Discovering the regulatory roles of transcription during erythropoiesis, one nucleotide at a time

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PROJECT SUMMARY / ABSTRACT The process of cellular differentiation hinges on well-coordinated and regulated gene expression, and at the heart of this developmental process is transcriptional control. Missteps in transcription regulation result in many human diseases, including cancers and developmental disorders. The more information we gain about transcription regulation during differentiation, the clearer it becomes that this process is quite complex. For example, specialized regulatory regions, called enhancers, have been proposed as the primary determinant of cell type–specific gene expression . Non-coding RNA transcripts, such as eRNA, are speculated to be involved in the activation of some target genes. Additionally, transcription elongation and other post-initiation events, such as promoter-proximal pausing, are emerging as crucial regulatory steps in the transcriptional control of gene expression patterns. Yet, studies of gene expression patterns during differentiation have often focused on transcription initiation; and the stage-specific function of regulatory regions, such as enhancers, has not been well investigated over the course of differentiation into a specific lineage. Therefore, there is a significant lack of knowledge about the temporal relationship between regulatory regions, non-coding RNAs, and post-initiation transcriptional events and consequently how protein-encoding genes are expressed during differentiation. A critical first step to understanding how the genome regulates itself during differentiation is to clarify the relationship between transcriptional events and chromatin accessibility. We propose using a new high- resolution technology, native elongating transcript sequencing (NET-seq), — coupled with DNase-seq — to simultaneously map transcription and regions of open chromatin, and to study erythrocyte differentiation proceeding from human adult hematopoietic stem/progenitor cells to definitive erythroblasts in order to establish a temporal relationship between transcriptional events during differentiation. NET-seq interrogates the 3' ends of nascent RNA, providing a direct visualization of active gene transcription one nucleotide at a time. Additionally, erythropoiesis is an ideal system for studying temporal relationships between transcriptional events during differentiation as many enhancers involved in erythropoiesis show a delay between accessibility and transcribing target genes. However, there has not been systematic analysis coupling transcription elongation events and the chromatin landscape during erythropoiesis. Through this research, we expect to see novel aspects of transcription elongation during erythropoiesis that have not been previously observed. Thus, expanding our understanding of transcriptional control during development beyond events occurring at gene promoters and clarifying how inaccuracies in transcription regulation result in blood cancers and other associated diseases; as well as putting us one step closer to understanding how the genome regulates itself during differentiation.