PROJECT SUMMARY Elucidating mechanisms that regulate gene expression in red blood cell production is fundamental to understanding cellular differentiation, and to developing new therapies for red cell disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression. Recently, we and others have identified erythroid-specific enhancer elements through genome-scale profiling of chromatin features. Further analysis has uncovered GATA1-interacting coactivators and their combinations as candidate drivers of enhancer function. These studies established a comprehensive catalog of erythroid enhancer elements, yet the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. Given that enhancers are frequently targeted by disease-associated genetic variations, it is imperative to fill this gap in knowledge. The objective of this project is to determine the protein complexes and long-range DNA interactions responsible for enhancer assembly and function in situ during erythropoiesis. The central hypothesis is that erythroid enhancers are assembled by specific combinations of lineage-regulating transcription factors such as GATA1 to recruit transcriptional coactivators and to initiate long-range chromatin interactions for gene transcription. This hypothesis has been formulated on the basis of our preliminary studies of an erythroid-specific super-enhancer comprised of three individual enhancers that regulates the SLC25A37 gene, a mitochondrial iron transporter essential for iron metabolism and heme synthesis, and an innovative approach to identify macromolecules associated with a single genomic locus using the endonuclease-deficient Cas9 (dCas9) and single guide RNA (sgRNA) components of the CRISPR system. Guided by these preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify and characterize SLC25A37 enhancer-associated proteins in situ by dCas9 affinity purification, followed by quantitative proteomic analysis and validation studies. 2) Determine SLC25A37 enhancer long-range DNA interactions using dCas9-mediated chromatin interaction analysis by paired-end sequencing (dCas9-ChIA-PET). By comparing the frequency and location of chromatin interactions between individual SLC25A37 enhancers and their temporal changes during erythropoiesis, these analyses will establish functional links between chromatin looping and enhancer function. 3) Define the functional requirement of 41 erythroid disease-associated, evolutionarily conserved super-enhancers using an engineered dCas9-LSD1 repressor complex in a dCas9 knockin mouse model. Together these studies will not only elucidate the mechanisms for the genetic control of a principal regulator of erythroid iron metabolism, but also provide new tools for in situ analysis of enhancer- regulating components. Such results are expected to advance our understanding of the composition and function of enhancers in coordinating erythroid gene expression. Ultimately, such knowledge has the potential to inform the design of therapeutic strategies to target non-coding regulatory elements in red cell disorders.