heavy chain (MHC-α) in non-dissected sibling embryos.</p

Functional analysis of xSuv4-20h HMTases.

<p>(A) Transiently transfected eGFP-tagged Suv4-20h1 and h2 enzymes from frog or mouse re-establish H4K20me3 marks in heterochromatic foci of Suv4-20h1/h2 DKO MEFs. (B–D) Bulk histones from tadpoles (NF30-33) injected with morpholinos targeting translation of endogenous xSuv4-20h1 and h2 mRNA show a strong reduction in H4K20me2 and H4K20me3 levels and a concomitant increase in the H4K20me1 mark. (B) Western Blot analysis of uninjected embryos, control morphants (ctrl-MO), and double morphants with antibodies against H4K20 mono-, di- and trimethylation. PanH3 antibody was used as loading control. (C) Western Blot quantification of three independent biological experiments; data represent mean values, error bars indicate SEM. (D) Immuno-histochemistry on xSuv4-20h double morphant tadpoles. Panels show details from neural tubes stained with antibodies against the histone epitopes indicated on the side. Whole sections shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003188#pgen.1003188.s005" target="_blank">Figure S5A</a>.</p

Suv4-20h double-null ES cells have elevated Oct4 and lower CXCR4 protein levels before and during differentiation.

<p>Wild-type and Suv4-20h DKO ES cells were grown undifferentiated in LIF-containing medium, or differentiated <i>in vitro</i> by embryoid body formation. Wt26 - isogenic wild-type ES cell line; B4-2, B7-1 – independent Suv4-20h DKO ES cell lines. (A) Morphological appearance (scale bar 100 µm). Top row: undifferentiated ES cells (day 0); middle: embryoid bodies at day 6; bottom: cells from embryoid bodies, replated for 24h. (B) Before (day 0) and during (day 6) differentiation, cell lines were stained for Oct4 protein and 2×10⁴ cells from each condition were subjected to FACS analysis. Red graph: fluorescence of non-specific isotype control; black and green graphs represent the Oct4 protein levels in wild-type and Suv4-20h DKO ES cell lines, respectively. (C) Suv4-20h DKO cells have higher Oct4 protein levels compared to wild-type ES cells and maintain these during differentiation. Median fluorescence intensity was calculated from data in panel (B), error bars indicate SEM. (D) Suv4-20h DKO cells show a reduction in the percentage of CXCR4 + cells at differentiation day 6. Data represent normalized values of percentage of CXCR4+ cells as means of three independent experiments, error bars indicate SEM.</p

xSuv4-20h double morphants fail to silence Oct-25 expression in deep-layer ectoderm due to reduced H4K20me3 enrichment at Oct-25 promoter.

<p>RNA <i>in situ</i> hybridization analysis for Oct-25, Oct-60 and Oct-91 in embryos (A) and animal caps (B) for ctrl-morphants or xSuv4-20h double-morphant embryos, injected unilaterally at two-cell stage and fixed at midneurula stage (NF15). Injected sides were defined by coinjected Alexa-fluorescence prior to <i>in situ</i> hybridisation. (A) Dorsal views of stained embryos with anterior to the left. For Oct-25 Vibratome cross-sections, ctrl-MO and double-morphant embryos are shown. (B) Comparative expression analysis for Oct-25, Oct-60 and Oct-91 in animal caps from bilaterally injected embryos, fixed at midneurula stage (NF15). (C) qRT-PCR profiles for Oct-25 and Oct-91 in ctrl-MO and xSuv4-20h double-morphant embryos. Data represent normalized ratios of mRNA levels as means of four independent experiments, error bars indicate SEM. (D, E) Chromatin immunoprecipitation (ChIP) analysis on Oct-25, GAPDH, thra, thibz genes and major satellite repeat region 3 (MSAT3). (D) H4K20me3 levels on the indicated genes in normal embryos, and (E) in uninjected <i>versus</i> double-morphant embryos. Fold enrichment was calculated as the ratio between H4K20me3 precipitated material over negative control (No antibody sample). Data represent mean values of three to five independent experiments, error bars indicate SEM.</p

H3K9me3-heterochromatin loss at protein-coding genes enables developmental lineage specification

Gene silencing by chromatin compaction is integral to establishing and maintaining cell fates. Trimethylated histone 3 lysine 9 (H3K9me3)-marked heterochromatin is reduced in embryonic stem cells compared to differentiated cells. However, the establishment and dynamics of closed regions of chromatin at protein-coding genes, in embryologic development, remain elusive. We developed an antibody-independent method to isolate and map compacted heterochromatin from low-cell number samples. We discovered high levels of compacted heterochromatin, H3K9me3-decorated, at protein-coding genes in early, uncommitted cells at the germ-layer stage, undergoing profound rearrangements and reduction upon differentiation, concomitant with cell type-specific gene expression. Perturbation of the three H3K9me3-related methyltransferases revealed a pivotal role for H3K9me3 heterochromatin during lineage commitment at the onset of organogenesis and for lineage fidelity maintenance