Recruitment of transcriptional machinery following DNA replication

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Project Summary/Abstract Maintenance of transcriptional programs during cell division is the key role of epigenetics. For cells to transmit transcriptional memory during division, it is believed that certain proteins mark active and repressed genomic loci. For the most part, these factors include chromatin-associated proteins whose function is to remain stably associated with DNA throughout the cell cycle such that transcriptional patterns are remembered. The two main phases of the cell cycle where this is critical are S-phase and M-phase, due to transcriptional interruption by DNA duplication and chromatin condensation, respectively. The typical candidates for epigenetic marks are modified histones, which are believed to be transferred to nascent DNA and to then recruit chromosomal proteins to the two daughter strands. Recently, however, it was shown that many chromatin-associated proteins are not displaced from DNA during replication, challenging the long held belief that replication is inherently disruptive to chromatin structure. This raises two questions: are transcriptional proteins also potentially impervious to replication machinery, and if not, how quickly do they reassemble on newly synthesized DNA? The goal of this project is to investigate the fate of transcriptional proteins, including RNA polymerase II (Pol II) and associated factors, during S-phase, and in particular to understand their recruitment to nascent DNA, since maintenance of transcriptional programs is the paramount function of epigenetics. To track the recruitment of these proteins I will use a novel immunofluorescent assay developed by our lab, called the Chromatin Assembly Assay (CAA). The power of using CAA is that it is amenable to a pulse-chase work flow to track protein recruitment to DNA in cultured cells. In order to understand mechanistically how these proteins are being recruited, CAA will be combined with super resolution microscopy, to study how these interactions spatially occur with regard to replication and transcription factories. Additionally, I will use sequential chromatin immunoprecipitation (re-ChIP) assays designed to investigate protein recruitment to nascent DNA at specific genomic loci. These assays will help to validate the CAA findings, and to further interrogate these initial results at the gene specific level. Furthermore, the power of gene-specific assay will allow further inquiry recruitment and/or retention of transcriptional proteins at different types of genes, including active, paused, and poised genes. These analyses will provide a better understanding of how epigenetics functions to maintain transcriptional programs, and will be vital in understanding how cells alter these programs during cellular differentiation and the development of disease.