SAT-029 Understanding The Functions Of Parp-1 In Embryonic Stem Cells

Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear enzyme that plays diverse roles in a variety of nuclear processes, including DNA damage detection and repair, chromatin remodeling, transcriptional regulation, and RNA processing. PARP-1 functions both as an enzyme, through its NAD+-dependent ADP- ribosyltransferase catalytic activity, and a chromatin structural protein, through an NAD+-independent DNA binding activity. We have observed a loss of the maintenance of pluripotency in murine embryonic stem cells (mESCs) lacking PARP-1. Additionally, we have seen a dramatic induction of PARP-1 catalytic activity during the early stages of mESC differentiation. From these observations, we sought to understand the regulation of PARP-1 catalytic activity during differentiation and its effect. We hypothesize that the onset of ADP-ribosylation events by PARP-1, regulated by compartmentalized NAD+, generates site-specific and target-specific modifications (ADP-ribosylation) that assists differentiation. Proteins ADP-ribosylated during this period of enhanced PARP-1 catalytic activity were previously unknown. Using analog NAD+-sensitive PARP-1 technology developed in the Kraus lab, we have identified the PARP-1 ADP-ribosylated proteome of mESCs during the onset of differentiation. These modified proteins appear to be highly associated with specific pathways including - mRNA processing, transcription regulation, and development - shedding light on additional layers of regulation of the differentiation process. We are now using biochemical, molecular, and cellular assays to determine the functional relevance of site-specific ADP-ribosylation events in the differentiation process. This work is supported by theNSF-Graduate Research Fellowship toAJ and agrantfrom the NIH/NIDDK to WLK.