RNA polymerase II promotes the organization of chromatin following DNA replication.

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription. However, RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin via pathways that are not observed in steady-state chromatin. These include ATP-dependent remodellers, transcription factors and histone methyltransferases. We also identify a set of DNA repair factors that may handle transcription-replication conflicts during normal transcription in human non-transformed cells. Our study reveals that transcription plays a greater role in the organization of chromatin post-replication than previously anticipated.

TALK-1-mediated alterations of ß-cell mitochondrial function and insulin secretion impair glucose homeostasis on a diabetogenic diet.

Mitochondrial Ca2+ ([Ca2+]m) homeostasis is critical for ß-cell function and becomes disrupted during the pathogenesis of diabetes. [Ca2+]m uptake is dependent on elevations in cytoplasmic Ca2+ ([Ca2+]c) and endoplasmic reticulum Ca2+ ([Ca2+]ER) release, both of which are regulated by the two-pore domain K+ channel TALK-1. Here, utilizing a novel ß-cell TALK-1-knockout (ß-TALK-1-KO) mouse model, we found that TALK-1 limited ß-cell [Ca2+]m accumulation and ATP production. However, following exposure to a high-fat diet (HFD), ATP-linked respiration, glucose-stimulated oxygen consumption rate, and glucose-stimulated insulin secretion (GSIS) were increased in control but not TALK1-KO mice. Although ß-TALK-1-KO animals showed similar GSIS before and after HFD treatment, these mice were protected from HFD-induced glucose intolerance. Collectively, these data identify that TALK-1 channel control of ß-cell function reduces [Ca2+]m and suggest that metabolic remodeling in diabetes drives dysglycemia.

αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.