Genome-wide methods for mapping transcription factor binding sites in vivo, by ChIP-seq and ChIP-chip methods, have led to an explosion of information about the deployment of regulatory factors across the genome in living cells. The parts list for developmental systems biology now includes not only regulatory factors but also many candidate cis-regulatory target sites. Still, a major problem for understanding transcriptional regulation in development is how to match transcription factor site occupancy with sites of actual functional impact. Static transcription factor binding snapshots derived fro cell lines or terminally differentiated states may indicate sites where a factor is working in thos cells. However, in fine-scale examination of actual developmental systems, dynamic changes in factor occupancy occur in parallel with changes in RNA expression. The functional impact of factor binding at many sites is obscure, and many sites of factor occupancy may be superfluous for transcriptional control. This underlines the urgency of finding better methods to predict functionality of transcription factor engagement at specific sites. This project is based on a global strategy for elucidating relationships between genome-wide factor binding and transcriptional function, in a well-established developmental system in which multipotent blood-cell precursors become committed to develop as T cells. Our previous work has defined the transitions in this process at both cellular and molecular levels, including a recent global analysis of stepwise transcriptome changes, changes in transcription factor gene expression, local chromatin marking changes, and changes in magnitude and sites of binding occupancies across the genome by specific transcription factors. The multistage information about regulatory components of this process makes it possible to analyze transcription factor action dynamically, not only in terms of where binding is seen, but also in terms of the changes over time in binding occupancy at each site, which can then be compared to expression changes in the linked genes. To evaluate whether the transcription factor binding changes actually cause these gene expression changes, functional perturbations are required. Here, we propose to apply a systematic program of fast, stage-specific perturbations to determine the mechanisms of action of two essential transcription factors, PU.1 and Runx1. An important set of tools will be obligate repressor constructs used as competitors for endogenous factors to distinguish direct vs. indirect activation and repression. Quick gain and loss of function can thus be applied at particular stages and the effects analyzed by RNA-seq to identify affected genes system-wide, even if effects of a factor change magnitude or sign between developmental stages. The sites and site combinations thus suggested to have functional impact will be examined for distinctive co-clustered sites, histone modification rules, and transcription factor exchange rates. These features will then be tested for generality by functional assays of new, specifically predicted cis regulatory site modules within the gene network.

Public Health Relevance

Active regulation of gene expression by proteins called transcription factors is essential for development of all embryos as well as for development of cells, like blood cells, that are produced from stem cells throughout our adult lives. New methods have made it possible to catch 'snapshots' of transcription factors binding to DNA, but much remains to be understood about how they work and how they choose what genes they will actually control. This proposal is to develop and test new tools to understand where and how the factors are actually working in immature mouse blood cells. This information is crucial in order to be able to correct disease-related mistakes in gene regulation eventually without dangerous side effects.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
4R01HD076915-04
Application #
9052794
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Coulombe, James N
Project Start
2013-07-05
Project End
2018-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
4
Fiscal Year
2016
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Ungerbäck, Jonas; Hosokawa, Hiroyuki; Wang, Xun et al. (2018) Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1). Genome Res 28:1508-1519
Hosokawa, Hiroyuki; Ungerbäck, Jonas; Wang, Xun et al. (2018) Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding. Immunity 49:782
Hosokawa, Hiroyuki; Ungerbäck, Jonas; Wang, Xun et al. (2018) Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding. Immunity 48:1119-1134.e7
Hosokawa, Hiroyuki; Romero-Wolf, Maile; Yui, Mary A et al. (2018) Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16. Nat Immunol 19:1427-1440
Hosokawa, Hiroyuki; Rothenberg, Ellen V (2018) Cytokines, Transcription Factors, and the Initiation of T-Cell Development. Cold Spring Harb Perspect Biol 10:
Rothenberg, Ellen V; Kueh, Hao Yuan; Yui, Mary A et al. (2016) Hematopoiesis and T-cell specification as a model developmental system. Immunol Rev 271:72-97
Rothenberg, Ellen V (2016) Eric Davidson: Steps to a gene regulatory network for development. Dev Biol 412:S7-19
Coskun, Ahmet F; Cai, Long (2016) Dense transcript profiling in single cells by image correlation decoding. Nat Methods 13:657-60
Rothenberg, Ellen V; Ungerbäck, Jonas; Champhekar, Ameya (2016) Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control. Adv Immunol 129:109-74
Champhekar, Ameya; Damle, Sagar S; Freedman, George et al. (2015) Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1. Genes Dev 29:832-48

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