No abstract

SUMMARY Members of the Sirtuin (SIRT) family of NAD+-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened lifespan and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-κB signaling. SIRT6 interacts with the NF-κB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-κB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy, and enhanced NF-κB-dependent modulation of gene expression, apoptosis and cellular senescence. Computational genomics analyses revealed increased activity of NF-κB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-κB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-κB signaling may contribute to premature and normal aging.

SUMMARY Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here we show that targeted deletion of mouse Hotair lncRNA leads to de-repression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA-seq and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19, without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2 that methylates histone H3 at lysine 27 (H3K27) and Lsd1 complex that demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA to enforce silent chromatin state at Hox and additional genes.

Gene expression signatures encompassing dozens to hundreds of genes have been associated with many important parameters of cancer, but mechanisms of their control are largely unknown. Here we present a method based on genetic linkage that can prospectively identify functional regulators driving large-scale transcriptional signatures in cancer. Using this method we show that the wound response signature, a poor-prognosis expression pattern of 512 genes in breast cancer, is induced by coordinate amplifications of MYC and CSN5 (also known as JAB1 or COPS5). This information enabled experimental recapitulation, functional assessment and mechanistic elucidation of the wound signature in breast epithelial cells.

IntroductionThe present study was aimed to test the role of endothelin-1 (ET-1) as a possible autocrine/paracrine growth factor for cardiac fibroblasts, and to examine its interaction with cardiac natriuretic hormones. Expression of preproET-1 (ppET-1) mRNA by cultured cardiac fibroblasts from neonatal rats was demonstrated by Northern blot analysis using cDNA for rat ppET-1 as a probe. Although cardiomyocytes occupy -75% of the structural space of the heart, they constitute only one third of the total cell population (1, 2). The remaining non-myocytes consist mainly of cardiac fibroblasts in the interstitium. It has been recently shown that growth of cardiac fibroblast associated with enhanced collagen accumulation in the myocardial interstitium is involved in the remodeling process of left ventricular hypertrophy (3, 4). Several recent in vivo studies have suggested that angiotensin II (ANG I) 1 is a critical growth factor for cardiac hypertrophy. Treatment with a subdepressor dose of angiotensin-converting enzyme inhibitors (5) and angiotensin receptor antagonists (6) prevent an increase in left ventricular mass. It has been also reported that chronic infusion of a subpressor dose of ANG II into rats causes left ventricular hypertrophy(6). Since ANG II stimulates cellular proliferation (7) and several extracellular matrix gene expressions in cardiac fibroblasts (8), and induces cardiomyocyte hypertrophy (9, 10), ANG II is considered to act directly on both cardiomyocytes and fibroblasts.We (11)

Sé zary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL

Doravirine is a novel nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1 infection. In vitro studies were conducted to assess the potential for drug interactions with doravirine via major drug-metabolizing enzymes and transporters. Kinetic studies confirmed that cytochrome P450 3A (CYP3A) plays a major role in the metabolism of doravirine, with ϳ20-fold-higher catalytic efficiency for CYP3A4 versus CYP3A5. Doravirine was not a substrate of breast cancer resistance protein (BCRP) and likely not a substrate of organic anion transporting polypeptide 1B1 (OATP1B1) or OATP1B3. Doravirine was not a reversible inhibitor of major CYP enzymes (CYP1A2, -2B6, -2C8, -2C9, -2C19, -2D6, and -3A4) or of UGT1A1, nor was it a time-dependent inhibitor of CYP3A4. No induction of CYP1A2 or -2B6 was observed in cultured human hepatocytes; small increases in CYP3A4 mRNA (Յ20%) were reported at doravirine concentrations of Ն10 M but with no corresponding increase in enzyme activity. In vitro transport studies indicated a low potential for interactions with substrates of BCRP, P-glycoprotein, OATP1B1 and OATP1B3, the bile salt extrusion pump (BSEP), organic anion transporter 1 (OAT1) and OAT3, organic cation transporter 2 (OCT2), and multidrug and toxin extrusion 1 (MATE1) and MATE2K proteins. In summary, these in vitro findings indicate that CYP3A4 and CYP3A5 mediate the metabolism of doravirine, although with different catalytic efficiencies. Clinical trials reported elsewhere confirm that doravirine is subject to drug-drug interactions (DDIs) via CYP3A inhibitors and inducers, but they support the notion that DDIs (either direction) are unlikely via other major drugmetabolizing enzymes and transporters.

The sterol-independent regulatory element (SIRE) of the LDL receptor (LDLR) promoter mediates oncostatin M (OM)-induced transcription of the LDLR gene through a cholesterol-independent pathway. Our prior studies have detected specific associations of the zinc finger transcription factor Egr1 with the SIRE sequence in OM-stimulated HepG2 cells. Because the SIRE motif is composed of a c/EBP binding site and a cAMP response element, both of which are quite divergent from the classical GC-rich Egr1 recognition sequences, we hypothesized that Egr1 may regulate LDLR transcription through interacting with members of the c/EBP and CREB families. Here, we show that treating HepG2 cells with OM specifically leads to prominent increases of the levels of c/EBP␤ and Egr1 bound to the LDLR promoter in vivo. In vitro, the binding of Egr1 to the SIRE sequence is weak, but is strikingly enhanced in the presence of HepG2 nuclear extract. Mammalian two-hybrid assays demonstrate that the N-terminal transactivation domain of Egr1 specifically interacts with c/EBP␤ but not with c/EBP␣ or CREB. The OM treatment further enhances this interaction, resulting in a large increase in the Egr1 transactivating activity. The direct protein to protein contact between Egr1 and c/EBP␤ is also demonstrated by co-immunoprecipitation experiments. Furthermore, we show that a mutation of the phosphorylation motif of c/EBP␤ diminished the OM-stimulated interaction of Egr1 and c/EBP␤. Taken together, we provide strong evidence that Egr1 regulates LDLR transcription via a novel mechanism of protein-protein interaction with c/EBP␤. Regulation of the human LDL receptor (LDLR)1 gene transcription in liver cells involves cholesterol-dependent and -independent mechanisms. The sterol-regulatory element (SRE-1) plays a key role in the cholesterol-mediated negative feedback regulation of the LDLR transcription by interacting with the SRE-1-binding proteins (SREBPs), whose nuclear translocation is controlled by intracellular cholesterol levels (1). The sterol-independent regulatory element (SIRE) has been recently identified and shown to mediate the stimulatory effects of cytokine oncostatin M (OM), cAMP, and c/EBP␤ on LDLR transcription in HepG2 cells in a cholesterol-independent fashion (2). The SIRE motif (TGCTGTAAATGACGTGG) is located at the promoter region of Ϫ17 to Ϫ1, downstream of the SRE-1 and Sp1 binding sites. It is composed of a c/EBP binding site (Ϫ17 to Ϫ9) and a CRE site (Ϫ8 to Ϫ1). Mutations within the SIRE sequence do not affect the cholesterol-mediated suppression and only slightly lower basal promoter activity to levels 60 -80% of the wild-type sequence. However, alterations of nucleotides (even a single base) within the SIRE motif can totally prevent the OM-induced increase in the LDLR promoter activity.In vitro DNA binding assays (EMSA) have detected the binding of several basic leucine zipper DNA-binding proteins including c/EBP␤, CREB, ATF1, ATF2, ATF3, and c-Jun to the SIRE sequence (2). These DNA-protein complexes can be detected in unsti...