Epigenetic alteration to activate Bmp2-Smad signaling in Raf-induced senescence

AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts(MEFs) by infecting retrovirus to express oncogenic Raf(RafV 600E). RNA was collected from RafV 600 E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV 600 E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by sh RNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss.CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.     

Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo

The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.     

Polycomb-Dependent H3K27me1 and H3K27me2 Regulate Active Transcription and Enhancer Fidelity

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Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes

MLL complexes are homologs of yeast COMPASS capable of methylating histone H3 Lys4 (H3K4). ASH2L, RbBP5 and WDR5 are conserved subunits of MLL complexes with homology to the Cps40/Cps60, Cps50 and Cps30 subunits of COMPASS, respectively. We report that ASH2L differentially regulates MLL's catalysis of H3K4 trimethylation similarly to Cps40 and Cps60. Furthermore, WDR5 is required to maintain MLL complex integrity, including the stability of ASH2L within the complex. These findings offer insight into the molecular role of ASH2L, and by extension that of WDR5, in proper H3K4 trimethylation.     

Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS

In yeast, the macromolecular complex Set1/COMPASS is capable of methylating H3K4, a posttranslational modification associated with actively transcribed genes. There is only one Set1 in yeast; yet in mammalian cells there are multiple H3K4 methylases, including Set1A/B, forming human COMPASS complexes, and MLL1-4, forming human COMPASS-like complexes. We have shown that Wdr82, which associates with chromatin in a histone H2B ubiquitination-dependent manner, is a specific component of Set1 complexes but not that of MLL1-4 complexes. RNA interference-mediated knockdown of Wdr82 results in a reduction in the H3K4 trimethylation levels, although these cells still possess active MLL complexes. Comprehensive in vitro enzymatic studies with Set1 and MLL complexes demonstrated that the Set1 complex is a more robust H3K4 trimethylase in vitro than the MLL complexes. Given our in vivo and in vitro observations, it appears that the human Set1 complex plays a more widespread role in H3K4 trimethylation than do the MLL complexes in mammalian cells.     

Arabidopsis PWWP domain proteins mediate H3K27 trimethylation on FLC and regulate flowering time

LHP_1 mediates recruitment of the PRC_2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H_3 K_(27)me_3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain proteins(PDPs) interact with FVE and MSI5 to suppress FLC expression and thereby promote flowering. We demonstrated that FVE, MSI_5, and PDP_3 were co-purified with LHP_1. The H_3K_(27)me_3 level on FLC was decreased in the pdp mutants as well as in the fve/msi_5 double mutant. This study suggests that PDPs function together with FVE and MSI_5 to regulate the function of the PRC_2 complex on FLC.     

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.     

Dynamics of histone H3 lysine 27 trimethylation in plant development

The development of multicellular organisms is governed partly by temporally and spatially controlled gene expression. DNA methylation, covalent modifications of histones, and the use of histone variants are the major epigenetic mechanisms governing gene expression in plant development. In this review, we zoom in onto histone H3 lysine 27 trimethylation (H3K27me3), a repressive mark that plays a crucial role in the dynamic regulation of gene expression in plant development, to discuss recent advances as well as outstanding questions in the deposition, recognition, and removal of the mark and the impacts of these molecular processes on plant development.     

Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis

Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low–nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.     

Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation

Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ～25% of the trophectoderm cells and in ～7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.