In Situ Capture of the Molecular Composition of Erythroid Transcriptional Enhancers

Elucidating mechanisms that regulate gene transcription during erythropoiesis is critical for identifying fundamental principles of cellular differentiation, and for developing new therapies for blood disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression, yet the molecular processes controlling enhancer activation and deactivation during lineage differentiation remain largely unknown. Recently, we and others have identified human erythroid cell-specific enhancers by mapping histone modifications, transcription factor binding and transcriptomic changes in primary fetal and adult hematopoietic stem/progenitor cells and lineage-committed erythroid progenitors. While these studies establish a comprehensive catalog of erythroid transcriptional enhancers, the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. This is a major impediment for understanding the coordinated control of gene transcription in normal and diseased erythroid cells.Given that enhancers are frequently targeted by disease-associated genetic variations, there is a critical need to correct this gap in knowledge. Identifying the complete composition of a specific enhancer within its native chromatin can provide unprecedented insight into the molecular mechanisms regulating its activity. To facilitate the molecular characterization of enhancers within their native chromatin environment, we have developed a new method, CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements), to isolate enhancer-associated molecular interactions by repurposing the CRISPR/Cas9 system. By using the endonuclease-deficient Cas9 (dCas9) and enhancer-targeting guide RNA (sgRNA), the enhancer-associated protein, DNA and RNA complexes are isolated by affinity purification and identified by high-throughput sequencing and proteomics, respectively. Using these methods, we isolated chromatin-regulating protein complexes associated with several critical cis -regulatory elements within the human β-globin cluster such as the locus control region (LCR) in human erythroid cells, and identified many known factors such as GATA1, TAL1, NFE2, LDB1 and the SWI/SNF chromatin remodeling complexes involved in globin gene regulation. More importantly, we also identified several new factors, such as the nuclear pore proteins NUP98 and NUP153, which have not previously been implicated in globin gene regulation. Subsequent studies of these factors established their roles in the developmental regulation of enhancer activities for globin gene transcription. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes also establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of cis -element-mediated long-range chromatin interactions at disease-associated non-coding elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Therefore, the dCas9-mediated affinity purification has the potential to advance the characterization of chromatin-templated hierarchical events by providing a method to examine the complete molecular composition of transcriptional enhancers and how composition changes in cellular differentiation. Our studies not only help elucidate the underlying principles regulating lineage-defining enhancer elements, but also establish new approaches for in situ molecular dissection of disease-associated cis -regulatory elements in globin gene transcription and erythropoiesis.