Regulation of RNA polymerase II transcription by Spt4 in Saccharomyces cerevisiae

Transcription of RNA polymerase II (RNAPII) through chromatin is coordinated by numerous transcription elongation factors (TEFs). Spt4 and Spt5 are two of the most highly conserved TEFs in organisms with nucleosomes. Spt4 forms a complex with Spt5, known as the DSIF complex in humans, which was discovered several decades ago and extensively studied, yet its exact function is still not well understood. Early experiments showed that spt4 and spt5 mutants share similar phenotypes with histone chaperone mutants, spt6 and spt16. Recently, in vitro studies showed that the Spt4/5 complex reduces RNAPII stalling upon transcribing into nucleosomes and cryo-EM images indicated that Spt4/5 occupy a space between the DNA exit site on RNAPII and a nucleosome. These observations implicate the Spt4/5 complex in the regulation of the interplay between transcription and chromatin, but it has not been addressed in vivo.
To understand the precise role of Spt4 in budding yeast, various high throughput sequencing techniques and mass spectrometry were used. The position of the Spt4 on RNAPII was mapped at single-nucleotide resolution using TEF-seq. With the same technique, the position of Spt5 on RNAPII was investigated in wild type (WT) and spt4 knock-out (spt4∆) cells. RNAPII dynamics were examined using NET-seq in spt4∆ cells or cells in which Spt4 has been anchored away to the cytoplasm (Spt4 AA). For comparison, RNAPII was also mapped in cells in which Spt5 has been anchored away to the cytoplasm (Spt5 AA). Additionally, nucleosome positions were studied using MNase-seq and the composition of the transcription complex was investigated using affinity purification coupled mass spectrometry in WT and spt4∆ cells.
The data show that the interaction between the Spt4/5 complex and RNAPII periodically changes as RNAPII transitions through nucleosomes. The dynamic interaction of Spt5 with RNAPII is dependent on Spt4. In spt4∆ and Spt4 AA cells, RNAPII distribution is altered in a similar way compared to their respective controls. After transcribing into nucleosomes, RNAPII accumulates upstream of nucleosome dyads, especially around the +2 nucleosome. In Spt5 AA cells, the distribution and amount of RNAPII on genes are severely affected.
Nucleosome positions are altered in spt4∆ cells. Although the position of the +1 nucleosome remains unchanged, nucleosome spacing from this point is increased, and the level of increase in nucleosome spacing correlates with the level of RNAPII accumulation.
The composition of the transcription complex is changed in spt4∆ compared to WT cells. The levels of some transcription factors related to chromatin regulation are lowered, while the abundance of TFIIH subunits are significantly increased on the spt4∆ transcription complex.
Overall, this work suggests that Spt4 regulates nucleosome positioning by a mechanism related to transcription and promotes RNAPII movement through nucleosome barriers. The findings presented here add to the current understanding of the function of the Spt4/5 complex in transcription.

Keywords: Transcription
RNA polymerase II
Spt4
Saccharomyces cerevisiae
Advisor: Furger, A
Mellor, J