Adipogenic Transcriptional Regulation Mediated by PARP7 Through NAD+ Sensing Mechanism [ATAC-seq]

The process of differentiating from a preadipocyte into a mature, fat storing, adipocyte (adipogenesis) is a complex and tightly regulated process vital to human health. The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined. Here we have identified a mono(ADP-ribosyl)transferase (MART), PARP7, that plays a pivotal role in adipogenesis. ADP-ribosylation – the process whereby specific members of the poly(ADP-ribosyl)polymerase (PARP) family facilitate the covalent transfer of ADP-ribose moieties from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery. Herein we have found that PARP7 is required for modulation of the adipogenic transcriptional program during adipogenesis, and that the loss of PARP7 results in a decrease of the adipogenic process. Contrary to previous studies, the ability to modulate the adipogenic transcriptional program is independent of PARP7 catalytic activity. However, the catalytic activity of PARP7 plays a vital role in regulating protein stability, in a time and space specific manner, which is required for modulation of the adipogenic transcriptional program. This study has found a novel pathway which utilizes NAD+ compartmentalization to stabilize PARP7 at a specific time during the adipogenic process allowing for the regulation of adipogenic genes, through modulation of the well-known adipogenic transcription factor C/EBPß, in a time sensitive manner. Therefore, acting as another check and balance to the adipogenic differentiation process. Overall design: 3T3-L1 cells were exposed to various treatments and culture conditions for the experiments described herein. For treatment with MG-132 (10 µM, Sigma, M7449), the cells were grown as described above, cells were then treated with 10 µM MG-132 for 6 hours before collection. For treatment with RBN2397, the cells were grown as described above upon differentiation of 3T3-L1 cells using differentiation cocktail the cells were treated with 400 nM RBN2397. The treatment continued every other day with media changes until specified collection time. For PARP7 MAR immunoprecipitation assays, the cells were treated with RBN 2397 for 16hrs