The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1–50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.
Maria K Tsoumpra, Shun Sawatsubashi, Michihiro Imamura, Seiji Fukumoto, Shin’ichi Takeda, Toshio Matsumoto and Yoshitsugu Aoki
Ke-Hung Tsui, Li-Chuan Chung, Shyi-Wu Wang, Tsui-Hsia Feng, Phei-Lang Chang and Horng-Heng Juang
Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1α was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with H IF-1α expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at −1013 to −842 upstream of the translation initiation site. l-mimosine, an iron chelator, stabilized HIF-1α but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells.
SM Woodall, NS Bassett, PD Gluckman and BH Breier
The mechanisms that contribute to postnatal growth failure following intrauterine growth retardation (IUGR) are poorly understood. We demonstrated previously that nutritional deprivation in the pregnant rat leads to IUGR in offspring, postnatal growth failure and to changes in endocrine parameters of the somatotrophic axis. The present study examines the effects of maternal undernutrition (30% of the ad libitum available diet; IUGR group) throughout pregnancy on hepatic insulin-like growth factor-I (IGF-I), growth hormone receptor (GHR) and GH-binding protein (GHBP) gene expression using solution hybridisation/RNase protection assays (RPAs). Animals were killed at fetal (E22, term=23 days) and postnatal (birth, days 5, 9, 15, 21) ages, livers were collected and RNA extracted for RPAs. Results demonstrate the presence of all IGF-I mRNAs resulting from transcription start sites (ss) in exon 1 (ss1/2, ss3, ss2 spliced), exon 2, the two IGF-I E-domain variants (Ea and Eb) as well as GHR and GHBP mRNAs in hepatic tissue at E22 in both the ad libitum fed and IUGR offspring. In the postnatal liver, IGF-I ss1/2, ss3, ss2 spliced, Ea and Eb IGF-I variants as well as GHR and GHBP mRNA transcripts increased in abundance from birth to day 21. IGF-I exon 2 transcripts were relatively constant from E22 until postnatal day 15, then increased at postnatal day 21 in both the ad libitum fed and IUGR offspring. The expressions of all hepatic IGF-I leader exon ss and Ea domain variants were significantly reduced in IUGR offspring (P<0.05) from E22 to postnatal day 9. In contrast, relative abundance of hepatic IGF-I Eb variants, GHR and GHBP mRNAs were unaltered in IUGR offspring compared with the ad libitum fed animals. Whether these postnatal effects of undernutrition are a direct consequence of IUGR or whether they are related, in part, to differences in postnatal food intake remains to be investigated. In summary, we have demonstrated that hepatic IGF-I ss within exon 1 and exon 2 are coordinately regulated. Use of exon 1 ss increased during normal development and decreased with IUGR without changes in GHR or GHBP gene expression. Eb transcripts, thought to represent GH-dependent endocrine regulation of IGF-I, were unchanged in IUGR. These results suggest a possible postreceptor defect in GH action as a consequence of IUGR.
Nicola J Smith and Tim R Fenton
The interaction between human papillomaviruses (HPV) and the apolipoprotein-B mRNA-editing catalytic polypeptide-like (APOBEC)3 (A3) genes has garnered increasing attention in recent years, with considerable efforts focused on understanding their apparent roles in both viral editing and in HPV-driven carcinogenesis. Here, we review these developments and highlight several outstanding questions in the field. We consider whether editing of the virus and mutagenesis of the host are linked or whether both are essentially separate events, coincidentally mediated by a common or distinct A3 enzymes. We discuss the viral mechanisms and cellular signalling pathways implicated in A3 induction in virally infected cells and examine which of the A3 enzymes might play the major role in HPV-associated carcinogenesis and in the development of therapeutic resistance. We consider the parallels between A3 induction in HPV-infected cells and what might be causing aberrant A3 activity in HPV-independent cancers such as those arising in the bladder, lung and breast. Finally, we discuss the implications of ongoing A3 activity in tumours under treatment and the therapeutic opportunities that this may present.
Fraydoon Rastinejad, Pengxiang Huang, Vikas Chandra and Sepideh Khorasanizadeh
Nuclear receptors (NRs) are a major transcription factor family whose members selectively bind small-molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were focused exclusively on the individual ligand-binding domains (LBDs) or DNA-binding domains of NRs. These analyses revealed the basis for both ligand and DNA binding and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the peroxisome proliferator-activated receptor-γ–retinoid X receptor α (PPARγ–RXRα) heterodimer and hepatocyte nuclear factor (HNF)-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain–domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts.
Alexander Dierks, Urs D Lichtenauer, Simone Sackmann, Ariadni Spyroglou, Igor Shapiro, Marcel Geyer, Jenny Manonopoulou, Martin Reincke, Constanze Hantel and Felix Beuschlein
potassium-induced gene regulation In the first instance, we investigated long-term effects of potassium supplementation on murine adrenal morphology and transcriptional regulation of aldosterone synthase in vivo . Despite the fact that no significant
A J Tyson-Capper, E A Shiells and S C Robson
polyadenylation and associates directly with RNA polyadenylation elements within the PR-regulated gene, COX-2, suggesting additional roles in post-transcriptional gene regulation ( Hall-Pogar et al . 2007 ). Therefore, in this study, we investigated whether PSF
’Malley 2010 , Nautiyal et al . 2013 a , Dasgupta et al . 2014 , Stashi et al . 2014 ). These proteins are essential for gene regulation, by regulating functions of transcription factors including all members of the nuclear receptor superfamily in the
Aurimas Vinckevicius and Debabrata Chakravarti
some of the earlier methods that were instrumental in our current understanding of hormone action. We discuss several techniques for investigating protein–DNA interactions that fueled early research of gene regulation and explore the advancements that
Anne Wulf, Marianne G Wetzel, Maxim Kebenko, Meike Kröger, Angelika Harneit, Jennifer Merz and Joachim M Weitzel
that TR is able to bind and activate the mGPDH promoter but failed to activate the TSH promoter under identical experimental conditions. Of note, the investigated promoter fragment is identical to those showing a negative gene regulation in Fig. 1C and