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BBA - Gene Regulatory Mechanisms (v.1819, #11-12)

Editorial Board (pp. i).

The let-7 microRNA enhances heme oxygenase-1 by suppressing Bach1 and attenuates oxidant injury in human hepatocytes by Weihong Hou; Qing Tian; Nury M. Steuerwald; Laura W. Schrum; Herbert L. Bonkovsky (pp. 1113-1122).

The let-7 microRNA (miRNA) plays important roles in human liver development and diseases such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein oxidative stress accelerates the progression of these diseases. To date, the role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7 miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene, and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR, Western blot, and luciferase reporter assays. Dual luciferase reporter assays revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1 3′-untranslated region (3′-UTR)-dependent luciferase activity but not mutant Bach1 3′-UTR-dependent luciferase activity, whereas mutant let-7 miRNA containing base complementarity with mutant Bach1 3′-UTR restored its effect on mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein levels by 50–70%, and subsequently up-regulated HMOX1 gene expression by 3–4 fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts on the 3′-UTR of Bach1 and negatively regulates expression of this protein, and thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA family members may represent a novel approach to protecting human hepatocytes from oxidant injury.► let-7 miRNA directly binds to the 3’-UTR of Bach1 and negatively regulates Bach1 gene expression. ► let-7 miRNA thereby up-regulates the HMOX1 gene expression. ► Over-expression of let-7 miRNA attenuates oxidative injury in human hepatocytes.

Keywords: Abbreviations; BCA; bicinchoninic acid; bZip; basic leucine zipper; CLIP; cross-linking immunoprecipitation; CO; carbon monoxide; DMEM; Dulbecco's modified Eagle medium; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HITS; high-throughput sequencing; HMOX1; heme oxygenase1; MARE; Maf recognition element; miRNA; microRNA; MINC; microRNA inhibitor negative control; MMNC; microRNA mimic negative control; NQO1; NAD(P)H:quinone oxidoreductase; nt; nucleotide(s); PAGE; polyacrylamide gel electrophoresis; PVDF; polyvinylidene fluoride; qRT-PCR; quantitative reverse transcription-polymerase chain reaction; ROS; reactive oxygen species; SDS; sodium dodecyl sulfate; tBuOOH; tert-butyl hydroperoxide; UTR; untranslated region; WB; Western blot let-7; Heme oxygenase1; Bach1; MicroRNA; Oxidative stress; Huh-7 cell

Regulation of mGluR1 expression in human melanocytes and melanoma cells by Hwa Jin Lee; Brian A. Wall; Janet Wangari-Talbot; Suzie Chen (pp. 1123-1131).

We demonstrated that ectopic expression of metabotropic glutamate receptor 1 (mGluR1/Grm1) in mouse melanocytes was sufficient to induce melanoma development in vivo with 100% penetrance. We also showed that about 60% of human melanoma biopsies and cell lines, but not benign nevi or normal human melanocytes expressed mGluR1, suggesting that GRM1 may be involved in melanomagenesis. mGluR1 is expressed primarily in neurons. In various non-neuronal cells, mGluR1 expression is regulated via binding of Neuron-Restrictive-Silencer-Factor (NRSF) to a Neuron-Restrictive-Silencer-Element (NRSE). Here, we report on the possibility that aberrant mGluR1 expression in melanoma is due to alterations in NRSF and/or NRSE. We show that in human melanocytes, binding of NRSF to NRSE in the GRM1 promoter region is necessary for the suppression of mGluR1 expression. We also show that inhibiting the expression of the transcription factor Sp1 or interference with its ability to bind DNA can result in increased mGluR1 expression perhaps via its function as a negative regulator. In addition, we also provide evidence that demethylation within the promoter region of GRM1 may also be a mechanism for the derepression of mGluR1 expression in melanocytes that progress to cell transformation and tumor formation.► Binding of NRSF to NRSE at 5′ region of GRM1 ► Negative regulation by Sp1 ► Demethylation at the GRM1 promoter

Keywords: Melanoma; mGluR1; NRSF/NRSE; Sp1; Methylation

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells by Soichiro Iwaki; Shuhei Yamamura; Moyoko Asai; Burton E. Sobel; Satoshi Fujii (pp. 1132-1141).

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3′-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3′ terminus (+2177 to 3176nt) of the 3′-UTR of the 3.2kb PAI-1 mRNA [3′-UTR (+2177–3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3′-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3′-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.► We report S1P regulates PAI-1, a major fibrinolysis inhibitor, in liver cell line. ► S1P regulates PAI-1 expression at transcriptional and posttranscriptional levels. ► Regulation at posttranscriptional level involves the RNA binding protein SERBP1. ► Posttranscriptional PAI-1 diminution may serve as a defense against thrombosis.

Keywords: Abbreviations; PAI-1; plasminogen activator inhibitor type-1; S1P; sphingosine 1-phosphate; UTR; untranslated region; ARE; AU-rich element; ORF; open reading framePlasminogen activator inhibitor-1; Posttranscriptional regulation; RNA binding protein; S1P

The bZIP repressor proteins, c-Jun dimerization protein 2 and activating transcription factor 3, recruit multiple HDAC members to the ATF3 promoter by Ilona Darlyuk-Saadon; Keren Weidenfeld-Baranboim; Kazunari K. Yokoyama; Tsonwin Hai; Ami Aronheim (pp. 1142-1153).

JDP2, is a basic leucine zipper (bZIP) protein displaying a high degree of homology with the stress inducible transcription factor, ATF3. Both proteins bind to cAMP and TPA response elements and repress transcription by multiple mechanisms. Histone deacetylases (HDACs) play a key role in gene inactivation by deacetylating lysine residues on histones. Here we describe the association of JDP2 and ATF3 with HDACs 1, 2–6 and 10. Association of HDAC3 and HDAC6 with JDP2 and ATF3 occurs via direct protein–protein interactions. Only part of the N-terminal bZIP motif of JDP2 and ATF3 basic domain is necessary and sufficient for the interaction with HDACs in a manner that is independent of coiled-coil dimerization. Class I HDACs associate with the bZIP repressors via the DAC conserved domain whereas the Class IIb HDAC6 associates through its C-terminal unique binder of ubiquitin Zn finger domain. Both JDP2 and ATF3 are known to bind and repress the ATF3 promoter. MEF cells treated with histone deacetylase inhibitor, trichostatin A (TSA) display enhanced ATF3 transcription. ATF3 enhanced transcription is significantly reduced in MEF cells lacking both ATF3 and JDP2. Collectively, we propose that the recruitment of multiple HDAC members to JDP2 and ATF3 is part of their transcription repression mechanism.► The bZIP repressors, JDP2 and ATF3, associate with Class I and II HDAC members. ► bZIP repressors' association with HDAC is direct and independent of a functional leucine zipper. ► Class I HDACs associate with the bZIP repressors via their DAC domain. ► Class II HDAC association with the bZIP repressors is DAC domain independent. ► The ATF3 promoter is subjected to HDAC regulation which is partially bZIP repressors dependent.

Keywords: Abbreviations; ATF3; Activating transcription factor 3; bZIP; Basic leucine zipper; HDAC; Histone deacetylase; HEK-293T; Human embryonic kidney 293T; JDP2; c-Jun dimerization protein 2; qRT-PCR; quantitative real time polymerase chain reaction; TSA; Trichostatin AHDAC; bZIP; JDP2; ATF3; Histone acetylation; Repressor

Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells by Nina Pettersen Hessvik; Santosh Phuyal; Andreas Brech; Kirsten Sandvig; Alicia Llorente (pp. 1154-1163).

Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes.► RWPE-1 cells secrete less exosomal RNA than PC-3 cells. ► Exosomes from PC-3 cells contain miRNAs. ► The profile of exosomal miRNA resembles the profile of the parent cells. ► Some miRNAs are selected into exosomes.

Keywords: Abbreviations; MVBs; multivesicular bodies; miRNA; microRNA; snRNA; small nuclear RNA; FC; fold change; LNA; locked nucleic acidBiomarker; Exosome; Microvesicle; miRNA; Prostate cancer; Prostasome

Suspected leukemia oncoproteins CREB1 and LYL1 regulate Op18/STMN1 expression by Serban San-Marina; YouQi Han; Jian Liu; Mark D. Minden (pp. 1164-1172).

Stathmin (STMN1) is a microtubule destabilizing protein with a key role in cell cycle progression and cell migration that is up-regulated in several cancers and may contribute to the malignant phenotype. However, the factors that regulate its expression are not well understood. Loss as well as gain-of-function p53 mutations up-regulate STMN1 and in acute myelogenous leukemia where p53 is predominantly wild‐type, STMN1 is also over-expressed. Here we show regulatory control of STMN1 expression by the leucine zipper transcription factor (TF) CREB1 and the basic helix–loop–helix TF LYL1. By ChIP-chip experiments we demonstrate in vivo the presence of LYL1 and CREB1 in close proximity on the STMN1 promoter and using promoter assays we reveal co-regulation of STMN1 by CREB1 and LYL1. By contrast, TAL1, another suspected oncoprotein in leukemia and close relative of LYL1, exerts no regulatory effect on the STMN1 promoter. NLI, LMO2 and GATA2 are previously described co-activators of Tal1/Lyl1-E47 transcriptional complexes and potentiate Lyl1 activation of the STMN1 promoter while having no effect on TAL1 transactivation. Promoter mutations that abrogate CREB1 proximal binding or mutations of the DNA-binding domain of CREB1 abolish LYL1 transcriptional activation. These results show that CRE and Ebox sites function as coordinated units and support previous evidence of joint CREB1-and LYL1 transcription events activating an aberrant subset of promoters in leukemia. CREB1 or LYL1 shRNA knock-down down-regulate STMN1 expression. Because down-regulation of STMN1 has been shown to have anti-proliferative effects, while CREB1 and LYL1 are suspected oncoproteins, interference with CREB1–LYL1 interactions may complement standard chemotherapy and yield additional beneficial effects.► AML microarrays show co-regulation of Op18/Stmn1 by Creb1/Lyl1 but not Tal1/p53.. ► Creb1-Lyl1 interact on the Op18/Stmn1 promoter. ► Adjacent Ebox and Cre sites are required in the minimal Op18/Stmn1 promoter. ► Cre site or Creb1 mutations abrogate Op18/Stmn1 regulation by Lyl1. ► shRNA-mediated Creb1 or Lyl1 knockdown downregulate Op18/Stmn1 expression.

Keywords: Transcription factor; AML; Lyl1; Creb1; Stmn1/Op18; Creb1–Lyl1 interaction

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells by Emanuele Valli; Stefania Trazzi; Claudia Fuchs; Daniela Erriquez; Renata Bartesaghi; Giovanni Perini; Elisabetta Ciani (pp. 1173-1185).

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G₀/G₁ phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5.► CDKL5 enhances neuronal differentiation. ► CDKL5 arrests cell cycle of neuronal precursor cells. ► MYCN directly represses transcription of CDKL5. ► These results may help explain the neurological symptoms of RTT patients.

Keywords: Rett's syndrome; CDKL5; MYCN; Neurogenesis; Differentiation

Plasmodium falciparum Prp16 homologue and its role in splicing by Prashant Kumar Singh; Shivani Kanodia; Chethan Jambanna Dandin; Usha Vijayraghavan; Pawan Malhotra (pp. 1186-1199).

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N‐terminal domain (1–80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature‐sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes.Display Omitted► mRNA splicing and splicing factors are not well understood in P. Falciparum. ► Biochemical and functional characterization of an important splicing factor, PfPrp16. ► PfPrp16 acts at second step of splicing. ► Biochemical properties of PfPrp16 may be modulated by its C-terminal region. ► Study will facilitate understanding of RNA splicing machinery in P. Falciparum.

Keywords: Malaria; Parasite; Helicase; Prp16; Splicing

Histone demethylase JMJD2B-mediated cell proliferation regulated by hypoxia and radiation in gastric cancer cell by Joong-Gook Kim; Joo Mi Yi; Seong-Joon Park; Joong-Sun Kim; Tae Gen Son; Kwangmo Yang; Mi-Ae Yoo; Kyu Heo (pp. 1200-1207).

Histone modifying factors are functional components of chromatin and play a role in gene regulation. The expression level of JMJD2B, a histone demethylase, is notably up-regulated in cancer tissues. Upregulation of JMJD2B promotes cancer cell proliferation under hypoxic conditions through target gene expression. Here, we describe the patterns of histone methylation and JMJD2B expression under various stressed conditions, such as hypoxia and radiation, in a gastric cancer cell line. JMJD2B expression in AGS cells was actively regulated by hypoxia and radiation. Chromatin immunoprecipitation experiments demonstrated that binding of JMJD2B on the cyclin A1 (CCNA1) promoter resulted in CCNA1 upregulation under hypoxic conditions. Furthermore, we confirmed that AGS cell proliferation was directly affected by JMJD2B and CCNA1 expression by performing experiments with JMJD2B depleted cells. Interestingly, the effects of JMJD2B on cell growth under hypoxia were remarkably repressed after gamma-ray irradiation. These results suggest that JMJD2B may play a central role in gastric cancer cell growth and might constitute a novel therapeutic target to overcome hypoxia-induced radio-resistance, thereby improving the efficiency of radiation therapy.► We described the patterns of histone methylations under hypoxia and radiation in gastric cancer. ► The induced protein expression of JMJD2B through hypoxia was notably diminished after gamma-ray irradiation ► JMJD2B expression modulated by hypoxia and radiation regulated AGS cell proliferation through CCNA1 regulation. ► JMJD2B depletion caused the decrease of AGS proliferation under hypoxia

Keywords: JMJD2B; Hypoxia; Gamma-ray irradiation; AGS cell; CCNA1; Histone methylation

αNAC interacts with histone deacetylase corepressors to control Myogenin and Osteocalcin gene expression by Toghrul Jafarov; James W.M. Alexander; René St-Arnaud (pp. 1208-1216).

In the nucleus of differentiated osteoblasts, the DNA-binding αNAC protein acts as a transcriptional coactivator of the Osteocalcin gene. Chromatin immunoprecipitation-microarray assays (ChIP-chip) showed that αNAC binds the Osteocalcin promoter but also identified the Myogenin promoter as an αNAC target. Here, we confirm these array data using quantitative ChIP and further detected that αNAC binds to these promoters in myoblasts. Since these genes are differentially regulated during osteoblastogenesis or myogenesis, these results suggest cell- and promoter-context specific functions for αNAC. We hypothesized that αNAC dynamically recruits corepressors to inhibit Myogenin expression in cells committing to the osteoblastic lineage or to inhibit Osteocalcin transcription in differentiating myoblasts. Using co-immunoprecipitation assays, we detected complexes between αNAC and the corepressors HDAC1 and HDAC3, in myoblasts and osteoblasts. Sequential ChIP confirmed HDAC1 recruitment by αNAC at the Osteocalcin and Myogenin promoters. Interaction with the corepressors was detectable in pre-osteoblasts and in myoblasts but disappeared as the cells differentiate. Treatment with an HDAC inhibitor caused de-repression of Osteocalcin expression in myoblasts. Overexpression of αNAC in myoblasts inhibits expression of Myogenin and differentiation. However, overexpression of an N-terminus truncated αNAC mutant allowed myoblasts to express Myogenin and differentiate, and this mutant did not interact with HDAC1 or HDAC3. This study identified an additional DNA-binding target and novel protein–protein interactions for αNAC. We propose that αNAC plays a role in regulating gene transcription during mesenchymal cell differentiation by differentially recruiting corepressors at target promoters. ► Myogenin was identified as a novel αNAC target gene. ► αNAC interacts with the corepressors HDAC1 and HDAC3. ► αNAC recruits HDAC1/3 to the promoter of silent, but not expressed, target genes. ► A mutant αNAC not interacting with HDACs cannot inhibit Myogenin or myogenesis. ► We propose new roles for αNAC in transcriptional control during differentiation.

Keywords: Abbreviations; αNAC; nascent polypeptide associated complex And Coactivator alpha; HDAC; histone deacetylase; ChIP; chromatin immunoprecipitation; RT-qPCR; reverse transcription-quantitative polymerase chain reaction; NaBu; sodium butyrateαNAC; Histone deacetylase; Osteoblastogenesis; Myogenesis; Osteocalcin; Myogenin

Long distance relationships: Enhancer–promoter communication and dynamic gene transcription by Judith Marsman; Julia A. Horsfield (pp. 1217-1227).

The three-dimensional regulation of gene transcription involves loop formation between enhancer and promoter elements, controlling spatiotemporal gene expression in multicellular organisms. Enhancers are usually located in non-coding DNA and can activate gene transcription by recruiting transcription factors, chromatin remodeling factors and RNA Polymerase II. Research over the last few years has revealed that enhancers have tell-tale characteristics that facilitate their detection by several approaches, although the hallmarks of enhancers are not always uniform. Enhancers likely play an important role in the activation of genes by functioning as a primary point of contact for transcriptional activators, and by making physical contact with gene promoters often by means of a chromatin loop. Although numerous transcriptional regulators participate in the formation of chromatin loops that bring enhancers into proximity with promoters, the mechanism(s) of enhancer–promoter connectivity remain enigmatic. Here we discuss enhancer function, review some of the many proteins shown to be involved in establishing enhancer–promoter loops, and describe the dynamics of enhancer–promoter contacts during development, differentiation and in specific cell types.► Enhancers influence tissue-specific gene expression by interacting with promoters. ► Developmentally important genes can be prepatterned or poised for activation. ► CTCF, cohesin and various transcription factors establish and alter chromatin loops.

Keywords: Enhancer; Chromatin looping; Cohesin; CTCF; Gene transcription

Corrigendum to “The dual lives of bidirectional promoters” [Biochim. Biophys. Acta. 1819 (2012) 688–693] by Clay Wakano; Jung S. Byun; Li-Jun Di; Kevin Gardner (pp. 1228-1229).

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BBA - Gene Regulatory Mechanisms (v.1819, #11-12)