| Gene expression involves the processes of chromatin remodeling, RNA polymerase II (pol II) and general transcription factor recruitment, mRNA processing and mRNA export from the nucleus. All of these steps are interdependent and there are many factors with multiple roles to coordinate these processes. A thorough understanding of the dynamic processes of gene expression will lead to a better understanding of its regulatory mechanisms. Although these processes have been extensively studied at the biochemical level, in vivo studies at the single cell level are needed to characterize aspects of gene expression at high spatial and temporal resolution.;In my research, I elaborated on an in vivo live cell imaging system to study the dynamic process of transcription, a key component of gene expression. Transcription is silenced during cell division; however, it must be re-activated upon exit from mitosis to maintain cell lineage and cell cycle progression. The transmission of the gene expression program from mother to daughter cells has been suggested to be mediated by gene bookmarking, which may involve transcription factors, silencing proteins, histone modifications/variants, and/or DNA methylation. However, the mechanism by which gene bookmarking mediates rapid post-mitotic transcriptional re-activation remains unclear. Here, I used a real-time gene expression system to quantitatively demonstrate that transcriptional activation of the same genetic locus occurs with a significantly more rapid kinetics in post-mitotic cells versus interphase cells. Pol II and Bromodomain Protein 4 (BRD4) were recruited to the locus in a different sequential order upon interphase initiation versus post-mitotic re-activation resulting from the recognition by BRD4 of increased levels of histone H4 lysine 5 acetylation (H4K5Ac) on the previously activated locus. BRD4 accelerated the dynamics of mRNA synthesis in post-mitotic cells by decondensing chromatin and hence facilitating transcriptional reactivation. Together, using a real-time quantitative approach, I identified differences in the kinetics of transcriptional activation between interphase and post-mitotic cells that are mediated by a H4K5Ac-BRD4-dependent mechanism. In summary, my studies have provided significant insight into the kinetics of gene expression and have advanced our understanding of gene bookmarking in this process. |
