Regulation of hematopoiesis by epigenetic timing control

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Project Summary: The timing of stem cell fate decisions is critical for multicellular tissue size and function. In many developmental processes, stem cells differentiate only after a long time-delay spanning multiple days and cell divisions, allowing rare populations of multipotent progenitors to expand exponentially. Variation in differentiation timing generates dramatic changes in tissue size and morphology and likely contributes to human development and disease. However, the mechanism underlying timing control during differentiation remains unclear. This proposal will use hematopoiesis a model system to understand how epigenetic regulation contributes to the timing of cell fate decisions. Recent evidence suggests that epigenetic mechanisms acting at individual genomic loci in cis can change slowly over the course of multiple days and cell divisions, implementing rate-limiting steps to developmental gene activation. However, it remains unclear if this is a paradigm for timing control broadly utilized during cell differentiation. This proposal aims to determine the prevalence of epigenetic timing mechanisms (Aim 1) and uncover how they can be regulated by non-coding DNA elements (Aim 2). Epigenetic regulation, unlike transcription factor regulation in trans, functions at each gene copy independently in the same cell (e.g. X-chromosome inactivation). Therefore, to investigate epigenetic timing control prevalence and mechanisms, this proposal will use mouse models in which the activity of individual gene copies can be monitored separately. In Aim 1, a F1 hybrid mouse strain that harbors frequent single nucleotide polymorphisms will be used to analyze individual alleles genome-wide by CUT&Tag. This single-cell genomics approach can distinguish epigenetic states by quantifying of protein-DNA interactions and will reveal the generality of epigenetic timing mechanisms during hematopoiesis. In Aim 2, a two-copy gene reporter system will be used to provide a highly sensitive readout for epigenetic regulation in hematopoietic progenitors undergoing differentiation. CRISPR/Cas9 approaches will be used to efficiently identify functional non-coding DNA elements and elucidate how they contribute to epigenetic timing control. This fellowship will provide an opportunity for training in computational genomics and CRISPR/Cas9 genome and epigenome editing. Experts in these fields will serve as mentors for skills training and career development. This training plan incorporates tailored courses, seminars and workshops that will enhance the training environment and facilitate the transition to a postdoctoral research fellowship. The expected outcome of this proposal will establish a new model for timing control during tissue development and homeostasis. It will also provide new insights into blood disease pathogenesis and inspire new approaches for epigenetic reprogramming in cell-based therapies.