Two Widespread DNA Binding Factors Mediate Differential Regulation of Promoter-proximal Pausing in Drosophila.

The pausing of RNA polymerase (Pol) II at the 5’ end of mammalian and Drosophila genes represents an important regulatory checkpoint during transcription, but its mechanistic basis is not understood. Since the DNA within paused Pol II is single-stranded and thus hyperreactive to permanganate, we developed a permanganate ChIP-seq assay to map the Pol II transcription bubbles across the Drosophila genome. This method provides a near base-pair resolution and definitive assessment of transcriptionally engaged Pol II. With these powers, I have determined that the majority of promoter-associated Pol II on protein-coding genes is paused in a region from 20-60 bp downstream of the transcription start site. In contrast, Pol II associated with snRNA genes appears to be concentrated in an open complex at the transcription start. Our permanganate ChIP-seq data also suggest pausing is tightly linked to the occupancy of GAGA factor (GAF), a widespread DNA binding factor that has been previously implicated in chromatin remodeling and transcription initiation. I developed a biochemical system for promoter proximal pausing, and demonstrate that GAF exerts its control on pausing before transcription initiation, as well as afterward. I also collected genetic and biochemical evidence indicating that pausing is a kinetic consequence of competition between the rate of elongation and the rate at which NELF associates with the elongation complex. Here, I show that GAF recruits the pausing factor NELF to the promoter and tips the kinetic competition in favor of pausing. A different mechanism is likely to contribute to the establishment of paused Pol II on GAF-less promoters. On these promoters, pausing relies on the coordinated actions of DNA binding factors that recruit Pol II and the first nucleosome (+1 nucleosome) downstream from the start site. An example of the factors that recruit Pol II on GAF-less promoters is a novel DNA binding protein I identified, which I call M1BP. M1BP recognizes the previously identified core iv promoter element Motif 1, and associates with about 2000 promoters in Drosophila. Remarkably, M1BP is highly enriched at promoters that associate with Pol II but lack GAF. Transcriptome analyses and ChIP assays indicated M1BP functions in transcription activation and Pol II recruitment on its target genes. Evidence for the +1 nucleosome being involved in pausing on GAF-less genes comes from analyses of published nucleosome mapping data. I found distinct nucleosome distributions on GAF-associated and GAF-less promoters. Based on the location relationship of the +1 nucleosome and paused Pol II, I propose that pausing on GAF-less genes involves a collision between Pol II and the +1 nucleosome. Although both GAF and M1BP are highly enriched on promoters with paused Pol II, they regulate genes that are involved in distinct biological processes. M1BP-associated genes are involved in basic cellular processes and tend to be evenly expressed in various tissues and at different stages in development. GAF-associated genes are involved in developmental control, and exhibit considerable variation in expression in various tissues and at different developmental stages. Genomic analyses reveal that M1BPand GAF-associated promoters are strikingly different in their Pol II distribution and their dependencies on gene-specific regulators. In addition, M1BP associates with the insulator protein, CP190, which has been implicated in establishing the transcriptional independence of promoters. I conclude that M1BP and GAF orchestrate distinct mechanisms of transcriptional control and that M1BP functions as a master regulator of constitutively expressed genes while GAF potentiates dynamic patterns of expression. In summary, my doctoral work provides a clear assessment of the status of Pol II at Drosophila promoters. It also indicates pausing is controlled by a kinetic competition between the elongation rate and the rate at which NELF associates with the elongation complex. This competition provides a mechanistic basis for how sequence specific transcription factors and chromatin structure affect the location and efficiency of promoter proximal pausing. This work v also compares two widespread DNA binding factors GAF and M1BP that are highly enriched at paused promoters and demonstrates how these two factors differentially regulate transcription on their target genes.