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Genome-wide determinants of sequence-specific DNA binding

ABSTRACT: Genome-wide determinants of sequence-specific DNA binding

PROVIDER: PRJNA361490 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5179180_1.fastq.gz	Fastqsanger.gz
	SRR5179180_2.fastq.gz	Fastqsanger.gz
	SRR5179190_1.fastq.gz	Fastqsanger.gz
	SRR5179190_2.fastq.gz	Fastqsanger.gz
	SRR5179200_1.fastq.gz	Fastqsanger.gz

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Genome-wide determinants of sequence-specific DNA binding

Project description:DNA binding protein are generally thought to bind specific DNA sequences through selective interactions with DNA bases. However, it is now becoming more widely appreciated that DNA shape, which may not be specified by a unique base sequence, also contributes to site-specific binding. Here we elucidate how DNA sequence and shape confer site specificity on a genomic scale, and relate this to specificity imparted indirectly through occlusion of sequences by the in vivo environment. For simplicity, we focus on the set of General Regulatory Factors (GRFs) that do not rely on other factors for binding. They also serve a related function in organizing chromatin. Remarkably, we find that GRFs will not bind to their cognate motif if the DNA surrounding that sequence lacks a specific shape. While proper DNA sequence/shape properties tend to be restricted to promoter regions, weaker sites that are still binding-competent reside in gene bodies, but are prevented from binding by resident chromatin. Thus, site-specificity is achieved across a genome in vivo by the combined action of favorable DNA sequence and shape interactions, and occlusion by chromatin.

2018-01-16 | GSE93662 | GEO

Genome-wide determinants of sequence-specific DNA binding of general regulatory factors.

Project description:General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. It is assumed that sequence recognition via direct base readout suffices for specificity and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-base pair (bp) resolution using several ChIP-exo-based assays. We find that computationally predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the nonunique portions of GRF motifs and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels.

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Genome-wide analysis reveals determinants of redundant and specific binding within a transcription factor family

Project description:A central question in transcription factor biology is how a specific member of a transcription factor family occupies a promoter in vivo, when all family members bind the same consensus site in vitro. To uncover the mechanisms regulating DNA binding specificity within transcription factor families, we have used the techniques of chromatin immunoprecipitation coupled with genome-wide microarray analysis to query the occupancy of three members of the ETS transcription factor family in a human T-cell line. Unexpectedly, redundant occupancy was frequently detected while specific occupancy was less likely. An unbiased bioinformatics approach correlated redundant binding with consensus ETS binding sequences near transcription start sites, whereas specific binding sites diverged dramatically from the consensus, were coupled with a site for a cooperative binding partner, and were found further from transcription start sites. The specific and redundant DNA binding modes illustrate the regulation of transcription factor specificity in vivo and suggest two distinct roles for members of the ETS transcription factor family. Keywords: ChIP-chip Chromatin IP from uninduced asynchronous Jurkat T cells, or HT29 colon cells using antibodies to three ETS transcription factors (ETS1, ELF1, and GABPa) or the transcription factors RUNX1 or E2F4. IP and whole input DNAs are amplified (WGA2 kit sigma) and labeled and compared by promoter microarrays (Agilent). Each experiment requires 2 microarrays to cover the genome. Proximal promoter arrays assay 1kb surrounding transcription start sites, extended promoter arrays assay 7kb surrounding transcription start sites. Each microarray is performed as two biological replicates with the exception of ETS1 in HT29 proximal promoter array which were done once, and ETS1 in Jurkat proximal promoter array which was done three times.

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