The Regulation of RNA Polymerase II Transcription and Co-Transcriptional Processes

Transcription by RNA polymerase II (Pol II) is one of the most highly regulated processes within the cell and is coupled to multiple co-transcriptional events. Furthermore, many different classes of coding and non-coding transcripts exist, and each is subject to distinct regulatory mechanisms. To explore the mechanisms coupling transcription to co -transcriptional processes we identified the full complement of factors that interact with Pol II during the different stages of transcription. This was achieved by purifying factors associated with different phosphorylation states of the Pol II C-terminal domain (CTD) repeat (Y1S2P3T4S5P6S7) and by isolating complexes from specifically the 5ʹ or 3ʹ regions of a gene. Through this work we identify hundreds of enriched factors revealing unappreciated links between CTD phosphorylation and regulation of co-transcriptional processes. We go on to define roles for CTD Ser5 and CTD Thr4 in the regulation of co and post-transcriptional splicing respectively. We further demonstrate that CTD Thr4 is required for proper transition between 3′ end processing and termination genome-wide. Comparative analysis of Pol II complexes from the 5ʹ and 3ʹ regions of genes reveals that canonical transcription termination factors also regulate transcription elongation and that the ubiquitin ligase Bre1 is important for proper Pol II dynamics during the latter stages of transcription. To investigate mechanisms regulating a large class of non-coding transcripts, known as antisense transcripts, we analyzed production of nascent RNA genome-wide using native elongating transcript sequencing (NET-seq) in a number of transcription and chromatin related mutants. NET-seq analysis in these mutants revealed that factors regulating histone acetylation are key modulators of antisense transcription levels. Furthermore we demonstrate that hyperacetylation of histone H3 results in increased antisense transcription genome-wide. This work establishes new mechanisms coupling transcription to RNA processing as well as mechanisms regulating antisense transcription and thus provides new insights into the vast network of transcriptional regulation.Medical Science