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Inferring gene regulatory circuitry from functional genomics data

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

SEN, SHURJO KUMAR

项目受资助机构

COLUMBIA UNIV NEW YORK MORNINGSIDE

立项年度

2020

立项时间

未公开

项目编号

5R01HG003008-15

项目级别

国家级

研究期限

未知 / 未知

受资助金额

560259.00美元

学科

Biotechnology;Genetics;Human Genome

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

BUSSEMAKER, HARMEN J

参与机构

NATIONAL HUMAN GENOME RESEARCH INSTITUTE

项目标书摘要：PROJECT SUMMARY It has long been known that methylation of genomic DNA correlates with gene expression. However, the structural mechanisms that underlie these observations remain obscure. In this project, we will pursue several innovative strategies for studying how methylation affects transcription factor (TF) binding. First, we will use Methyl-SELEX-seq ? a novel experimental method developed in the previous cycle of this grant that uses barcoded mixtures of methylated and unmethylated DNA ligands ? to create detailed maps of the effect of methylation on binding affinity for a broad panel of human transcription factors from various structural families. Second, we will perform detailed computational analyses and follow-up experiments to test the hypothesis that methylation causes local changes in DNA shape, which in turn modify TF binding affinity. We have shown that adding a methyl group in the major groove changes the geometry of the minor groove and enhances the electrostatic interaction between negative charges in the DNA minor groove and positively charged amino- acids in the TF. We will extend these analyses to other DNA modifications, as well as a wider range of DNA shape parameters and associated flexibility parameters. By building interpretable TF-DNA recognition models that integrate base, shape, and flexibility features using a powerful new machine learning framework developed in the previous funding cycle, we will make specific predictions regarding sequence and methylation readout mechanisms, and validate these using SELEX experiments with mutated TFs. To assess to what extent our quantitative models for binding to naked DNA built from SELEX data are predictive of binding to genomic DNA in the context of the living cell, we will perform detailed parallel analyses of SELEX and ChIP- seq data for Hox proteins and other TFs. Finally, to study the relationship between DNA binding and gene expression control in human cell lines, we will exploit Survey of Regulatory Elements (SuRE-seq), a novel massively parallel reporter assay that provides unique information about the autonomous transcriptional activity for each of >108 overlapping genomic fragments.

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  • 1.Low-Affinity Binding Sites and the Transcription Factor Specificity Paradox in Eukaryotes

    • 关键词：
    • PROTEIN-DNA INTERACTIONS; EVOKES LATENT DIFFERENCES; LIQUID PHASE-SEPARATION; CHIP-SEQ DATA; IN-VIVO; GENE-EXPRESSION; HOX SPECIFICITY; CRO REPRESSOR; SEQUENCE; RECOGNITION
    • Kribelbauer, Judith F.;Rastogi, Chaitanya;Bussemaker, Harmen J.;Mann, Richard S.
    • 《ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 35》
    • 2019年
    • 会议

    Eukaryotic transcription factors (TFs) from the same structural family tend to bind similar DNA sequences, despite the ability of these TFs to execute distinct functions in vivo. The cell partly resolves this specificity paradox through combinatorial strategies and the use of low-affinity binding sites, which are better able to distinguish between similar TFs. However, because these sites have low affinity, it is challenging to understand how TFs recognize them in vivo. Here, we summarize recent findings and technological advancements that allow for the quantification and mechanistic interpretation of TF recognition across a wide range of affinities. We propose a model that integrates insights from the fields of genetics and cell biology to provide further conceptual understanding of TF binding specificity. We argue that in eukaryotes, target specificity is driven by an inhomogeneous 3D nuclear distribution of TFs and by variation in DNA binding affinity such that locally elevated TF concentration allows low-affinity binding sites to be functional.

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