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Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

新生转录本测序揭示的转录控制机制

基本信息

批准号：
10584193
负责人：
Lee Stirling Churchman
金额：
$ 59.22万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584193
关键词：
Adenine Binding Binding Proteins Biochemical Cell Line Cell Nucleus Cells Chromatin Chromatin Fiber Complex Couples Cytoplasm DNA Data Data Set Detection Diagnosis Disease Down-Regulation Elements Enhancers Event Fiber Gene Expression Gene Expression Regulation Genes Genetic Transcription Genomics Heat-Shock Response Human Genome Introns Kinetics Knowledge Label Location Malignant Neoplasms Maps Measures Messenger RNA Metabolic Methods Methylation Methyltransferase Mission Modeling Molecular Monitor Nuclear Nuclear Export Nucleoplasm Nucleosomes Nucleotides Play Polymerase Positioning Attribute Process Production Promoter Regions Public Health RNA RNA Editing RNA Polymerase II RNA Processing RNA Splicing RNA chemical synthesis RNA purification RNA-Binding Proteins Regulator Genes Research Residencies Resolution Role Shapes Site Techniques Technology Testing Third Generation Sequencing Time Transcript Transcription Elongation Transcription Initiation Transcriptional Regulation U1 Small Nuclear Ribonucleoprotein United States National Institutes of Health Work base base editor biological adaptation to stress blind design epigenomics functional genomics genome-wide genomic data histone modification human disease mRNA Precursor nano new technology novel strategies nucleobase premature promoter recruit technology development termination factor tool transcription factor transcriptome transcriptome sequencing

项目摘要

Transcription initiation is highly regulated by genomic elements in promoters and enhancers that bind transcription factors and recruit RNA polymerase II (Pol II). After Pol II escapes from the promoter, many regulatory steps occur during transcription elongation that control Pol II progression, RNA processing, and finally release of the RNA from chromatin for nuclear export. These steps are tightly controlled by genomic and epigenomic features, such as histone modifications, nucleosome positions, and cis-elements that bind transcription factors and RNA-binding proteins (RBPs). Current functional genomic technologies tend to provide a static, averaged view of genomic function that obscure the transient roles that many genomic elements play in shaping the transcriptome. To identify and characterize all functional genomic elements, we need to develop approaches that quantitatively monitor the many layers of gene expression regulation, including transient interactions and short-lived events. In the proposed research, we will create a suite of genome-wide tools to map Pol II across single chromatin fibers, track the association of RBPs, and follow RNAs from transcription initiation to nuclear export. These techniques will highlight unappreciated connections between genomic elements, molecular events on chromatin, and post-initiation regulatory events. Our rationale is that by creating tools to quantify short-lived steps in mRNA production, unappreciated functional roles for genomic elements can be revealed. To demonstrate how these technologies reveal unappreciated gene regulatory mechanisms, we will apply them to the heat shock response (HSR), a stress response that has served as a model to study the many regulatory layers in gene expression. In Aim 1 we will develop a strategy that reveals Pol II locations across single long chromatin fibers. Fiber-seq is a method that identifies regions of accessible DNA across long (up to ~20 kb) chromatin fibers one molecule at a time. We will extend this approach to identify Pol II footprints, analyze the distributions of Pol II complexes across gene bodies, and characterize the interactions between Pol II complexes and nucleosomes. In Aim 2 we will measure the timing of binding of proteins across long pre-mRNA molecules. RBP binding will be marked by RNA editing and long- read direct RNA sequencing of nascent RNA (nano-COP) will enable us to resolve transient binding events that impact pre-mRNA processing and nuclear export. These events are transient, as many RBPs bind to introns and are released from RNA in the cytoplasm immediately after nuclear export. In Aim 3, we will create an approach to measure kinetics of RNA production, chromatin release, and nuclear export that couples 4sU metabolic labeling of newly synthesized RNAs with biochemical purification of RNAs from chromatin, nucleoplasm, and cytoplasm. The data from this technique will allow us to estimate the rates of release of RNA from chromatin, nuclear export of RNA, and cytoplasmic degradation of every RNA. Using public datasets from ENCODE and elsewhere, we will identify and test hypotheses for the determinants of RNA lifetimes.

转录起始受到结合启动子和增强子的基因组元件的高度调控