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1.
Nat Struct Mol Biol ; 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38956169

ABSTRACT

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

2.
Hum Mol Genet ; 27(14): 2409-2424, 2018 07 15.
Article in English | MEDLINE | ID: mdl-29659838

ABSTRACT

Alterations of DNA methylation landscapes and machinery are a hallmark of many human diseases. A prominent case is the ICF syndrome, a rare autosomal recessive immunological/neurological disorder diagnosed by the loss of DNA methylation at (peri)centromeric repeats and its associated chromosomal instability. It is caused by mutations in the de novo DNA methyltransferase DNMT3B in about half of the patients (ICF1). In the remainder, the striking identification of mutations in factors devoid of DNA methyltransferase activity, ZBTB24 (ICF2), CDCA7 (ICF3) or HELLS (ICF4), raised key questions about common or distinguishing DNA methylation alterations downstream of these mutations and hence, about the functional link between the four factors. Here, we established the first comparative methylation profiling in ICF patients with all four genotypes and we provide evidence that, despite unifying hypomethylation of pericentromeric repeats and a few common loci, methylation profiling clearly distinguished ICF1 from ICF2, 3 and 4 patients. Using available genomic and epigenomic annotations to characterize regions prone to loss of DNA methylation downstream of ICF mutations, we found that ZBTB24, CDCA7 and HELLS mutations affect CpG-poor regions with heterochromatin features. Among these, we identified clusters of coding and non-coding genes mostly expressed in a monoallelic manner and implicated in neuronal development, consistent with the clinical spectrum of these patients' subgroups. Hence, beyond providing blood-based biomarkers of dysfunction of ICF factors, our comparative study unveiled new players to consider at certain heterochromatin regions of the human genome.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Helicases/genetics , Immunologic Deficiency Syndromes/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Chromosomal Instability/genetics , DNA Methylation/genetics , Female , Genome, Human/genetics , Genotype , Heterochromatin/genetics , Humans , Immunologic Deficiency Syndromes/physiopathology , Male , Mutation , Neurogenesis/genetics , DNA Methyltransferase 3B
3.
Nature ; 530(7588): 113-6, 2016 Feb 04.
Article in English | MEDLINE | ID: mdl-26814966

ABSTRACT

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.


Subject(s)
Chromatin Assembly and Disassembly , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genome/genetics , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Animals , DNA Helicases/metabolism , Histones/metabolism , Mice , Micrococcal Nuclease/metabolism , Mouse Embryonic Stem Cells/cytology , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , RNA Polymerase II/metabolism , Substrate Specificity , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription Initiation Site
4.
PLoS Genet ; 9(6): e1003536, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23754960

ABSTRACT

Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (ADAMTS17, CERS3) and abolishes a sequence for a long non-coding RNA (FLJ42289). Whereas mutations in ADAMTS17 have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for CERS3 (ceramide synthase 3) and FLJ42289 so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.


Subject(s)
ADAM Proteins/genetics , Genome-Wide Association Study , Ichthyosiform Erythroderma, Congenital/genetics , Sphingosine N-Acyltransferase/genetics , ADAMTS Proteins , Animals , Genes, Recessive , Homozygote , Humans , Mutation , Organ Specificity , Polymorphism, Single Nucleotide , RNA Splice Sites/genetics , RNA, Long Noncoding/genetics , Sphingolipids/metabolism , Sphingosine N-Acyltransferase/metabolism , Tunisia
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