Studies of the Mechanisms of TFIIH and Noncoding RNAs in Eukaryotic Transcription

摘要

The control of eukaryotic transcription is carefully orchestrated and involves many types of regulatory factors. Transcription is the underlying mechanism that controls all cellular processes and when left unchecked results in diseased cell states and cell death. Understanding the detailed mechanisms and processes of eukaryotic transcription is the goal of these studies. Inspired by our previous eukaryotic transcription kinetic studies, Chapter 2 describes identifying a factor that accelerates the rate of promoter escape. Spiking in vitro transcription assays with a nuclear extract resulted in an increase in the rate of in vitro transcription from the adenovirus major late promoter. With the understanding that many factors are involved in transcriptional regulation, we hypothesized that a factor could function to enhance the rate of transcription after being recruited to promoters. I set out to purify, identify, and characterize this factor. I developed a rate assay to monitor purification of the factor over several columns. The purified rate-accelerating factor was identified to be the general transcription factor TFIIH. Comparing my purified TFIIH to two standard TFIIH purifications revealed that high concentrations of TFIIH accelerated the rate of early transcription. Recent studies have identified thousands of long noncoding RNAs (lncRNAs) with the potential to regulate gene expression, some on a single gene level and others potentially regulating multiple genes through mechanisms controlling chromatin structure. At the time this work was started, there were no genome-wide methods to determine whether these lncRNAs interact directly with chromatin, and if so, where. I developed a method named ChOP-seq to identify the genomic regions with which the lncRNA HOTAIR associates. I was ultimately able to show RNA-dependent enrichment of specific genomic regions using the ChOP technique, identifying a diverse set of genes that may be regulated by HOTAIR. We are positioned to apply our new knowledge of ChOP assays to other ncRNAs. This method has the potential to extend our understanding of the mechanisms that contribute to epigenetic programming.