Large-Scale Functional Genomics Screen to Identify Modulators of Human ß-Cell Insulin Secretion.

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic ß-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-ßH1 cells, a human ß-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-ßH1 cells for large-scale siRNA screening to identify candidate genes regulating ß-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional ß-cell mass occurring in T2D.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy.

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217-mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217-mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.