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

项目总结/摘要 细胞分化的过程取决于良好协调和调控的基因表达，并且在细胞分化的过程中， 这个发育过程的核心是转录控制。转录调控的失误导致许多 人类疾病，包括癌症和发育障碍。我们得到的信息越多 在分化过程中的转录调控，它变得越清楚，这个过程是相当复杂的。为 例如，被称为增强子的专门调控区已被提议作为免疫调节的主要决定因素。 细胞类型特异性基因表达 .非编码RNA转录物，如eRNA，被推测参与 一些靶基因的激活。此外，本发明还 转录延伸和其它后起始事件， 如启动子近端暂停，正在成为转录控制的关键调控步骤， 基因表达模式然而，分化过程中基因表达模式的研究往往集中在 转录起始;和调节区的阶段特异性功能，如增强子，还没有被发现。 在分化成特定谱系的过程中被充分研究。因此，严重缺乏 关于调控区、非编码RNA和启动后的时间关系的知识 转录事件以及因此蛋白质编码基因在分化期间如何表达。 理解基因组在分化过程中如何调节自身的关键第一步是阐明 转录事件和染色质可及性之间的关系。我们建议使用新的高- 解析技术，天然延伸转录测序（NET-seq），-与DNase-seq -偶联， 同时绘制转录和开放染色质区域，并研究红细胞分化 从人成体造血干/祖细胞到定形成红细胞， 在分化过程中建立转录事件之间的时间关系。 NET-seq询问新生RNA的3'末端，提供活性基因转录的直接可视化 一次一个核苷酸此外，红细胞生成是研究时间关系的理想系统 在分化过程中转录事件之间的差异，因为许多参与红细胞生成的增强子显示， 可及性和转录靶基因之间的延迟。但是，还没有系统的分析 偶联转录延伸事件和红细胞生成过程中的染色质景观。通过这个 研究，我们希望看到在红细胞生成过程中转录延伸的新方面， 以前观察过。因此，扩展我们对发育过程中转录控制的理解， 发生在基因启动子上的事件，并阐明转录调控的不准确性如何导致血液中 癌症和其他相关疾病;以及让我们更接近了解如何 基因组在分化过程中自我调节。

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.