Adipogenic Transcriptional Regulation Mediated by PARP7 Through NAD+ Sensing Mechanism [RNA-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 seeded in 6-well plates and treated as described above. The cells and tissues were collected, and total RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer's protocols. Total RNA was reverse transcribed using oligo (dT) primers and MMLV reverse transcriptase (Promega) to generate cDNA. The cDNA samples were subjected to qPCR using gene-specific primers, as described below. For the reverse transcription quantitative real-time PCR (RT-qPCR) analyses, “relative expression” was determined in comparison to a value of the control sample.