Identification and Functional Characterization of a Novel Androgen Receptor Coregulator, EAP1

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression.

SUMO and Transcriptional Regulation: The Lessons of Large-Scale Proteomic, Modifomic and Genomic Studies

One major role of the eukaryotic peptidic post-translational modifier SUMO in the cell is transcriptional control. This occurs via modification of virtually all classes of transcriptional actors, which include transcription factors, transcriptional coregulators, diverse chromatin components, as well as Pol I-, Pol II- and Pol III transcriptional machineries and their regulators. For many years, the role of SUMOylation has essentially been studied on individual proteins, or small groups of proteins, principally dealing with Pol II-mediated transcription. This provided only a fragmentary view of how SUMOylation controls transcription. The recent advent of large-scale proteomic, modifomic and genomic studies has however considerably refined our perception of the part played by SUMO in gene expression control. We review here these developments and the new concepts they are at the origin of, together with the limitations of our knowledge. How they illuminate the SUMO-dependent transcriptional mechanisms that have been characterized thus far and how they impact our view of SUMO-dependent chromatin organization are also considered.

The Contribution of Transcriptional Coregulators in the Maintenance of β-cell Function and Identity

Islet β-cell dysfunction that leads to impaired insulin secretion is a principal source of pathology of diabetes. In type 2 diabetes, this breakdown in β-cell health is associated with compromised islet-enriched transcription factor (TF) activity that disrupts gene expression programs essential for cell function and identity. TF activity is modulated by recruited coregulators that govern activation and/or repression of target gene expression, thereby providing a supporting layer of control. To date, more than 350 coregulators have been discovered that coordinate nucleosome rearrangements, modify histones, and physically bridge general transcriptional machinery to recruited TFs; however, relatively few have been attributed to β-cell function. Here, we will describe recent findings on those coregulators with direct roles in maintaining islet β-cell health and identity and discuss how disruption of coregulator activity is associated with diabetes pathogenesis.

Activation of the Crtc2/Creb1 transcriptional network in skeletal muscle enhances weight loss during intermittent fasting

Exercise is a behavior modification indispensable for long-term weight loss. Exercise activates the Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators to drive Creb1-mediated anabolic transcriptional programs in skeletal muscle. Here, we show that induced overexpression of a skeletal muscle specific Crtc2 transgene in aged mice leads to greater weight loss during alternate day fasting, and selective loss of fat rather than lean mass. Transcriptional profiling revealed that fasting and weight loss downregulated most of the mitochondrial electron transport genes and other regulators of mitochondrial function that were substantially reversed in the Crtc2 mice, which maintained higher energy expenditure during fasting. The Crtc2 mice displayed greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity before the fast, suggesting muscle-intrinsic mechanisms in support of improved weight loss. This work reveals that Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb contribute to the effects of exercise on metabolism and weight loss.

Soya isoflavones, genistein and daidzein, induce differential transcriptional modulation in the ovary and testis of zebrafish Danio rerio

Most research into the effects of phytochemical isoflavones has focussed on endocrine disruptions, and especially on oestrogenic imbalances; however, little is known about their effects on other molecular signals such as transcriptional coregulators and choriolytic enzymatic pathways, which are also important in reproductive processes. In male and female zebrafish Danio rerio, the soya isoflavones genistein and daidzein (provided at 10 mg l-1 for 15 d) modulated the basal expression levels of oestrogen receptor transcripts (ERβ) in variable and differential ways. Exposure to genistein resulted in decreased levels of ERβ in the zebrafish ovary; conversely, this isoflavone increased the basal expression levels of the hatching enzyme (HE1) in both gonads. On the other hand, daidzein increased the basal expression levels of the bromodomain testis-specific gene (BRDT) in the male gonad, but not in the ovary. Both isoflavones also differentially modulated (up-down regulations) the basal expression patterns of the 3 molecular signals studied in other regions of the body (e.g. head, digestive system, skeletal musculature). Despite all these transcriptional imbalances, neither of the phytoestrogens modified gonadal histomorphology or the baseline histochemical pattern of proteins, carbohydrates and glycoconjugates distributed in either the vitelline structures or in the developing and maturing germ cells of Danio rerio.

OR14-03 The Transcriptional Coactivation Function of EHMT2 Restricts Chronic Glucocorticoid Exposure Induced Insulin Resistance

Glucocorticoids are required for metabolic adaptations during times of stress. However, chronic glucocorticoid exposure is associated with metabolic disorders such as insulin resistance. Glucocorticoids mainly convey their signals through an intracellular glucocorticoid receptor (GR). GR is a transcription factor that requires interactions with transcriptional coregulators to modulate the transcription of GR primary target genes, which in turn regulate specific aspects of physiology. Euchromatic Histone Methyltransferase 2 (Ehmt2) is a transcriptional coregulator for GR that can act as a corepressor or a coactivator. We found that glucocorticoid-induced insulin resistance was exacerbated when Ehmt2 levels were reduced in the liver. Intriguingly, this phenotype resulted from the transactivation function of Ehmt2. This is because a mutation at the lysine 182 automethylation site, which is required for the coactivation but not the corepression function of Ehmt2, results in similar exacerbated GC-induced insulin resistance. These results suggest that Ehmt2 coactivation dependent GR primary target genes restrict the extent of glucocorticoid-induced insulin resistance. Gene expression analysis identified Dusp4 (a.k.a. Mkp-2) as an Ehmt2 coactivation dependent GR-activated gene, which when overexpressed in liver, attenuated glucocorticoid-induced insulin resistance. Thus, we have identified a novel GR-Ehmt2-Dusp4 axis that plays a key role in controlling the extent of the development of insulin resistance. Notably, the classical view of how GC induce hepatic insulin resistance is that GR activates genes that inhibit insulin signaling and enhance hepatic gluconeogenesis. Our study, however, provides a revolutionary concept in which the extent of GC-induced insulin resistance is controlled by the balance of GR-activated genes that promote insulin sensitivity or insulin resistance.

YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

Blood vessels are integrated into different organ environments with distinct properties and physiology (Augustin and Koh, 2017). A striking example of organ-specific specialization is the bone vasculature where certain molecular signals yield the opposite effect as in other tissues (Glomski et al., 2011; Kusumbe et al., 2014; Ramasamy et al., 2014). Here, we show that the transcriptional coregulators Yap1 and Taz, components of the Hippo pathway, suppress vascular growth in the hypoxic microenvironment of bone, in contrast to their pro-angiogenic role in other organs. Likewise, the kinase Lats2, which limits Yap1/Taz activity, is essential for bone angiogenesis but dispensable in organs with lower levels of hypoxia. With mouse genetics, RNA sequencing, biochemistry, and cell culture experiments, we show that Yap1/Taz constrain hypoxia-inducible factor 1α (HIF1α) target gene expression in vivo and in vitro. We propose that crosstalk between Yap1/Taz and HIF1α controls angiogenesis depending on the level of tissue hypoxia, resulting in organ-specific biological responses.

Sex-Dependent Mechanisms of Glucocorticoid Regulation of the Mouse Hypothalamic Corticotropin-Releasing Hormone Gene

To limit excessive glucocorticoid secretion following hypothalamic-pituitary-adrenal (HPA) axis stimulation, circulating glucocorticoids inhibit corticotropin-releasing hormone (CRH) expression in paraventricular nucleus (PVN) neurons. As HPA function differs between sexes and depends on circulating estradiol (E2) levels in females, we investigated sex/estrous stage-dependent glucocorticoid regulation of PVN Crh. Using NanoString nCounter technology, we first demonstrated that adrenalectomized (ADX’d) diestrous female (low E2), but not male or proestrous female (high E2), mice exhibited a robust decrease in PVN CRH mRNA following 2-day treatment with the glucocorticoid receptor (GR) agonist RU28362. Immunohistochemical analysis of PVN CRH neurons in Crh-IRES-Cre;Ai14 mice, where TdTomato fluorescence permanently tags CRH-expressing neurons, showed similarly abundant co-expression of GR-immunoreactivity in males, diestrous females, and proestrous females. However, we identified sex/estrous stage-related glucocorticoid regulation or expression of GR transcriptional coregulators. Out of 17 coregulator genes examined using nCounter multiplex analysis, mRNAs that were decreased by RU28362 in ADX’d mice in a sex/estrous stage-dependent fashion included: GR (males = diestrous females > proestrous females), signal transducer and activator of transcription 3 (STAT3) (males < diestrous = proestrous), and HDAC1 (males < diestrous > proestrous). Steroid receptor coactivator 3 (SRC-3), nuclear corepressor 1 (NCoR1), heterogeneous nuclear ribonucleoprotein U (hnrnpu), CREB binding protein (CBP) and CREB-regulated transcription coactivator 2 (CRTC2) mRNAs were lower in ADX’d diestrous and proestrous females versus males. Additionally, most PVN CRH neurons co-expressed methylated CpG binding protein 2 (MeCP2)-immunoreactivity in diestrous female and male Crh-IRES-Cre;Ai14 mice. Our findings collectively suggest that GR’s sex-dependent regulation of PVN Crh may depend upon differences in the GR transcriptional machinery and an underlying influence of E2 levels in females.

C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment

Traumatic brain injury (TBI) induces an acute inflammatory response in the central nervous system that involves both resident and peripheral immune cells. The ensuing chronic neuroinflammation causes cell death and tissue damage and may contribute to neurodegeneration. The molecular mechanisms involved in the maintenance of this chronic inflammation state remain underexplored. C-terminal binding protein (CtBP) 1 and 2 are transcriptional coregulators that repress diverse cellular processes. Unexpectedly, we find that the CtBPs can transactivate a common set of proinflammatory genes both in lipopolysaccharide-activated microglia, astrocytes and macrophages, and in a mouse model of the mild form of TBI. We also find that the expression of these genes is markedly enhanced by a single mild injury in both brain and peripheral blood leukocytes in a severity- and time-dependent manner. Moreover, we were able to demonstrate that specific inhibitors of the CtBPs effectively suppress the expression of the CtBP target genes and thus improve neurological outcome in mice receiving single and repeated mild TBIs. This discovery suggests new avenues for therapeutic modulation of the inflammatory response to brain injury.