Wednesday, April 2, 2025
4/2/2025
Homeobox protein Hhex in early T cell development - Project Summary The proline-rich homeobox protein Hhex is important in hematopoiesis, in embryonic development, and in cancer. A critical regulator of early embryonic hematopoiesis and other embryonic tissues, it is also known to be required for hematopoietic stem cells to maintain the ability to give rise to T and B cell progeny. It is intimately involved in several aspects of B cell development, and it is crucial for establishment of memory B cells. However, how it enables stem cells to produce T cells has remained unclear and paradoxical. Normally, Hhex is silenced during T cell lineage commitment and remains off in most mature T cells, sharply distinguishing T cells from nearly all other hematopoietic cell types. Yet not only is Hhex needed to support T lineage potential, but also forced expression of Hhex in T lineage cells can promote increased thymocyte self- renewal. Is its role positive or negative, or both sequentially? We propose that in fact Hhex roles can be reconciled as results of the need of early T-cell precursors to pass through a “phase 1” progenitor-like regulatory state en route to a “phase 2” T-lineage-definitive regulatory state, even though these states are mutually antagonistic. Our recent results show that Hhex at natural endogenous levels is a potent regulator of T lineage differentiation speed, and suggest that it may initially delay the onset of commitment by repressing a key T-lineage commitment regulator, Bcl11b. The role of Hhex in Bcl11b regulation could provide much-needed insight into a control mechanism for large-scale epigenetic transformation in development. Unexpectedly, our preliminary evidence also suggests that Hhex positively regulates other factors expressed in the precommitment stage. This proposal thus grows from new data from our group and exploits newly enhanced experimental systems that enable us to interrogate the functions of transcription factors like Hhex, targeting specific stages from prethymic hematopoietic progenitors through T- lineage commitment. This will empower us to reveal the specific target genes and mechanisms that Hhex uses to control them when it is operating properly in T-cell precursors. This will be undertaken through the following aims: 1. Determine the target genes in early T lineage cells affected by acute loss of Hhex: separate analyses for cells pre and post contact with T-inductive Notch signaling microenvironment 2. Quantify the impact of Hhex on cell population survival and proliferation before and after contact with Notch signaling and lineage commitment 3. Define Hhex functional targeting sites based on binding of epitope-tagged Hhex, and on ATAC accessibility, H3K27 trimethylation, and TLE corepressor recruitment changes in early T lineage cells under acute Hhex perturbation. Identify Hhex impact on chromatin of the Bcl11b gene
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