PROJECT SUMMARY Deciphering mechanisms that control lineage-specific gene transcription during hematopoiesis is critical for identifying fundamental principles of cellular differentiation and new therapies for blood disorders. Enhancers are noncoding cis-acting DNA sequences that control cell fate by directing spatiotemporal gene expression. Despite the increasing availability of genome-wide profiles of putative enhancers, it remains difficult to elucidate the molecular processes controlling enhancer function during lineage differentiation, highlighting a major impediment for understanding the major determinants of normal and pathological hematopoiesis. The challenges we are now facing are to characterize the regulatory components of lineage-defining enhancers in physiologically relevant contexts and to interrogate their in vivo function during lineage specification. Addressing these challenges requires a shift from enhancer mapping studies to detailed characterization of their molecular composition in native chromatin during cellular differentiation. The objectives of this project are to determine the protein and RNA complexes controlling enhancer activation in situ, and to establish the in vivo function of lineage-specifying enhancers during erythroid cell differentiation and hematopoiesis. The central hypothesis is that enhancers are assembled by combinations of tissue-specific transcription factors, chromatin regulators, and RNA complexes to direct long-range chromatin interactions for lineage-specific gene transcription. This hypothesis has been formulated on the basis of the substantial progresses made during the prior funding period of this project, including the development of CRISPR/dCas9-based affinity capture of locus-specific chromatin interactions and enhancer-targeting epigenetic perturbation systems for in vivo interrogation of enhancer function. Using these approaches, we uncovered the organizational principles controlling erythroid lineage-specific super-enhancers and established new molecular links between hierarchical organization of 3D chromatin structures and in vivo enhancer function. Guided by these preliminary data, our hypothesis will be tested by three specific aims: 1) Determine enhancer-regulating chromatin complexes during cellular differentiation by dCas9-based proximity labeling. 2) Identify and characterize chromatin-associated RNAs that control enhancer structure and function. 3) Elucidate the in vivo function of “hub” enhancers in hematopoiesis by single-cell CRISPR epigenetic perturbation. Together these studies will not only elucidate mechanisms for the transcriptional control of principal determinants of cellular differentiation, but also establish new tools for the analysis of enhancer structure-function in situ and in vivo. Such results are expected to advance our mechanistic understanding of the causal relationships between enhancer composition and in vivo function during hematopoiesis. Ultimately, these fi...