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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [Cut&Run]

ABSTRACT: Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [Cut&Run]

PROVIDER: PRJNA856256 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20000600_1.fastq.gz	Fastqsanger.gz
	SRR20000600_2.fastq.gz	Fastqsanger.gz
	SRR20000601_1.fastq.gz	Fastqsanger.gz
	SRR20000601_2.fastq.gz	Fastqsanger.gz
	SRR20000602_1.fastq.gz	Fastqsanger.gz

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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [Cut&Run]

Project description:Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro – the hypoxia response element (HRE) – but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions (IDR). Using orthogonal genomic approaches such as Cut-and-Run and RNA-seq, we also confirmed IDR-dependent binding and activation for a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.

2022-07-08 | GSE207573 | GEO

Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

Project description:Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

Project description:Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

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Bulk Cut&run and Cut&tag

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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [RNA-seq]

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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [RNA-seq]

Project description:Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors [RNA-seq]

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Activation of g-globin expression by hypoxia-inducible factor 1a [CUT&RUN]

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CUT&RUN HUDEP1 OE SOX6-Flag

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Project description:Here we describe successful implementation of CUT&RUN for profiling protein-DNA interactions in zebrafish embryos. We apply modified a CUT&RUN method to generate high resolution maps of enrichment for H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II during zebrafish gastrulation. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, and we demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences with reduced mappability. Our work demonstrates the power of combining CUT&RUN with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.

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