Role of PARP-1 in Mediating Inflammatory Gene Transcription

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Overproduction of inflammatory mediators can lead to major organ failure and damage. The transcription of a defined set of inflammatory genes requires the simultaneous specific DNA-protein and protein-protein interactions in highly ordered complexes called enhanceosome. Poly(ADP-ribose) polymerase-1 (PARP-1) regulates transcription factors responsible for inflammatory gene expression, and importantly PARP-1-deficient (PARP-1-/-) mice were shown to be resistant to inflammatory diseases. The overall HYPOTHESES are that PARP-1 is recruited to the promoter, modifies transcription regulators with poly(ADP-ribose), and mediates recruiting transcription regulators to assemble the enhanceosome for synergistic gene transcription. Our SPECIFIC AIMS are to (1) determine whether PARP- 1 binds the iNOS promoter in a sequence-specific manner; (2) investigate if PARP-1 is recruited to the iNOS promoter by interacting with a sequence-specific transcription factor(s) and increasing its DMA binding activity; (3) investigate whether p42/44MAPK directly or indirectly phosphprylates and induces PARP-1 catalytic activity in response to LPS; (4) determine whether PARP-1 targets transcription regulators with poly(ADP-ribose) for the efficient gene transcription in response to LPS;(5) characterize PARP-1 mediating the recruitment of transcription regulators to the promoters (i) by comparing wild-type and PARP-1-/- cells, (ii) by simultaneously analyzing PARP-1-mediated recruitments, and (iii) of iNOS, TNFalpha, IL-1beta, and IL-6; and (6) definitively determine the specificity of PARP-1 and its catalytic activity on inflammatory gene regulation by expressing wild-type PARP-1 and catalytically inactive PARP-1 mutant in PARP-1-/- cells. The SIGNIFICANCE of the proposed studies is to offer new insights into the mechanism(s) by which PARP-1 may regulate and be regulated to achieve synergistic inflammatory gene transcription. The inflammatory gene transcription is critical for immune responses and for determining the onset of inflammatory diseases such as septic shock, acute lung inflammation, Alzheimer's disease, multiple sclerosis, and HIV-associated dementia.