The goal of this proposal is to investigate the mechanisms by which poly ADP-ribosylation (pADPr) of proteins regulates gene expression. The level of protein pADPr reflects the relative activities of the poly(ADP-ribose) polymerase (PARP) enzyme, which utilizes NAD to create pADPr-modified proteins, and the poly(ADP-Ribose) glycohydrolase (PARG) enzyme, which removes pADPr moieties. My studies in Drosophila first revealed vital roles for PARP protein in the establishment of silent chromatin domains as well as in the chromatin loosening and transcriptional activation of a subset of inducible chromosomal loci. Subsequently, increased expression of inactive PARP1 protein has been implicated in the formation of condensed and silent chromatin domains, whereas upon the stimulation of PARP enzymatic activity, chromatin decondenses and becomes transcriptionally active. At present, the main gaps in our understanding of the PARP1-dependent transcriptional regulation are (1) the mechanism of PARP protein targeting to specific chromatin domains, and (2) the mechanism of local PARP activation. In preliminary studies, we have successfully identified novel chromatin-associated PARP1 partners by use of a Tandem Affinity Purification (TAP) strategy together with sucrose gradient purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Candidate interactors arising from this approach were functionally analyzed for influence on PARP1, using standard genetic approaches combined with immunostaining and confocal microscopy. Based on this work, we have identified the variant histone H2Av as a protein that promotes targeting of PARP1 to chromatin. Recent studies of histone H2Av by other groups have suggested this protein triggers DNA repair, apoptosis and heterochromatin formation. Ser137- phosphorylated H2Av has been shown to co-localize with foci of local PARP1 activation in vivo. In this proposal, we will focus on two aspects of PARP1 targeting to chromatin. First, we will characterize the mechanism through which H2Av controls PARP1 protein incorporation to chromatin and regulates chromatin-directed PARP activity. Second, we will evaluate functions of the PARP1 protein domains, defining which specify PARP1 interactions with chromatin. We are will address these questions with the following specific aims:
Aim 1. To characterize the mechanism by which H2Av targets PARP1 to chromatin.
Aim 2. To determine the role of core histones in the PARP1 protein binding to chromatin.
Aim 3. To investigate the roles of automodification and phosphorylation in regulating PARP1 targeting to chromatin in vivo. Relevance to Public Health Statement: Understanding how an organism can utilize PARP protein ability to modulate chromatin and transcription will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis. ? ? ? ? ? ?
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