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Project description:Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. We investigated the role of Taf4 in the murine intestinal epithelium using tissue-specific inactivation. Taf4 inactivation during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells, ultimately leading to death. In adults, Taf4 loss perturbed the stem cell compartment and the associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge and potentiating Apc-driven tumorigenesis. In addition, Taf4 inactivation ex vivo in organoids prevented budding formation and maintenance. Combining immunohistology with bulk RNA-seq, ATAC-seq and single-cell RNA-seq, showed that Taf4 loss caused broad chromatin remodeling, and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). In line with this observation, treatment of Taf4-mutant organoids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Our results reveal the critical role of Taf4 in intestinal development and homeostasis and a novel mechanism by which Taf4 acts to maintain the stem/progenitor compartment by counteracting PRC2 repression of the stem cell gene expression program.
2023-01-05 | GSE205329 | GEO
Project description:Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. We investigated the role of Taf4 in the murine intestinal epithelium using tissue-specific inactivation. Taf4 inactivation during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells, ultimately leading to death. In adults, Taf4 loss perturbed the stem cell compartment and the associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge and potentiating Apc-driven tumorigenesis. In addition, Taf4 inactivation ex vivo in organoids prevented budding formation and maintenance. Combining immunohistology with bulk RNA-seq, ATAC-seq and single-cell RNA-seq, showed that Taf4 loss caused broad chromatin remodeling, and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). In line with this observation, treatment of Taf4-mutant organoids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Our results reveal the critical role of Taf4 in intestinal development and homeostasis and a novel mechanism by which Taf4 acts to maintain the stem/progenitor compartment by counteracting PRC2 repression of the stem cell gene expression program.
2023-01-05 | GSE205326 | GEO
Project description:Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. We investigated the role of Taf4 in the murine intestinal epithelium using tissue-specific inactivation. Taf4 inactivation during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells, ultimately leading to death. In adults, Taf4 loss perturbed the stem cell compartment and the associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge and potentiating Apc-driven tumorigenesis. In addition, Taf4 inactivation ex vivo in organoids prevented budding formation and maintenance. Combining immunohistology with bulk RNA-seq, ATAC-seq and single-cell RNA-seq, showed that Taf4 loss caused broad chromatin remodeling, and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). In line with this observation, treatment of Taf4-mutant organoids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Our results reveal the critical role of Taf4 in intestinal development and homeostasis and a novel mechanism by which Taf4 acts to maintain the stem/progenitor compartment by counteracting PRC2 repression of the stem cell gene expression program.
2023-01-05 | GSE205441 | GEO
Project description:Wapl protein regulates binding of the cohesin complex to chromosomes during interphase and helps remove cohesin from chromosomes at mitosis. We isolated a dominant mutation in wapl (wapl(AG)) in a screen for mutations that counteract silencing mediated by an engrailed Polycomb-group response element. wapl(AG) hemizygotes die as pharate adults and have an extra sex combs phenotype characteristic of males with mutations in Polycomb-group (PcG) genes. The wapl gene encodes two proteins, a long form and a short form. wapl(AG) introduces a stop codon at amino acid 271 of the long form and produces a truncated protein. The expression of a transgene encoding the truncated Wapl-AG protein causes an extra-sex-comb phenotype similar to that seen in the wapl(AG) mutant. Mutations in the cohesin-associated genes Nipped-B and pds5 suppress and enhance wapl(AG) phenotypes, respectively. A Pds5-Wapl complex (releasin) removes cohesin from DNA, while Nipped-B loads cohesin. This suggests that Wapl-AG might exert its effects through changes in cohesin binding. Consistent with this model, Wapl-AG was found to increase the stability of cohesin binding to polytene chromosomes. Our data suggest that increasing cohesin stability interferes with PcG silencing at genes that are co-regulated by cohesin and PcG proteins.
| S-EPMC3478686 | biostudies-literature
Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.
| S-EPMC2730368 | biostudies-literature
Project description:Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation, we developed an in vivo targeting approach and found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover, targeting MSK1 to the α-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly, we discovered a mechanism whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes, but it also induces a methyl-acetylation switch of the adjacent K27 residue. Our findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism.
| S-EPMC3041124 | biostudies-other
Project description:Breast cancer progression is associated with aberrant DNA methylation and expression of genes that control the epithelial-mesenchymal transition (EMT), a critical step in malignant conversion. Although the genes affected have been studied, there is little understanding of how aberrant activation of the DNA methylation machinery itself occurs. Using a breast cancer cell-based model system, we found that cells that underwent EMT exhibited overactive transforming growth factor beta (TGFbeta) signaling and loss of expression of the CDH1, CGN, CLDN4, and KLK10 genes as a result of hypermethylation of their corresponding promoter regions. Based on these observations, we hypothesized that activated TGFbeta-Smad signaling provides an "epigenetic memory" to maintain silencing of critical genes. In support of this hypothesis, disrupting Smad signaling in mesenchymal breast cancer cells resulted in DNA demethylation and reexpression of the genes identified. This epigenetic reversal was accompanied by an acquisition of epithelial morphology and a suppression of invasive properties. Notably, disrupting TGFbeta signaling decreased the DNA binding activity of DNA methyltransferase DNMT1, suggesting that failure to maintain methylation of newly synthesized DNA was the likely cause of DNA demethylation. Together, our findings reveal a hyperactive TGFbeta-TGFbetaR-Smad2 signaling axis needed to maintain epigenetic silencing of critical EMT genes and breast cancer progression.
| S-EPMC2946209 | biostudies-literature
Project description:Heterochromatin formation is important for genome stability and transposon silencing. The evolutionally conserved Heterochromatin Protein 1 (HP1) recognizes H3K9me3 and maintains heterochromatin from fission yeast to mammalians. However, it is still questioned about the presence of HP1 protein in plants. Here, we discovered a multivalent H3K9me2 reader ADCP1 in Arabidopsis through biophysical and structural studies, and agenet domain in ADCP1 has the similar function of chromo domain in HP1. We further showed that ADCP1 was essential for heterochromatin maintenance through modulating H3K9me2 and DNA methylation levels. Similar to human HP1alpha and fly HP1a, ADCP1 was also able to mediate heterochromatin phase separation both in vitro and in vivo. Our results demonstrate the plant specific and evolution conserved ADCP1 functions as the HP1 equivalent protein in plant.
2018-11-15 | GSE118063 | GEO
Project description:Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ? involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ? is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells.
| S-EPMC4937302 | biostudies-literature
Project description:Vernalization, the acceleration of flowering by winter, involves cold-induced epigenetic silencing of Arabidopsis FLC. This process has been shown to require conserved Polycomb Repressive Complex 2 (PRC2) components including the Su(z)12 homologue, VRN2, and two plant homeodomain (PHD) finger proteins, VRN5 and VIN3. However, the sequence of events leading to FLC repression was unclear. Here we show that, contrary to expectations, VRN2 associates throughout the FLC locus independently of cold. The vernalization-induced silencing is triggered by the cold-dependent association of the PHD finger protein VRN5 to a specific domain in FLC intron 1, and this association is dependent on the cold-induced PHD protein VIN3. In plants returned to warm conditions, VRN5 distribution changes, and it associates more broadly over FLC, coincident with significant increases in H3K27me3. Biochemical purification of a VRN5 complex showed that during prolonged cold a PHD-PRC2 complex forms composed of core PRC2 components (VRN2, SWINGER [an E(Z) HMTase homologue], FIE [an ESC homologue], MSI1 [p55 homologue]), and three related PHD finger proteins, VRN5, VIN3, and VEL1. The PHD-PRC2 activity increases H3K27me3 throughout the locus to levels sufficient for stable silencing. Arabidopsis PHD-PRC2 thus seems to act similarly to Pcl-PRC2 of Drosophila and PHF1-PRC2 of mammals. These data show FLC silencing involves changed composition and dynamic redistribution of Polycomb complexes at different stages of the vernalization process, a mechanism with greater parallels to Polycomb silencing of certain mammalian loci than the classic Drosophila Polycomb targets.
| S-EPMC2579339 | biostudies-literature