Abstract
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.
Highlights
Embryonic development is controlled by fine-tuned differential gene expression
Compared to uninjected controls or embryos injected with an unrelated control Morpholino oligonucleotides (MO), the double morphants contained significantly less H4K20me2 (p = 0.0011) and H4K20me3 (p = 0.0164), which was coupled to an increase in H4K20me1 (p = 0.0034) (Figure 1B and 1C)
We have investigated the developmental functions of the histone- methyltransferases Suv4-20h1 and h2 during frog embryogenesis, which are responsible for the establishment of the H4K20 di- and trimethylated states
Summary
Embryonic development is controlled by fine-tuned differential gene expression. A succession of regulatory protein networks unfolds the zygotic gene expression program along a hierarchy of decisions, leading from the embryonic ground state to the epiblast and to germ layers, which become patterned into cell type and organ precursor territories. Recent studies have revealed that alterations in chromatin structure, dynamics and composition represent fundamental processes, which define the epigenetic landscape that directs cell type specification along this hierarchy [2,3]. The transition from pluripotent to differentiated cells is characterized by a progressive increase in heterochromatin formation, in a process that changes the hyperdynamic open chromatin structure into a less accessible architecture [1,8]. In vivo studies have shown that dynamic alterations in the levels of histone modifications characterize early stages of development both in mammals [9,10,11] and other vertebrates [12,13,14]
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