? ? The goal of this multi-investigator proposal is to determine how different kinds of new epigenetic marks are distributed and stabilized specifically in mitotic chromatin. While many transcription factors, co-regulators, and histone modifications exist in interphase chromatin and are important for gene regulation, those which occur in mitotic chromatin can clearly confer non-mutational, epigenetic inheritance. Histone modifications are broadly associated with active or inactive genes, but do not set or re-set precise gene expression states. Such states can be set by transcription factors and co-regulators, but most which have been investigated do not occupy mitotic chromatin and hence cannot be epigenetic. However, we recently discovered that FoxA transcription factors, which possess intrinsic chromatin opening properties, and poly-ADP ribose polymerase (PARP), which loosens local chromatin, along with poly(ADP ribose) (pADPr) itself, are distinct from many other factors in that they stably occupy mitotic chromatin. Furthermore, we discovered that FoxA factors and PARP interact with one another and have many genetic targets in common. Our recent mechanistic studies using fluorescence recovery after photobleaching (FRAP) show that FoxA and PARP binding to chromatin is far more stable than that of most other transcription factors. Also, we developed an assay to monitor nascent chromatin sites modified by PARP. Given the ability of these factors to remain stably bound to chromatin through mitosis and their ability to establish a transcriptionally competent state, we propose that FoxA, PARP, and pADPr are a new class of epigenetic mark. We propose the following aims to investigate these epigenetic marks in mitosis: 1. To determine genomic sites of FoxA and PARP1 occupancy and polyADP ribosylation which are specific to mitotic chromatin, compared to interphase chromatin. Such sites would represent a distinctive subset of regulatory sequences for epigenetic inheritance. Such sites will be identified by genomic ChIP approaches and tested for regulatory function. Motif analysis of flanking sequences and TAP-tag studies in mitotic cells will reveal cooperating factors which act at mitotic epigenetic control elements. 2. To determine how FoxA, PARP1, and polyADP ribosylation stably persist in mitotic chromatin and affect local chromatin structure. FRAP studies of FoxA and PARP1, and mutants thereof, and of FoxA and PARP1 partners in mitotic chromatin will be performed to understand how these epigenetic factors remain engaged in mitosis under the extraordinary degree of mitotic chromosome compaction. We will investigate whether FoxA or PARP occupancy, or polyADP ribosylation may induce special structural configurations at epigenetic marks in mitotic chromatin. ? ? ?
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