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BBA - Gene Regulatory Mechanisms (v.1809, #3)

Editorial Board (pp. i).

Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter by Barbara Renga; Marco Migliorati; Andrea Mencarelli; Sabrina Cipriani; Claudio D'Amore; Eleonora Distrutti; Stefano Fiorucci (pp. 157-165).

Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1- b][1,3]thiazole-5-carbaldehyde- O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.Display Omitted► FXR negatively regulates the expression of MRP4 in vitro and in vivo. ► FXR and CAR exert antagonistic effects at the MRP4 promoter. ► Human MRP4 promoter contains an overlapping binding site for FXR and CAR.

Keywords: Cholestasis; Multidrug resistance associated protein-4; Farnesoid X receptor; Constitutive androstane receptor; Overlapping binding site

Identification of novel alternative splicing variants of interferon regulatory factor 3 by Yong Li; Xiuhua Hu; Yuqin Song; Zheming Lu; Tao Ning; Hong Cai; Yang Ke (pp. 166-175).

Interferon regulatory factor 3 (IRF-3) plays a crucial role in host defense against viral and microbial infection as well as in cell growth regulation. IRF-3a is the only structurally and functionally characterized IRF-3 splicing variant and has been established to antagonize IRF-3 activity. Here, five novel splicing variants of IRF-3, referred to as IRF-3b, -3c, -3d, -3e, and -3f, were identified and shown to be generated by deletion of exons 2, 3, or 6 or some combination thereof. RT-PCR examination revealed that these novel splicing variants were more frequently expressed in human liver, esophagus, and cervical tumor tissues than in their normal counterparts. Additionally, electrophoretic mobility shift assay and subcellular localization showed only IRF-3 and IRF-3e were capable of binding the PRDI/III element of interferon-beta (IFNβ) promoter in vitro and underwent cytoplasm-to-nucleus translocation following Poly(I:C) stimulation. Coimmunoprecipitation assay revealed that only IRF-3c (3f) of novel splicing variants associated with IRF-3 in vivo. Further luciferase assay showed IRF-3c (3f) and IRF-3e failed to transactivate PRDI/III-containing promoter but appeared to inhibit transactivation potential of IRF-3 to varying degrees. Taken together, our findings suggest novel splicing variants may function as negative modulators of IRF-3 and may be correlated with pathogenesis of human tumors.►Novel IRF-3 splicing variants are identified and named as IRF-3b, -3c, -3d, -3e, -3f. ►Novel IRF-3 splicing variants demonstrate obvious cancer-specific expression pattern. ►IRF-3 and -3e are capable of binding with the PRDI/III element of IFNβ promoter. ►IRF-3 and -3e translocate from cytoplasm to nucleus following Poly(I:C) stimulation. ►Novel splicing variants inhibit transactivation potential of IRF-3 to varying degrees.

Keywords: Abbreviations; AIE; autoinhibitory element; DBD; DNA-binding domain; HPV; human papillomavirus; IAD; IRF-associated domain; IFNβ; interferon-beta; IRF-3; interferon regulatory factor 3; ISREs; IFN-stimulated response elements; NES; nuclear export signal; NLS; nuclear localization signal; TAD; transactivation domainIRF-3; IRF-3a; IFNβ; Splicing variant; Cancer

Correlation of dysfunction of nonmuscle myosin IIA with increased induction of Cyp1a1 in Hepa-1 cells by Masayuki Ebina; Masahiko Shibazaki; Kyoko Kudo; Shuya Kasai; Hideaki Kikuchi (pp. 176-183).

The aryl hydrocarbon receptor (AhR) is one of the best known ligand-activated transcription factors and it induces Cyp1a1 transcription by binding with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Recent focus has been on the relationship of AhR with signaling pathways that modulate cell shape and migration. In nonmuscle cells, nonmuscle myosin II is one of the key determinants of cell morphology, but it has not been investigated whether its function is related to Cyp1a1 induction. In this study, we observed that (−)-blebbistatin, which is a specific inhibitor of nonmuscle myosin II, increased the level of CYP1A1-mRNA in Hepa-1 cells. Comparison of (−)-blebbistatin with (+)-blebbistatin, which is an inactive enantiomer, indicated that the increase of CYP1A1-mRNA was due to nonmuscle myosin II inhibition. Subsequent knockdown experiments observed that reduction of nonmuscle myosin IIA, which is only an isoform of nonmuscle myosin II expressed in Hepa-1 cells, was related to the enhancement of TCDD-dependent Cyp1a1 induction. Moreover, chromatin immunoprecipitation assay indicated that the increase of Cyp1a1 induction was the result of transcriptional activation due to increased binding of AhR and RNA polymerase II to the enhancer and proximal promoter regions of Cyp1a1, respectively. These findings provide a new insight into the correlation between the function of nonmuscle myosin II and gene induction.► Cyp1a1 induction is increased by inhibition of NMII with (−)-blebbistatin. ► Among nonmuscle myosin II, NMIIA is only expressed in Hepa-1. ► Knockdown of NMIIA enhances TCDD-induced Cyp1a1 induction. ► Binding of AhR and RNAPII to their target regions is increased by NMIIA dysfunction.

Keywords: Abbreviations; TCDD; 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR; aryl hydrocarbon receptor; XRE; xenobiotic responsive element; NMIIA; nonmuscle myosin heavy chain IIA or MYH9; qPCR; Quantitative real-time PCR; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; LDH; lactase dehydrogenase; RNAPII; RNA polymerase II; ChIP; chromatin immunoprecipitationKeyword; TCDD; AhR; Transcription; NMIIA

Transcription factor Sp1 regulates basal transcription of the human DRG2 gene by Hyo Jeong Kim; Myoung Seok Ko; Hong Kyung Kim; Wha Ja Cho; Seon Ho Lee; Byung Ju Lee; Jeong Woo Park (pp. 184-190).

Developmentally regulated GTP-binding protein 2 (DRG2) is an evolutionarily conserved GTP-binding protein. DRG2 mRNA expression has been confirmed in many animal and human tissues. DRG2 is thought to play an essential role in the control of cell growth and differentiation. However, transcriptional regulation of DRG2 is largely unknown. To investigate the mechanisms controlling DRG2 expression, we cloned 1509bp of the 5′-flanking sequence of this gene. Deletion analysis showed that the region between −113 and −70 is essential for the basal level expression of the DRG2 gene in K562 human erythroleukemic cells. Mutation of a putative stimulating protein 1 (Sp1) regulatory site located at position −108 resulted in a significant decline in DRG2 promoter activity. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that Sp1 binds to this site. Knockdown of Sp1 expression using siRNA inhibited the promoter activation as well as the endogenous DRG2 transcriptional level. Taken together, these results demonstrate that basal expression level of DRG2 is regulated by the Sp1 transcription factor.► In K562 cells, transcription factor Sp1 binds to GC-rich region of DRG2 promoter. ► Downregulation of Sp1 decreases the expression of DRG2. ► Point mutation of the Sp1 binding motif blocks the DRG2 promoter activity. ► Thus, the Sp1 is responsible for the basal expression of DRG2.

Keywords: DRG2; Basal transcription; Promoter; Sp1

Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP by Cote-Velez Antonieta Cote-Vélez; Perez-Maldonado Adrián Pérez-Maldonado; Joel Osuna; Benito Barrera; Jean-Louis Charli; Patricia Joseph-Bravo (pp. 191-199).

Expression of hypophysiotropic TRH, that controls thyroid axis activity, is increased by cold exposure; this effect is mimicked in rat hypothalamic cells incubated with norepinephrine or cAMP analogs. TRH proximal promoter contains three putative CRE: Site-4 or CRE-1 that overlaps an element recognized by thyroid hormone receptors, CRE-2 with adjacent sequences GC box or CACCC recognized by Sp/Krüppel factors (extended CRE-2), and AP-1 sites flanking a GRE_1/2. To evaluate the role of each element in the cAMP response, these sites were mutated or deleted in rat TRH promoter linked to luciferase gene (TRH-luc) and co-transfected with β-gal expression vector in various cell lines; C6 cells gave the highest response to forskolin. Basal activity was most affected by mutations or deletion of CRE-2 site, or CACCC (50–75% of wild type—WT). Forskolin-induced 3× stimulation in WT which decreased 25% with CRE-1 or AP-1 deletions, but 50% when CRE-2 or its 5′ adjacent GC box was altered. SH-SY5Y cells co-transfected with CREB-expression vector increased dB-cAMP response in the wild type but not in the CRE-2 mutated plasmid; cotransfecting CREB-A (a dominant negative expression vector) strongly diminished basal or cAMP response. Primary cultures of hypothalamic cells transfected with plasmids containing deletions of CRE-1, CRE-2, or extended CRE-2 failed to respond to forskolin when CRE-2 was modified. These results corroborate the CRE-2 site as the main cAMP-response element of rat TRH promoter, not exclusive of transcription factors of hypothalamic cells, and stress the relevance of adjacent Sp-1 sites, important mediators of some metabolic hormones.►Forskolin or cAMP stimulates TRH transcription in various cell types. ►Mutations in CRE-1 (Site-4) had little effect on basal or cAMP-induced transcription. ►CRE-2 (TGCCGTCA) of TRH promoter is the most important site for cAMP response. ►Sp/KFL sites adjacent to CRE-2 participate in basal or forskolin-induced activity. ►Transcription was reduced by expression of a dominant negative CREB.

Keywords: Abbreviations; TRH; thyrotropin releasing hormone; GRE; glucocorticoid response element; cAMP; cyclic AMP; Luc; luciferase; Fsk; forskolin; PVN; paraventricular nucleus of the hypothalamus; TPA; 12-O-tetradecanoylphorbol-13-acetate; β-gal; β-galactosidase; WT; wild type; LPS; lipopolysacharideTRH; CRE; cAMP; Sp/KFL; Hypothalamus; Transcription

δEF1 promotes osteolytic metastasis of MDA-MB-231 breast cancer cells by regulating MMP-1 expression by Fen Hu; Chuan Wang; Shaocong Guo; Wei Sun; Dong Mi; Yang Gao; Jie Zhang; Tianhui Zhu; Shuang Yang (pp. 200-210).

Breast cancer metastasis is supposed to involve several stages in which epithelial–mesenchymal transition (EMT) is regarded as the mechanistic basis for the behavior of cancer cells. Our recent studies have implicated that δEF1, a member of the zinc finger-homeodomain transcription factor family, is required for governing both breast cancer EMT and bone remodeling, highlighting a potential role of δEF1 in modulating bone metastasis of breast cancer. In the present study, we further demonstrated that conditioned medium derived from δEF1-overexpressing MDA-MB-231 cells significantly induces osteoclast maturation and concurrently represses osteoblast differentiation and mineralization. On the contrary, conditioned medium derived from δEF1-interfered MDA-MB-231 cells exhibits an opposite effect, thus confirming the effect of δEF to mediate osteolytic metastasis of breast cancer. Furthermore, we found that, during this process, δEF1 remarkably up-regulates matrix metalloproteinase-1 (MMP-1) expression at both mRNA and protein levels in MDA-MB-231 cells. Importantly, the results of luciferase and CHIP assays indicated that δEF1 activates MMP-1 promoter activity through the AP-1 response element. Overexpression of δEF1 in MDA-MB-231 cells significantly increases the recruitment of the AP-1 components, c-Jun/c-Fos, to the endogenous MMP-1 promoter, which in effect could be mediated via the MAPK signaling pathway. In conclusion, these observations suggest a potent role of δEF1 to promote breast cancer metastasis to bone by regulating secretion of growth factors in the tumor microenvironment.► δEF1 promotes cell migration and invasion in MDA-MB-231 breast cancer cells. ► δEF1 expression in MDA-MB-231 facilitates its osteolytic bone metastasis. ► δEF1 modulates MDA-MB-231-mediated bone cell activity by inducing MMP-1 expression. ► δEF1 stimulates the promoter activity of MMP-1 through the MAPK/AP-1 pathway.

Keywords: δEF1; MMP-1; AP-1; Breast cancer; Bone metastasis

Biochemical characterization of enterovirus 71 3D RNA polymerase by Hongbing Jiang; Leiyun Weng; Na Zhang; Minetaro Arita; Renqing Li; Lijuan Chen; Tetsuya Toyoda (pp. 211-219).

An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3D^pol) showed polymerase activity with an Mn²⁺. Little activity was detected with Co²⁺, and no activity was detected with Mg²⁺, Ca²⁺, Cu²⁺, Ni²⁺, Cd²⁺, or Zn²⁺. It is a primer-dependent polymerase, and the enzyme functioned with both di- and 10-nucleotide RNA primers. DNA primer, dT15, increased primer activity, similar to other enterovirus 3D^pol. However, EV71 3D^pol initiated de novo transcription with a poly(C) template and genome RNA. Its RNA binding activity was weak. Terminal nucleotidyl transferase and reverse transcriptase activity were not detected. The Km and Vmax for EV71 3D^pol were calculated from classic Lineweaver–Burk plots. The Km values were 2.35±0.05 (ATP), 5.40±0.93 (CTP), 1.12±0.10 (GTP) and 2.81±0.31 (UTP), and the Vmax values were 0.00078±0.00005/min (ATP), 0.011±0.0017/min (CTP), 0.050±0.0043/min (GTP) and 0.0027±0.0005/min (UTP). The Km of EV71 3D^pol was similar to that of foot and mouth disease virus and rhinovirus. Polymerase activity of BrCr-TR strain and a strain from a clinical isolate in Beijing, 2008 were similar, indicating the potential for 3D^pol as an antiviral drug target.► We analyzed biochemical character of enterovirus (EV) 71 3D RNA polymerase (3D^pol). ► Since 2008 China has suffered from unusual EV71 epidemic. ► 3D^pol of the prototype strain and the Chinese isolate showed the similar activity. ► Our EV71 3D^pol showed polymerase activity only with Mn²⁺. ►Our EV71 3D^pol showed low template RNA binding activity.

Keywords: Abbreviations; 3D; ^pol; 3D polymerase; BJ; Beijing; cre; cis; -acting replication element; CV; coxsackie virus; E. coli; Escherichia coli; EV71; enterovirus 71; FBS; fetal bovine sera; FMDV; foot-and-mouth disease virus; HCV; hepatitis C virus; HFMD; hand foot mouth disease; HRV; human rhino virus; IRES; internal ribosome entry site; MEM; minimal essential media; MuLV; murine leukemia virus; PAGE; polyacrylamide gel electrophoresis; RD; rhabdomyosarcoma; RTase; reverse transcriptase; RdRp; RNA dependent RNA polymerase; SPR; surface plasmon resonance; Tm; melting temperature; knt; kilo-nucleotide; nt; nucleotideEnterovirus 71; 3D protein; RNA polymerase; Transcription; K; m; V; max

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BBA - Gene Regulatory Mechanisms (v.1809, #3)