We propose to use an innovative multiplex fluorescent in situ method for determining transcript numbers from multiple genes in the same cells, seqFISH, in a model system to address the general problem of how transcription factor effects are exerted on different classes of target genes in a dynamic developmental system. The proposal is a Revision for the existing, funded project, """"""""Genomic site binding rules and regulatory factor function in developing T cells"""""""" (R01HD076915), and it adds a crucial single-cell analysis component to clarify the interpretation and extend the insights from the project. In parallel, the biological system offers many advantages for enhancing the power and demonstrating the utility of the seqFISH technique. We exploit a well-characterized framework of developmental events through which multipotent hematopoietic progenitors undergo commitment to become T-cell precursors, a system that can be studied in parallel in vivo and in vitro and which is highly defined at the cellular level and in terms of patterns of gene expression The project focuses on the crucial but enigmatic role of the transcription factor PU.1, which is needed to support the early stages in T-cell development but does so apparently at the price of maintaining a regulatory bridge to an alternative set of developmental fates, i.e. macrophage, granulocyte, and dendritic cell fates. PU.1 has lineage-specific differences in its patterns of genomic occupancy in different hematopoietic precursors, but many of its binding sites do not appear to be linked with function. The parent proposal combines acute perturbation assays, genome-wide RNA-seq, and diagnostic ChIP-seq approaches to determine the rules that relate PU.1 occupancy to PU.1 regulatory functions in this early T-cell context. However, it is important to determine how homogeneous each baseline state is, and how uniform PU.1 actions are on all the cells at a given early T-cell stage, to resolve which PU.1 target genes are actually responding to PU.1 in the same regulatory-state context and which are responding in a different one. To answer this question and reveal in detail how different target genes """"""""process"""""""" changes in activity of the same regulator, we propose a new collaboration between the Ellen Rothenberg and Long Cai groups.
Specific aim 1 : Use seqFISH to characterize the variation in PU.1 expression in T-cell precursors and variation in levels of known and suspected PU.1 target genes, relative to expression of genes that can modulate PU.1 effects.
Specific aim 2 : Optimize seqFISH technology for detection of transcripts from >20 different genes per cell in individual cells.
Specific aim 3 : Analyze the effects of PU.1 deletion and PU.1 antagonism on expression of different classes of candidate target genes in single cells, and determine their correlation wit levels of different modulating factors in those cells.

Public Health Relevance

This project is a basic science collaboration to reveal the way powerful gene regulation proteins work and how their activities can be channeled to different target genes, through developing and extending new methods for visualizing exactly how strongly different genes are being expressed at a given time in the same cells. This project uses the new visualization approach to understand how a particular gene regulation protein can be assigned to control different genes, as it works to guide stem cells to branch out to make different kinds of immune cells. Both the visualization method and the general principles of gene control are relevant to a wide range of mechanisms in health and disease, from immune deficiency to cancer and in many stem-cell based forms of development.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Special Emphasis Panel (ZRG1-CB-R (50))
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Coulombe, James N
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California Institute of Technology
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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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