1) We have established a reliable approach to profile mitogen-induced changes in the genome-wide association of p300 with proximal promoters in human T-cells. Through polyclonal antibodies we have generated and characterized against p300 and pol II and for use in chromatin immuno-precipitation and ChIP/Chip studies we have developed a molecular screening strategy to profile genome wide targeting by transcriptional co-regulators. This approach was used to identify a class of human proximal promoters that rapidly co-assemble p300 and pol II following mitogen stimulation (Figure 1). Correlation of this data set with gene expression data demonstrated that many members of these p300 associated genes were immediate early genes including numerous well known proto-oncogenes such as c-fos and c-jun. This work represents one of the first analyses of the genome-wide distribution of p300 in an inducible mammalian system. It is also the first identification of a common link between gene-specific p300 targeting and immediate early gene expression. 2) Using an assay system to profile the time and position dependent changes in recruitment and elongation events at target genes in vivo by quantitative ChIP, we have leveraged information gleaned from our p300 ChIP/Chip studies to show that mitogen-induced recruitment of pol II and p300 at the proximal promoters of immediate early genes, like c-fos, is necessary but not sufficient for gene expression. Instead we have found that p300 participates in a regulatory module at immediate early genes that licenses acetylation-dependent elongation events distinct from histone acetylation and linked to different nuclear protein complexes that facilitate and enforce transcriptional elongation. These findings are the first demonstration of a linkage between p300 function and transcriptional elongation in vivo, and signify an emerging paradigm shift that will define expanded roles for p300 and other co-regulators in the transcription cycle beyond basal factor recruitment and histone acetylation. 3) We have found that the bromodomain containing protein BRD4, shuttles in a complex with positive transcriptional elongation factors (pTEFb) to the promoter of the fos oncogene following mitogen stimulation. We also show that BRD4 recruitment is acetylation dependent and facilitates transcriptional elongation at the fos promoter. This is the first report of targeted inducible recruitment of BRD4 to the promoter region of a mammalian gene and has broad implications for BRD4 in the control of signal regulated transcription events through modulation of gene expression at the level of elongation. The known association of BRD4 with mitotic chromosomes implies added roles for this protein in post-mitotic processes that transmit regulatory memory of premitotic events. 4) We have found that the elongation factor eleven-nineteen lysine rich leukemia protein (ELL) is rapidly recruited to the promoters of numerous immediate early genes and assembles and travels with the elongating polymerase following mitogen stimulation. The association is acetylation dependent, regulated by p300, and is the first demonstration of a mitogen inducible association of ELL with the elongating polymerase at a mammalian gene. 5) We have found that the c-fos promoter undergoes a dynamic series of acetylation dependent changes in histone 3 lysine 4 tri-methylation (H3K4Me3) following mitogen stimulation that is associated with the recruitment of MLL to the c-fos locus. In addition we find that the H3K4Me3 modification at the c-fos promoter functions as an epigenetic mark for proliferative cell cycle entry that is retained or remembered after return to G0 and superinduced after subsequent challenge. These findings have implications for understanding the molecular events that could facilitate mechanisms of immunological memory in B and T-cells. 6) We have found that there is a mitogen-induced shift in the availability of active elongation factor complexes containing p-TEFb. These findings suggest that transcriptional elongation events may be influenced indirectly by signal transduction pathways that titrate the sequestered pool of pTEFb/HEXIM1 complexes. 7) We have found that p300 is a preferred client for nuclear chaperones and that, like glucocorticoid receptor, p300 mobility in the nucleus is highly energy dependent and involves multiple interactions with chaperones that are qualitatively and quantitatively distinct from CBP. These findings have major implications for our thinking about the co-factors and mechanisms that facilitate the assembly of p300 in multi-component complexes that control chromatin structure and transcription. 8) To facilitate the genomic approaches we employ in the lab to characterize mechanisms of p300 function, we have developed a number of genome based computational tools to help integrate the molecular, biological and bioinformatics methods employed in each project. These efforts were complementary to the in depth analysis provided by our more experienced collaborators, specifically tailored to our needs and necessary to interpret and filter the large volume of data typically generated by such approaches so that our interaction with genomic, statistic and bioinformatic experts were better targeted to address the most salient molecular and biologically relevant aspects of the projects. These tools include a batchwise oriented genome-wide promoter retrieval tool, a rapid and interactive database containing primers designed for quantitative RT-PCR detection of spliced mature mRNA transcripts, a computational tool to determine statistical enrichment of transcription factor binding site designed for high throughput batchwise analysis and a tool that designs primer sets to amplify any specified genomic region through the genome. As a result we have approached our projects with better insights and these tools have led to more effective interaction with our collaborators and multiple publications submitted and in press.
| Cui, Wenwu; Taub, Dennis D; Gardner, Kevin (2007) qPrimerDepot: a primer database for quantitative real time PCR. Nucleic Acids Res 35:D805-9 |
| Idelman, Gila; Taylor, James G; Tongbai, Ron et al. (2007) Functional profiling of uncommon VCAM1 promoter polymorphisms prevalent in African American populations. Hum Mutat 28:824-9 |
