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1.
Development ; 150(21)2023 11 01.
Article in English | MEDLINE | ID: mdl-37818613

ABSTRACT

The transcriptional co-regulator SIN3 influences gene expression through multiple interactions that include histone deacetylases. Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability and autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here, we show that loss of SIN-3, the single SIN3 homolog in Caenorhabditis elegans, results in maternal-effect sterility associated with de-regulation of the germline transcriptome, including de-silencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific histone deacetylases and show that they differentially contribute to fertility. Single-cell, single-molecule fluorescence in situ hybridization reveals that in sin-3 mutants the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells, suggesting a role for SIN-3 in its silencing. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN-3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Histone Deacetylases , Sin3 Histone Deacetylase and Corepressor Complex , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Germ Cells/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/metabolism , In Situ Hybridization, Fluorescence , X Chromosome/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex/genetics , Sin3 Histone Deacetylase and Corepressor Complex/metabolism
2.
Life Sci Alliance ; 5(3)2022 03.
Article in English | MEDLINE | ID: mdl-34893559

ABSTRACT

Changes in histone post-translational modifications are associated with aging through poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation at promoters is deposited by SET1 family methyltransferases acting within conserved multiprotein complexes known as COMPASS. Previous work yielded conflicting results about the requirement for H3K4 methylation during aging. Here, we reassessed the role of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespan and fertility by generating set-2(syb2085) mutant animals that express a catalytically inactive form of SET-2, the C. elegans SET1 homolog. We show that set-2(syb2085) animals retain the ability to form COMPASS, but have a marked global loss of H3K4 di- and trimethylation (H3K4me2/3). Reduced H3K4 methylation was accompanied by loss of fertility, as expected; however, in contrast to earlier studies, set-2(syb2085) mutants displayed a significantly shortened, not extended, lifespan and had normal intestinal fat stores. Other commonly used set-2 mutants were also short-lived, as was a cfp-1 mutant that lacks the SET1/COMPASS chromatin-targeting component. These results challenge previously held views and establish that WT H3K4me2/3 levels are essential for normal lifespan in C. elegans.


Subject(s)
Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Fertility/genetics , Histone-Lysine N-Methyltransferase/deficiency , Longevity/genetics , Nuclear Proteins/deficiency , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Catalysis , Enzyme Activation , Histones/metabolism , Methylation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism
3.
Nucleic Acids Res ; 47(21): 11164-11180, 2019 12 02.
Article in English | MEDLINE | ID: mdl-31602465

ABSTRACT

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Multiprotein Complexes/physiology , Sin3 Histone Deacetylase and Corepressor Complex/metabolism , Trans-Activators/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , DNA-Binding Proteins/genetics , Embryo, Nonmammalian , Histone-Lysine N-Methyltransferase/physiology , Multiprotein Complexes/metabolism , Protein Binding
4.
Genes Dev ; 29(24): 2547-62, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26637281

ABSTRACT

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.


Subject(s)
Breast Neoplasms/enzymology , Carcinogenesis/genetics , Gene Expression Regulation, Neoplastic/genetics , Polycomb Repressive Complex 2/metabolism , Animals , Animals, Genetically Modified , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Cell Line, Tumor , Disease Models, Animal , Enhancer of Zeste Homolog 2 Protein , Female , Histones/metabolism , Homeostasis/genetics , Humans , Male , Polycomb Repressive Complex 2/genetics , Prognosis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics
5.
Mol Cell ; 57(5): 769-783, 2015 Mar 05.
Article in English | MEDLINE | ID: mdl-25620564

ABSTRACT

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.


Subject(s)
Cell Differentiation , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Polycomb Repressive Complex 2/metabolism , Animals , Cell Line , Chromatin/genetics , Chromatin/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Enhancer of Zeste Homolog 2 Protein , Female , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Humans , Lysine/genetics , Lysine/metabolism , Methylation , Mice, Knockout , Models, Genetic , Mutation , Polycomb Repressive Complex 2/genetics , RNA Interference
6.
Cell ; 152(4): 859-72, 2013 Feb 14.
Article in English | MEDLINE | ID: mdl-23415232

ABSTRACT

Histone modifications are key regulators of chromatin function. However, little is known to what extent histone modifications can directly impact on chromatin. Here, we address how a modification within the globular domain of histones regulates chromatin function. We demonstrate that H3K122ac can be sufficient to stimulate transcription and that mutation of H3K122 impairs transcriptional activation, which we attribute to a direct effect of H3K122ac on histone-DNA binding. In line with this, we find that H3K122ac defines genome-wide genetic elements and chromatin features associated with active transcription. Furthermore, H3K122ac is catalyzed by the coactivators p300/CBP and can be induced by nuclear hormone receptor signaling. Collectively, this suggests that transcriptional regulators elicit their effects not only via signaling to histone tails but also via direct structural perturbation of nucleosomes by directing acetylation to their lateral surface.


Subject(s)
Gene Expression Regulation , Histone Code , Histones/metabolism , Transcriptional Activation , Acetylation , Animals , Cell Line, Tumor , Eukaryota/metabolism , Fibroblasts/metabolism , Humans , Mice , Models, Molecular , Nucleosomes/metabolism , Receptors, Estrogen/metabolism , Schizosaccharomyces/metabolism , Transcription Initiation Site , p300-CBP Transcription Factors/metabolism
7.
Nucleic Acids Res ; 40(2): 712-25, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21937513

ABSTRACT

Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.


Subject(s)
Mitochondria/enzymology , Reverse Transcription , Telomerase/metabolism , Cells, Cultured , DNA, Mitochondrial/isolation & purification , Humans , Membrane Potential, Mitochondrial , Mitochondria/ultrastructure , Protein Transport , RNA/isolation & purification , RNA/metabolism , RNA, Mitochondrial , RNA, Transfer/isolation & purification , Telomerase/isolation & purification
8.
EMBO J ; 30(14): 2817-28, 2011 Jun 21.
Article in English | MEDLINE | ID: mdl-21694722

ABSTRACT

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.


Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation , Histone-Lysine N-Methyltransferase/genetics , Promoter Regions, Genetic/genetics , RNA Polymerase II/genetics , Transcription, Genetic , Animals , Blotting, Western , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Immunoprecipitation , Lysine , Methylation , RNA Polymerase II/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
9.
Aging Cell ; 9(2): 203-19, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20089117

ABSTRACT

Telomerase is a reverse transcriptase specialized in telomere synthesis. The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria. Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation. Exactly why telomerase moves between subcellular compartments is still unclear. In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro. Overexpression of the mutant protein in primary fibroblasts is associated with telomere-based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53. Mitochondria function is also impaired in the cells. We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA. Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest. Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Mitochondria/enzymology , Mutation , Telomerase/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Biocatalysis , Cell Line , Cell Survival , Cellular Senescence , Diploidy , Humans , Microscopy, Electron , Mitochondria/ultrastructure , Molecular Sequence Data , Telomerase/genetics
10.
Proc Natl Acad Sci U S A ; 100(23): 13235-40, 2003 Nov 11.
Article in English | MEDLINE | ID: mdl-14581615

ABSTRACT

The mitochondrial ATP synthase is made of a membrane-integrated F0 component that forms a proton-permeable pore through the inner membrane and a globular peripheral F1 domain where ATP is synthesized. The catalytic mechanism is thought to involve the rotation of a 10-12 c subunit ring in the F0 together with the gamma subunit of F1. An important and not yet resolved question is to define precisely how the gamma subunit is connected with the c-ring. In this study, using a doxycycline-regulatable expression system, we provide direct evidence that the rest of the enzyme can assemble without the delta subunit of F1, and we show that delta-less mitochondria are uncoupled because of an F0-mediated proton leak. Based on these observations, and taking into account high-resolution structural models, we propose that subunit delta plays a key role in the mechanical coupling of the c-ring to subunit gamma.


Subject(s)
Mitochondrial Proton-Translocating ATPases/chemistry , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/metabolism , Catalysis , Cell Membrane/metabolism , Doxycycline/pharmacology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Hydrolysis , Microscopy, Fluorescence , Mitochondria/metabolism , Oxygen Consumption , Promoter Regions, Genetic , Protein Conformation , Protein Structure, Tertiary , Proton-Translocating ATPases/chemistry , Time Factors
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