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Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes

ABSTRACT: Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes

PROVIDER: PRJNA245820 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1265780.fastq.gz	Fastqsanger.gz
	SRR1265781.fastq.gz	Fastqsanger.gz
	SRR1265772.fastq.gz	Fastqsanger.gz
	SRR1265782.fastq.gz	Fastqsanger.gz
	SRR1265773.fastq.gz	Fastqsanger.gz

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Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes.

Project description:Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.

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Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes

Project description:This SuperSeries is composed of the SubSeries listed below.

2014-04-30 | GSE57168 | GEO

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Project description:Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.

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Epigenome editing strategies for the functional annotation of topological insulators

Project description:The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs), within which the majority of gene-enhancer interactions occur. Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries3,4. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, mitotically-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing with dCas9-KRAB and dCas9-DNMT3A3L. Finally, we apply these strategies to activate PDGFRA expression in glioblastoma stem cells, thus simulating an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

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Project description:By CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation in silico and genetic deletions in vivo revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.

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Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [VII]

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