Project description: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 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 amphibian and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.

Project description: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 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 amphibian and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state. Total RNA samples from Xenopus laevis embryos. Transcript levels were analyzed after injection of control or Suv420 morpholinos into blastomeres.

Project description:Interaction of the Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta protein with the cellular Notch signaling effector, Recombination Signaling Protein (RBP)-Jk (aka CSL and CBF-1), is essential for viral reactivation from latency. We previously showed that Rta binds to a DNA motif repeated in the viral Mta promoter (called “CANT” or Rta-c) to stimulate RBP-Jk DNA binding, and distinguished Rta from the activated Notch-1 protein. To determine whether Rta’s mechanism would apply generally to other viral promoters, we employed chromatin immunoprecipitation/deep sequencing (ChIP/Seq) to identify Rta and RBP-Jk binding sites across the KSHV genome. We show that RBP-Jk binds nearly exclusively to unique genome sites during latency and reactivation. Many, but not all, reactivation-specific RBP-Jk peaks were associated with Rta bound to single Rta-c motifs. Other motifs that were over-represented with stimulated RBP-Jk DNA binding including those for the cellular DNA binding proteins BCL11A, MNT, MAF B, and TCF12. Four of the top seven motifs that were most over-represented with inhibition of RBP-Jk DNA binding were putative binding sites for the Pit/Oct/Unc (POU) family of proteins, including POU3F3 (Oct-8) and POU5F1 (Oct-4). Interestingly, two other POU motifs, including one for the POU2F1 (Oct-1) protein, are associated with inhibition of RBP-Jk DNA binding unless Rta bound to a nearby Rta-c motif. The relative distances between the Rta-c, POU, and RBP-Jk motifs were conserved at reactivation-specific RBP-Jk peaks in three promoters that Rta transactivated, and the Rta-c and Oct-1 motifs overlapped in two of those. The proximity of the Rta-c/Oct-1 motif to an RBP-Jk motif is critical for Rta transactivation of the ORF50AS/KbZIP promoter, and knockdown of Oct-1 protein debilitated reactivation and production of infectious virus. Our data suggest a broad role for POU proteins in regulating DNA binding of RBP-Jk and its associated transactivators.

Dataset Information

Comparison of the expression profiles of wild type (WT) and Oct-6-deficient (Oct-6-/-) macrophages in response to poly(I:C) treatment

Publications

Octamer-binding factor 6 (Oct-6/Pou3f1) is induced by interferon and contributes to dsRNA-mediated transcriptional responses.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets