Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

PARPs and ADP-ribosylation in RNA biology: from RNA expression and processing to protein translation and proteostasis.

ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents.

Quercetin and Quercetin-Rich Red Onion Extract Alter Pgc-1α Promoter Methylation and Splice Variant Expression.

Pgc-1α and its various isoforms may play a role in determining skeletal muscle mitochondrial adaptations in response to diet. 8 wks of dietary supplementation with the flavonoid quercetin (Q) or red onion extract (ROE) in a high fat diet (HFD) ameliorates HFD-induced obesity and insulin resistance in C57BL/J mice while upregulating Pgc-1α and increasing skeletal muscle mitochondrial number and function. Here, mice were fed a low fat (LF), high fat (HF), high fat plus quercetin (HF + Q), or high fat plus red onion extract (HF + RO) diet for 9 wks and skeletal muscle Pgc-1α isoform expression and DNA methylation were determined. Quantification of various Pgc-1α isoforms, including isoforms Pgc-1α-a, Pgc-1α-b, Pgc-1α-c, Pgc-1α4, total NT-Pgc-1α, and FL-Pgc-1α, showed that only total NT-Pgc-1α expression was increased in LF, HF + Q, and HF + RO compared to HF. Furthermore, Q supplementation decreased Pgc-1α-a expression compared to LF and HF, and ROE decreased Pgc-1α-a expression compared to LF. FL-Pgc-1α was decreased in HF + Q and HF + RO compared to LF and HF. HF exhibited hypermethylation at the -260 nucleotide (nt) in the Pgc-1α promoter. Q and ROE prevented HFD-induced hypermethylation. -260 nt methylation levels were associated with NT-Pgc-1α expression only. Pgc-1α isoform expression may be epigenetically regulated by Q and ROE through DNA methylation.

A Review of Mitochondrial-derived Fatty Acids in Epigenetic Regulation of Obesity and Type 2 Diabetes.

Type 2 diabetes, the leading metabolic disease, is characterized by insulin resistance and is associated with obesity. The onset of type 2 diabetes is largely due to environmental inputs, such as high dietary fat content and decreased levels of exercise. Insulin resistance resulting from high fat diet is associated with skeletal muscle mitochondrial dysfunction, leading to alterations in lipid accumulation and specific species of intracellular fatty acids; whereas, exercise training augments insulin resistance while improving skeletal muscle mitochondrial function and producing beneficial fatty acid profiles. Additionally, high fat diets and exercise alter epigenetic modifications, including DNA methylation and histone acetylation, to produce differences in metabolic gene expression that are associated with insulin resistance and sensitivity, respectively. Recent evidence suggests that short chain fatty acids that act as histone deacetylase inhibitors prevent and ameliorate obesity and insulin resistance. Here, we discuss the potential of mitochondrial-derived fatty acids, especially short chain fatty acids, to epigenetically regulate obesity and type 2 diabetes.