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1.
Nucleic Acids Res ; 48(9): 4601-4613, 2020 05 21.
Article in English | MEDLINE | ID: mdl-32266374

ABSTRACT

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.


Subject(s)
Histones/physiology , Muscle, Skeletal/metabolism , Transcription, Genetic , Transcriptional Activation , Animals , Cell Differentiation , Cells, Cultured , Chromatin , Chromatin Immunoprecipitation Sequencing , Histones/genetics , Histones/metabolism , Mice , Muscle Fibers, Skeletal , Muscle, Skeletal/cytology , RNA-Seq , Repetitive Sequences, Nucleic Acid , Transcription Initiation Site
2.
Sci Rep ; 9(1): 14212, 2019 10 02.
Article in English | MEDLINE | ID: mdl-31578361

ABSTRACT

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.


Subject(s)
Chromatin Assembly and Disassembly/physiology , Fungal Proteins/physiology , Multiprotein Complexes/physiology , Nucleosomes/physiology , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell-Free System , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/physiology , DNA Footprinting , DNA, Bacterial/metabolism , Deoxyribonucleases, Type II Site-Specific , Histones/genetics , Histones/metabolism , Microscopy, Atomic Force , Nucleosomes/ultrastructure , Plasmids/chemistry , RNA-Binding Proteins/physiology , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/ultrastructure , Xenopus laevis/genetics
3.
Biochem Cell Biol ; 95(4): 491-499, 2017 08.
Article in English | MEDLINE | ID: mdl-28177753

ABSTRACT

H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity.


Subject(s)
Fibroblasts/metabolism , Histones/metabolism , Mitosis/genetics , Animals , Cell Death , Cell Nucleus/metabolism , Cell Proliferation , Cells, Cultured , Chromosome Segregation , Genome , Mice , Mice, Inbred C57BL , Mice, Mutant Strains
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