Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids

Housekeeping metabolic pathways such as glycolysis are active in all cell types. In addition, many types of cells are equipped with cell-specific metabolic pathways. To properly perform their functions, housekeeping and cell-specific metabolic pathways must function cooperatively. However, the regulatory mechanisms that couple metabolic pathways remain largely unknown. Recently, we showed that the steroidogenic cell-specific nuclear receptor Ad4BP/ SF-1, which regulates steroidogenic genes, also regulates housekeeping glycolytic genes. Here, we identify cholesterogenic genes as the targets of Ad4BP/SF-1. Further, we reveal that Ad4BP/SF-1 regulates Hummr, a candidate mediator of cholesterol transport from endoplasmic reticula to mitochondria. Given that cholesterol is the starting material for steroidogenesis and is synthesized from acetyl-CoA, which partly originates from glucose, our results suggest that multiple biological processes involved in synthesizing steroid hormones are governed by Ad4BP/SF-1. To our knowledge, this study provides the first example where housekeeping and cell-specific metabolism are coordinated at the transcriptional level.This work was supported by Grants 16H05142 (K.M.), 17H06427 (K.M.), 16K08593 (T.B.), and 17J03270 (M.I.) from the Japan Society for the Promotion of Science (JSPS) KAKENHI; The Uehara Memorial Foundation (K.M.); Takeda Science Foundation (T.B.); The Shin-Nihon of Advanced Medical Research (T.B.).Supplementary information accompanies this paper at https://doi.org/10.1038/s42003-018-0020-z

Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20^ protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes

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Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus. Nuclear magnetic resonance analysis demonstrated that ARIP4 interacts directly with the ubiquitin-Associated (UBA) domain of p62. ARIP4 and ubiquitin both bind to similar amino acid residues within UBA domains; therefore, these proteins may possess a similar surface structure at their UBA-binding interfaces. We also found that p62 is required for the regulation of ARIP4 protein levels under nutrient starvation conditions. We propose that p62 is a novel binding partner for ARIP4, and that its binding regulates the cellular protein level of ARIP4 under conditions of metabolic stress

Replacing the HMG box of Sox9 abolished the inhibitory effect of Exp4 on Sox9 transcription.

<p>(A) Schematic representation of GAL4-fused Sox proteins (GAL4-Sox9) used in this study. The TA domain is shown as a light gray box (406–507 a.a.). (B) The effect of Exp4 on Sox9-mediated transcription of the Col2a1 reporter gene (Col2a1x3-tk-Luc). HEK293 cells were transiently transfected with the reporter gene and the indicated amounts (ng) of Sox9 or Exp4. The relative fold changes in the luciferase activities are shown: luciferase activity in the presence of 50 ng of Sox9 and the absence of Exp4 is set at 100. Values are the mean ± SD of at least three experiments. (C) The effect of Exp4 on GAL4-Sox9 mediated transcription of the GAL4 reporter gene (UAS_Gx4-TK-LUC). HEK293 cells were transiently transfected with the reporter gene and the indicated amounts (ng) of GAL4-Sox9 or Exp4. The relative fold changes in the luciferase activities are shown: luciferase activity in the presence of 50 ng of GAL4-Sox9 and the absence of Exp4 is set at 100. Values are the mean ± SD of at least three experiments.</p

Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage

Identification of Exp4 as a major interaction partner of Sox9.

<p>(A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.</p