BME PhD Dissertation Defense - James Chuang

Title: “Genomic analyses of transcription elongation factors and intragenic transcription”

Committee: Fred Winston, PhD – Harvard Medical School, Department of Genetics (Advisor) Ahmad Khalil, PhD – BU BME (Co-Advisor) John Ngo, PhD – BU BME (Chair) Wilson Wong, PhD – BU BME L. Stirling Churchman, PhD – Harvard Medical School, Department of Genetics

Abstract: Transcription of protein-coding genes in eukaryotic cells is carried out by the protein complex RNA polymerase II (Pol II). During the elongation phase of transcription, Pol II associates with transcription elongation factors which modulate transcriptional activity and are required for co-transcriptional processes. Using yeasts as model eukaryotes, we studied the in vivo requirement for the transcription elongation factors Spt6 and Spt5.

Spt6 is a transcription elongation factor thought to replace nucleosomes in the wake of transcription. One phenotype observed upon mutation of Spt6 is the expression of intragenic transcripts, transcripts which initiate from within gene bodies. Using high resolution genomic assays of transcription initiation, we catalog the full extent of intragenic transcription in the Saccharomyces cerevisiae Spt6 mutant spt6-1004, and show for the first time on a genome-wide scale that this intragenic transcription is largely explained by new transcription initiation in the mutant. We also find that canonical genic transcription is greatly reduced in spt6-1004. By profiling chromatin structure genome-wide, we show that these changes occur in the context of a global depletion and disordering of nucleosomes. Altogether, we propose that the increase in intragenic transcription and decrease in genic transcription in spt6-1004 is explained by a competition for transcription initiation factors between genic and intragenic promoters, which is made possible by decreased nucleosome protection of intragenic promoters.

Spt5 is another transcription elongation factor, important for the processivity of the transcription complex and many transcription-related processes. To study the requirement for Spt5 in vivo, we applied multiple genomic assays to Schizosaccharomyces pombe cells depleted of Spt5. Our results reveal and accumulation of Pol II over the 5’ ends of genes, and a progressive decrease in transcript abundance towards the 3’ ends of genes. This is consistent with a model in which Spt5 depletion causes transcription elongation defects and increases early termination. We also unexpectedly discover that Spt5 depletion causes hundreds of antisense transcripts to be expressed across the genome.

The expression of intragenic transcripts when transcription elongation factors are disrupted suggests that cells have evolved to prevent spurious intragenic transcription. However, some cases of intragenic transcription are consistently detected in wild-type cells, and some of these cases are known to be important for different biological functions. To better understand the many ways in which intragenic transcription can function in wild-type cells, we identified intragenic transcription regulated by three stress conditions in S. cerevisiae. For the condition of oxidative stress, we show that most intragenic transcripts are expressed at a lower level than genic transcripts, and that many intragenic transcripts are likely to be translated at some level. Finally, we show that the expression of an oxidative-stress-induced transcript at the gene DSK2 is needed for S. cerevisiae to survive in conditions of oxidative stress.

Date: Wednesday, June 19th 2019

Start Time: 2:00pm

Location: 77 Avenue Louis Pasteur, NRB 350

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