Mammalian gene expression occurs via a series of transcriptional bursts, where convoys of 4-20 RNA Polymerase II (Pol II) molecules are loaded onto the promoter over the span of about 1 minute. In between these bursts, the gene promoter becomes non-permissive for transcription on timescales ranging from 1.5 to 30 minutes. Many genes also produce antisense transcripts that may regulate sense transcriptional bursting. However, it is not known how transient chromatin states involving histone acetylation and methylation can fine- tune these transcriptional bursts and transcription factor recruitment. This knowledge is critical for understanding how this process becomes dysregulated in diseases such as cancer. The p53 tumor suppression stress response system is ideal for studying transcriptional bursting in live cells. Our live cell imaging system will survey p53-dependent recruitment of histone acetylases (TIP60), deacetylases (HDAC1), methylases (EHMT1) and demethylases (PHF2) alongside sense and antisense transcriptional bursting. Preliminary data show 1.) transcription from the endogenous p21 locus occurs in sporadic bursts at low p53 levels, 2.) p53 induction leads to more frequent and longer transcriptional bursts, 3.) p53 occupancy rapidly oscillates on the p21 locus during transcriptional bursting, and 4.) in vitro, p53 dynamically binds target DNA sites that are wrapped in a nucleosome on the p21 promoter. Our long-term goal is to understand how gene expression is coordinately controlled by chromatin to regulate access of transcription factors to target DNA sites.
We aim to correlate sense and antisense transcriptional bursting and transcription factor binding with changes in histone modification at a single-gene locus in live cells. Based on our preliminary data and previous studies, we hypothesize that sense transcriptional bursting profiles at tumor suppression genes are regulated via oscillatory, p53-dependent recruitment of histone acetylases/deacetylases and methylases/demethylases. This hypothesis will be tested using cutting-edge single molecule live-cell microscopy in the following 3 specific aims: 1.) Establish a system to link transcription dynamics with changes in p53's DNA binding activity and Pol II recruitment in vivo. Live cell imaging will correlate sense and antisense transcriptional bursting with p53 and Pol II binding. 2.) Define how transcription bursting, p53:DNA binding and Pol II recruitment is regulated by histone acetylation at promoters in live cells. Live cell imaging will correlate transcriptional bursting with TIP60, HDAC1, p53 and Pol II occupancy/activity on a promoter. 3.) Define how transcription bursting, p53:DNA binding and Pol II recruitment is regulated by histone methylation at promoters in live cells. Live cell imaging will correlate sense and antisense transcriptional bursting with EHMT1, PHF2, p53 and Pol II occupancy on a promoter. These studies will provide key insights into how transcriptional bursting is modulated by the dynamic interplay between p53, chromatin-modification enzymes and Pol II in cells.
The proposed research is relevant to public health because it seeks to determine how gene expression is dynamically regulated by p53 and chromatin to direct critical cellular pathways involved in DNA repair, DNA replication and Cell division. This knowledge is important because it will serve as the foundation for development of therapeutics to help modulate cellular growth pathways in diseases such as cancer. Therefore this project is relevant to the part of NIH's mission of developing fundamental knowledge related to treatment of human disease.
| Limi, Saima; Senecal, Adrien; Coleman, Robert et al. (2018) Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process. J Biol Chem 293:13176-13190 |
