High-resolution studies of the chromatin and transcription landscape

Thesis (Ph.D.)--University of Washington, 2014Transcription from genomic DNA is regulated in many ways and governs cellular identity, growth, and homeostasis. If transcription becomes deregulated, it can serve a critical role in the development of cancer and other human pathologies, however complete understanding of this process is lacking. In eukaryotes, transcriptional regulation involves a balance between repressive packaging of the genome into nucleosomes and enabling access to regulatory proteins as well as RNA polymerase II (RNAPII). Nucleosomes are strong physical barriers to transcription in vitro that cause RNAPII to backtrack and arrest. Yet, in vivo RNAPII must transcribe across many nucleosomes for every gene at a very high rate. How this happens and what mechanisms enable RNAPII transit through nucleosomes has long remained unclear. Here we show that the nucleosomes of active genes have a distinct histone composition which involves the replacement of both canonical H2A histones with histone variant H2A.Z. These homotypic H2A.Z nucleosomes show evidence of disruption during transcriptional elongation, suggesting that they are formed through transcription mediated turnover and then replacement with H2A.Z. Homotypic H2A.Z nucleosomes have unique physical properties and interact with distinct chromatin remodelers from canonical nucleosomes. Hence, we developed a single-nucleotide resolution approach to map RNAPII genome-wide and determine the nature of transcription through these nucleosomes. We show that the entry site to the nucleosome is most refractory, contrary to existing models based on transcription in vitro, and that the first nucleosome from the transcription start site (+1) is a much larger barrier than downstream nucleosomes that causes RNAPII to backtrack. Nucleosome occupancy positively correlates with the magnitude of the barrier, however our results suggest that an evolved function of H2A.Z is to ease the inhibitory nature of nucleosomes on transcription. This helps to explain why H2A.Z is most enriched where the barrier is the largest and also why it is essential in development where transcriptional fidelity is critical