A long-standing biological mystery is how the same DNA genome can specify distinct cell types in an organism. Transcription, the synthesis of RNA on the DNA template, is the first step in gene expression that largely defines which genes will be active. The key unanswered question is how transcription is regulated across the genome to generate stable, cell-type-specific patterns of gene expression. By combining classic biochemical approaches with global sequencing, this project will determine how specific transcription patterns may be stabilized by universal mechanisms targeting the enzymes responsible for RNA synthesis. By studying the process of transcription, the project will tackle a general problem of how multiple layers of information could be packed into, and extracted from, the same DNA sequence.
This project deals with the phenomenon of promoter-proximal RNA polymerase II (Pol II) pausing, which occurs within the first 100 nucleotides of many metazoan genes and is controlled by factors involved in most if not all Pol II-transcribed promoters. Pausing is established by the Negative ELongation Factor (NELF) and, apart from the well-known productive elongation, can be alternatively released without mRNA synthesis via premature termination. This project will investigate the roles of factors responsible for pause establishment and release in human mesenchymal stem cells undergoing adipocyte differentiation. Objective 1 will focus on establishing pausing to determine how NELF defines genes to be paused. The question will be addressed by perturbing the levels of NELF at various time scales and monitoring Pol II pausing distribution and dynamics using our published as well as new methods including 5'-capped RNA- sequencing and multiplex permanganate footprinting. Objective 2 will focus on pause release to determine how the decision between mRNA production versus termination regulates expression of genes in response to a model heat shock stimulus. The question will be addressed by combining Pol II ChIP-sequencing and global run-on assays with novel analysis of termination-specific small RNAs derived from paused complexes. By exploring Pol II pausing as a prominent yet still poorly understood phenomenon, a new principle organizing genes into networks may be uncovered. Students engaged into ongoing research will make more informed decisions on whether or not to pursue careers in science, which will elevate the reputation of basic science among general public especially in rural communities.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
