The step-wise assembly of the spliceosome onto pre-mRNA occurs co-transcriptionally, while the nascent transcript is synthesized from RNA Polymerase II. The mechanisms underlying the molecular coupling and coordination of these co-transcriptional reactions have not been thoroughly elucidated in the context of the dynamic chromatin environment. The Johnson Lab previously discovered that the major yeast histone acetyltransferase (HAT) Gcn5 demonstrates genetic interactions with MSL1 and LEA1—which encode two core U2 small nuclear ribonucleoprotein particle (snRNP) components of the spliceosome. Additionally, the lab observed that Gcn5 HAT activity is required for proper co-transcriptional recruitment of the spliceosome in yeast where U2 snRNP association with the pre-mRNA and subsequent spliceosomal rearrangements are sensitive to Gcn5-dependent acetylation. While Gcn5-dependent histone acetylation is important for the fate of spliceosomal rearrangements of these two proteins, the genome-wide implications and overall mechanism underlying this result is not yet clear. Here, I employed RNA-seq in S. cerevisiae to identify mechanistic insights into how Gcn5-dependent histone acetylation can affect pre-mRNA splicing. I prepared libraries from the following yeast strains: wildtype, gcn5Δ and H3Δ9-16 (deletion of residues 9-16 from histone H3 N-terminal tail) such that genes for which splicing is similarly affected in both gcn5Δ and H3Δ9-16 will represent a defect specific to the absence of Gcn5-histone acetyltransferase (HAT) activity. Surprisingly, the results support a role for the Gcn5-HAT in decreasing gene expression of all intron-containing ribosomal protein genes (IC-RPGs). Consequently, gcn5Δ and H3Δ9-16 improved the splicing efficiency of a subset of IC-non-RPGs in a manner regulated by the competition of IC-RPGs for limited spliceosomes. Lastly, gcn5Δ and H3Δ9-16 resulted in the decreased splicing efficiency for another subset of IC-non-RPGs. With publicly available ChIP-seq and MNase-seq data, I show that despite the increased availability of limited spliceosomes via down-regulation of IC-RPGs, gcn5Δ and H3Δ9-16 dependent splicing outcomes of IC-non-RPGs are distinguished by differences in RNAPII, H3K9ac and MNase enrichment profiles. In this thesis, I uncover multiple effects of the gcn5Δ and H3Δ9-16 on pre-mRNA splicing genome-wide--encompassing mechanisms regarding the economics of limited spliceosome availability and distinct chromatin landscapes directly regulating splicing outcomes.

Clathrin-mediated trafficking is a conserved process during which clathrin coated vesicles transport cargo between the trans-Golgi Network (TGN) and the endosomes, and during the process of endocytosis. These studies identify a previously unrecognized sequence of assembly between adaptor-specific clathrin coated vesicles. At the TGN, we found that GGA-enriched vesicles form first, followed by the biogenesis of AP-1 enriched vesicles. We then identified the mechanism by which this process is temporally controlled. We have identified a novel direct physical interaction between the VHS domain of Gga2 and the yeast PI(4)-Kinase, Pik1p, that is important for the recruitment of Pik1p to the TGN membrane. Deletion of GGA proteins results in the delay of PI(4)P accumulation and Pik1p recruitment. Furthermore, we have mapped the regions through which Pik1p interacts with Gga2 and demonstrate that these binding sites are important for the Pik1p-GGA interaction in vivo. Disruption of these binding sites through mutation results in delayed PI(4)P accumulation, mislocalized Ent5p and delayed AP-1 recruitment. We also provide evidence that this Pik1/GGA interaction is regulated by GTP-Arf1. Arf1 binds to the GAT domain of GGA2, enabling Pik1p to directly bind to the VHS domain of Pik1p. We also find that this network of physical interactions is conserved in mammals.

Ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, coverts somatic cells directly into induced pluripotent stem cells (iPSCs), which are functionally equivalent to embryonic stem cells (ESCs). The discovery of iPSC has been reshaping the methodology of disease modeling and drug screening in the past decade, and provides tremendous promise for regenerative medicine. However, the mechanism underlying this conversion process, reprogramming, is not yet fully understood. I aimed to dissect the reprogramming process, by characterizing the functional domains of one reprogramming factor Klf4. The transcriptional activation domain (TAD) of Klf4 was revealed to be critical for reprogramming. To search for the factors that mediates the functionality of Klf4 TAD, I identified transcriptional coactivators CBP/p300 and Mediator complex as the physical interaction partners of Klf4 TAD, and further showed that this interaction is functionally required for Klf4 mediated transcriptional activation in reprograming. Clathrin heavy chain, initially identified as a physical interaction partner of Klf4 TAD, was shown to be not required for Klf4 transcriptional activation. Clathrin heavy chain was furthered characterized for its potential transcriptional activation activity in CHC-TFE3, a chromosomal fusion discovered in renal cell carcinoma. Clathrin heavy chain, as a conventional vesicle coat protein involved cellular membrane trafficking, was further studied with its accessory protein Irc6 in budding yeast.

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