Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY Both currently expressed transcription factors and inherited epigenetic states contribute to cell identity. However, the causal relationships between transcription factor action and changes in strongly acting chromatin states remain a central question in genomics of development. Despite much correlative data, it is poorly understood how to predict at what sites a chromatin state will prohibit transcription factor action, and under what conditions a transcription factor’s action will alter chromatin states, either transiently or long-term. This proposal takes advantage of Runx family transcription factors as illuminating tools to probe the rules underlying this relationship. The early stages of T cell development can be a particularly useful framework to do this. Runx family factors, especially Runx1 and Runx3, are vital for T cell development from the initiation of the T-lineage gene expression program. The proposed work is based on our recent evidence that despite nearly constant sustained levels of Runx activity across several stages of early T-cell development, the deployment of Runx factors to specific targets changes sharply at the specific transition of T cell development when the cells undergo lineage commitment. Either chromatin changes or changing partner factors could be responsible. Our latest results also indicate that Runx protein expression levels play a strong role in the timing of entry into the T-cell program, as higher levels promote access to key genomic sites prematurely, raising the hypothesis that Runx binding biophysics play a role in developmental timing. At some Runx-activated loci the appearance of new Runx binding sites is linked with major transformations in chromatin 3D interaction patterns of chromatin. The early T-cell development system is highly accessible through in vitro differentiation systems, well characterized as matched to in vivo development, and there is a rich body of data about the programmed expression and deployment of other transcription factors that may be potential Runx interaction partners, making this an ideal system to dissect cause-effect relationships between Runx factors and the epigenome. We propose to discover the rules through which Runx factors choose stage-specific target sites, by comparing Runx actions in cells as they go through successive transitions of early T cell development. In the first aim, we will develop a fine-scale, genome-wide temporal atlas for the binding of Runx factors relative to epigenetic states, both under normal conditions and under conditions when Runx levels are experimentally raised or lowered. This will characterize the epigenetic conditions that are most restrictive for Runx action and the conditions under which Runx can alter them. In the second aim, we will define the reciprocal impacts of Runx and transcription factors representing its candidate stage-specific interaction partners, examining how each affects the binding of the other and how different partners may compete for limited Runx. The third aim will focus on experimentally dissecting cis-elements needed for Runx control of Bcl11b and Ets1, where developmental regulation reflects large-scale chromatin as well as local factor binding changes.
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