Inr Element Research Articles

Molecular biology of the cell

Molecular biology of the cell

A reciprocal feedback of Myc and lncRNA MTSS1-AS contributes to extracellular acidity-promoted metastasis of pancreatic cancer.

Rationale: Previous studies have reported on the role of extracellular acidity in the metastasis of numerous cancers. However, the involvement of long noncoding RNA (lncRNA) in the extracellular acidity-induced cancer metastasis of pancreatic cancer (PC) remains unclear.Methods: Different expression levels of lncRNAs in PC cells under normal and acidic conditions were compared by RNA sequencing (RNA-seq). The effects of antisense lncRNA of metastasis suppressor 1 (MTSS1-AS) on acidic PC cells were assessed by gain- and loss-of-function experiments. Fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down, Western blot, luciferase reporter, and Chromatin immunoprecipitation assays were employed to determine the regulatory mechanisms of MTSS1-AS in the acidity-induced metastasis of PC cells. The expression of MTSS1-AS and associated pathways were compared in PC samples and peritumoral normal tissues.Results: RNA-seq demonstrated that MTSS1-AS was significantly downregulated in pancreatic cells cultured with the acidic medium. The overexpression of MTSS1-AS remarkably inhibited the acidity-promoted metastasis of PC cells by upregulating the expression of its sense gene metastasis suppressor 1 (MTSS1). Mechanistically, MTSS1-AS scaffolded the interaction between E3 ubiquitin-protein ligase STIP1 homology and U-box containing protein 1 (STUB1) and transcription regulator myeloid zinc finger 1 (MZF1), leading to ubiquitination-mediated degradation of MZF1. Further, MZF1 inhibited the expression of MTSS1 by binding to the MTSS1 promoter. Thus, the acidity-reduced MTSS1-AS facilitated the stability of MZF1 and its inhibitory effect on MTSS1 transcription, thereby promoting the metastasis of PC cells under acidic conditions. Moreover, MTSS1-AS was transcriptionally repressed by the binding of MYC proto-oncogene (Myc) with initiator (Inr) elements of the MTSS1-AS promoter. Meanwhile, MTSS1-AS mutually repressed the expression of Myc by impairing the MZF1-mediated transcription activation of Myc, thereby forming a negative feedback loop between MTSS1-AS and Myc in acidic PC cells. In accordance with the experimental results, MTSS1-AS and MTSS1 were downregulated in PC and correlated with poor overall survival.Conclusions: The results implicated that a reciprocal feedback loop between Myc and MTSS1-AS contributed to the extracellular acidity-promoted metastasis of PC, and indicated that MTSS1-AS was a valuable biomarker and therapeutic target for PC.

An in vitro characterisation of the Trichomonas vaginalis TATA box-binding proteins (TBPs).

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

Control of human gene expression: High abundance of divergent transcription in genes containing both INR and BRE elements in the core promoter.

BackgroundDNA sequence elements in the core promoter can play a central role in regulation of gene expression. Core elements (e.g. INR and TATA box) are located within ~50bp of the transcription start site and both upstream and downstream elements are known. Although all can affect the level of gene expression, their mechanism of action has yet to be fully defined. The studies described here are focused on two core promoter elements, INR and BRE, in the human genome. The locations of the two elements were determined in a large number of human promoters and the results were interpreted in terms of overall promoter function.ResultsA total of 13,406 promoters were collected from the reference version of the human genome and found to contain 62,891 INR sequences and 32,290 BRE. An INR sequence was found in the core region of 1231 (9.2%) promoters and a BRE in 2592 (19.3%); 121 promoters (0.9%) have both INR and BRE elements. Counts support the view that most human promoters lack an INR or BRE element in the core promoter. Further analysis was carried out with the aligned aggregate of promoters from each chromosome. The results showed distinct INR distributions in separate chromosome groups indicating a degree of chromosome specificity to the way core promoter elements are deployed in the genome. The rare promoters with both INR and BRE elements were found to be enriched among the genes with divergent transcription. Enrichment raises the possibility that core promoter elements can have a function in chromosome organization as well as in initiation of transcription.

Three newly identified Immediate Early Genes of Bovine herpesvirus 1 lack the characteristic Octamer binding motif- 1

Only three immediate early genes (IE) BICP0, BICP4 and BICP22 of Bovine herpesvirus 1 (BoHV-1) are known. These genes are expressed coordinately and their promoters are well characterized. We provide evidence for expression of three additional IE genes of BoHV-1 i.e. UL21, UL33 and UL34. These genes are expressed in the presence of cycloheximide (CH) at the same time as known IE genes. Surprisingly, the promoters of newly identified IE genes (UL21, UL33, UL34) lack the OCT-1 binding site, a considered site of transactivation of the BoHV-1 IE genes. The other difference in the promoters of the newly identified IE genes is the presence of TATA box at near optimal site. However, all the IE genes have similar spatial placements of C/EBPα, DPE and INR elements.

Differential context-specific impact of individual core promoter elements on transcriptional dynamics.

Eukaryotic transcription occurs in bursts that vary in size and frequency, but the contribution of individual core promoter elements to transcriptional bursting is not known. Here we analyze the relative contributions to bursting of the individual core promoter elements-CCAAT, TATAA-like, Sp1BS, and Inr-of an MHC class I gene in primary B-cells during both basal and activated transcription. The TATAA-like, Sp1BS, and Inr elements all function as negative regulators of transcription, and each was found to contribute differentially to the overall bursting pattern of the promoter during basal transcription. Whereas the Sp1BS element regulates burst size, the Inr element regulates burst frequency. The TATAA-like element contributes to both. Surprisingly, each element has a distinct role in bursting during transcriptional activation by γ-interferon. The CCAAT element does not contribute significantly to the constitutive transcriptional dynamics of primary B-cells, but modulates both burst size and frequency in response to γ-interferon activation. The ability of core promoter elements to modulate transcriptional bursting individually allows combinatorial fine-tuning of the level of MHC class I gene expression in response to intrinsic and extrinsic signals.

Distinct Role of Core Promoter Architecture in Regulation of Light-Mediated Responses in Plant Genes

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less (initiator-containing) promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr (Initiator) elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS (transcription start site), and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Identification of the minimal promoter for specific expression of the GABAρ1 receptor in retinal bipolar cells

γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter

BackgroundDLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized.ResultsThe goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression.ConclusionsOur results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.

A Novel E2F-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1-3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1-3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1-3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts.

A new bidirectional promoter from the human genome

Both human and other mammalian genomes contain a number of closely linked gene pairs transcribed in opposite directions. Bioinformatic analysis suggests that up to 10% of human genes are arranged in this way. This work reports cloning of a human genome fragment that separates two head-to-head oriented genes located at 2p13.1 and encoding hypothetical proteins with unknown functions: CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. The intergenic region CCDC142-TTC31 overlaps with a CpG island and contains a number of potential binding sites for transcription factors. This fragment functions as a bidirectional promoter in the system of luciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. Vectors containing oppositely oriented genes of two fluorescent proteins: green (EGFP) and red (DsRed2), separated by a fragment of the CCDC142-TTC31 intergenic region, were constructed. Transfection of HEK293 cells with these vectors resulted in simultaneous expression of both fluorescent proteins. The promoter activity was also determined for truncated versions of the intergenic region. The minimal promoter fragment contained Inr, BRE, and DPE elements characteristic for TATA-less promoters. Thus, a novel bidirectional promoter was cloned from the human genome; it can be used for simultaneous constitutive expression of two different genes in human cells.

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Myc interacts with Max and Miz1 to repress C/EBPδ promoter activity and gene expression

Background"Loss of function" alterations in CCAAT/Enhancer Binding Proteinδ (C/EBPδ) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPδ gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPδ exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing.We previously reported that C/EBPδ expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPα, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPδ expression.ResultsMyc represses C/EBPδ promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPδ promoter in proliferating cells, when C/EBPδ expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPδ promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPδ promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPδ promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPδ promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPδ expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPδ promoter activity.ConclusionMyc represses C/EBPδ expression by associating with the C/EBPδ proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPδ promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPδ, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.

Core promoters of the penicillin biosynthesis genes and quantitative RT-PCR analysis of these genes in high and low production strain of Penicillium chrysogenum

The transcription start points of the penicillin biosynthesis genes from Penicillium chrysogenum were mapped using the primer extension method. For each of the three genes consensus sequences of the core promoter elements were identified, supporting the notion that the basal transcription of these genes is mediated separately. Interestingly, transcription start of the pcbC gene is located within the potential Inr element with no TATA box-like sequence being found at expected position. This is in contrast to pcbAB and penDE genes with proposed TATA boxes or even to Aspergillus nidulans ipnA (pcbC) gene indicating possible differences in basal transcription regulation. Using the quantitative RT-PCR analysis the expression of all three biosynthesis genes was monitored in both the high and low production strain of P. chrysogenum during a 3-d cultivation under production conditions. The differences were found between the strains in time regulation and transcript levels of the biosynthesis genes. Furthermore, we showed that the effect of higher gene dosage on productivity in the production strain is amplified by more efficient transcription of the biosynthesis genes with the RNA levels approximately 37- and 12-times higher, respectively, than in a low production strain.

Delimitation and functional characterization of the bidirectional THOX–DUOXA promoter regions in thyrocytes

The THOX and DUOXA genes encode components of the oxidative machinery involved in thyroid hormone biosynthesis. Both of these genes are duplicated in mammalian genomes and are positioned in a head-to-head configuration, THOX1 facing DUOXA1 and THOX2 facing DUOXA2, respectively. The intergenic regions in both couples of genes exhibit dissimilar compositions, being highly GC-rich in the case of THOX1–DUOXA1 but not in the other case. In this study we localized precisely the transcription starts of all four genes using the RLM-RACE technique. It revealed that the distance between THOX1 and DUOXA1 transcription units is of about 70 bp only, whereas THOX2 and DUOXA2 transcription starts are separated by 170 bp. Analysis of these putative promoter regions revealed the presence of several potential binding sites for transcription factor Sp1 within the THOX1–DUOXA1 intergenic space, and of a TATA box and an Inr element in front of DUOXA2 and THOX2 genes, respectively. The putative promoter regions were inserted into a specifically designed vector harbouring two distinct reporter genes facing each other and their activity was investigated in transient transfection experiments in rat thyroid PCCl3 cells. Both regions exhibited bidirectional promoter activity in the assay. Gel shift experiments using extracts obtained from PCCl3 cells demonstrated the existence of at least one functional Sp1 binding site within the THOX1–DUOXA1 promoter. When Sp1 binding was abolished by mutation of the DNA sequence, a clear reduction in promoter activity in both THOX1 and DUOXA1 directions was observed in the functional assay. As these promoter sequences are well conserved in mammalian genomes, it appears very likely that the results we obtained here in the rat may be extended to the other species.

Promoter activity 5′ of Dβ2 is coordinated by E47, Runx1, and GATA-3

V(D)J recombination involves the stepwise assembly of B and T cell receptor genes as lymphocytes progress through the early stages of development. While the mechanisms that restrict each step in recombination to its appropriate developmental stage are largely unknown, they share many of the components that regulate transcription. For example, enhancer-dependent modifications in histone acetylation and methylation are essential for both germline transcription and rearrangement of antigen receptor genes. Promoters positioned proximal to individual D and J gene segments in Tcra, Tcrb, Tcrd, IgH, and Igk also contribute to antigen receptor gene assembly, though their effects appear more localized than those of enhancers. Tcrb assembly initiates with D-to-J joining at each of the two D–J–C gene segment clusters in DN1/2 thymocytes. DJ joints are fused with Vβ elements to complete Tcrb recombination in DN3 cells. We have previously shown that Dβ2 is flanked by upstream and downstream promoters, with the 5′ promoter being held inactive until D-to-J recombination deletes the NFκB-dependent 3′ promoter. We now report that activity of the 5′ promoter reflects a complex interplay among Runx1, GATA-3, and E47 transcription factors. In particular, while multiple E47 and Runx1 binding sites clustered near the Dβ2 5′RS and overlapping inr elements define the core 5′PDβ2, they act in concert with an array of upstream GATA-3 sites to overcome the inhibitory effects of a 110bp distal polypurine·polypyrimidine (R·Y) tract. The dependence of 5′PDβ2 on E47 is consistent with the reported role of E proteins in post-DN1 thymocyte development and V-to-DJβ recombination.

Acetylation of KLF5 Alters the Assembly of p15 Transcription Factors in Transforming Growth Factor-β-mediated Induction in Epithelial Cells

KLF5 plays important roles in a variety of cellular processes including proliferation and differentiation. Recently KLF5 was shown to reverse its function in proliferative and p15 regulation upon transforming growth factor-beta (TGFbeta)-mediated acetylation. To understand how KLF5 acetylation functions in TGFbeta-induced p15 transcription, we characterized the interactions of KLF5 with other transcription factors and promoter DNA elements in the context of TGFbeta. KLF5 interacted with Smad2-4 and Miz-1 in a TGFbeta-independent manner, but interacted with Myc only when TGFbeta was activated, and at least some of the interactions had an additive effect on TGFbeta-induced p15 transcription. Oligo pulldown assays showed that binding of Myc to the Inr element was KLF5-dependent, and TGFbeta could enhance the binding when more KLF5 was available. Furthermore, TGFbeta induced an interaction between KLF5 and the p300 acetylase, and acetylation of KLF5 was necessary for Smad4 to associate with p300. Failure in KLF5 acetylation not only prevented p300-assembled Smad4-KLF5 complex formation on p15 promoter but also affected the binding of Smad4 and FOXO3 on the p15 promoter in vivo. These findings suggest that without TGFbeta, some KLF5 associates with Smads in the nucleus and other KLF5 associates with Miz-1 on the p15 promoter to repress its transcription. Activation of TGFbeta recruits p300 to the KLF5-Smad complex to acetylate KLF5, and the complex with acetylated KLF5 binds to the Smad binding element and alters the binding of other factors to p15 promoter to induce its transcription.

Characterization of the 5'-flanking regions of the sea anemone ADP ribosylation factor 1 and actin genes

The 5'-flanking regions of the sea anemone, Aiptasia pulchella (ap) ARF1 gene showed the absence of a TATA box. The transcriptional start site determined by 5' rapid amplification of cDNA ends (5'RACE) is located 75 base pairs upstream of the translational start site. Transfection experiments in HeLa and COS-7 cells demonstrate that all the elements required to achieve significant basal transcription activity are located between position -208 and -88 relative to the transcriptional start site. There are three consensus initiator (Inr) elements for TATA-less promoter around the transcriptional start site of the apARF1 gene (+29, -158, and -226) that are likely to play roles in the regulation. For the apactin gene, the 5'-flanking region contains a TATA box located 30 base pairs upstream of the transcriptional start site. The transient transfection of apactin/luciferase deletion constructs revealed that the TATA box indeed is necessary for full expression.

Myocyte-Specific M-CAT and MEF-1 Elements Regulate G-Protein Gamma 3 Gene (γ3) Expression in Cardiac Myocytes

Little is known regarding the mechanisms that control the expression of G-protein alpha, beta, and gamma subtypes. We have previously shown that the G-protein gamma(3) gene is expressed in the heart, brain, lung, spleen, kidney, muscle, and testis in mice. We have also reported that the G-protein gamma(3) subunit is expressed in rat cardiac myocytes, but not in cardiac fibroblasts. Other studies have shown that the gamma(3) subunit couples to the angiotensin A1A receptor in portal vein myocytes, and has been shown to mediate beta-adrenergic desensitization in cardiac myocytes treated with atorvastatin. In the present study, we evaluated G-protein gamma(3) promoter-luciferase reporter constructs in primary myocytes to identify key regulatory promoter regions. We identified two important regions of the promoter (upstream promoter region [UPR] and downstream promoter region [DPR]), which are required for expression in cardiac myocytes. We observed that removal of 48 bp in the UPR diminished gene transcription by 75%, and that the UPR contains consensus elements for myocyte-specific M-CAT and myocyte enhancer factor 1 (MEF-1) elements. The UPR and DPR share transcription factor elements for myocyte-specific M-CAT element. We observed that cardiac myocyte proteins bind to gamma(3) oligonucleotides containing transcription factor elements for myocyte-specific M-CAT and MEF-1. Myocyte-specific M-CAT proteins were supershifted with transcriptional enhancer factor-1 (TEF-1) antibodies binding to the gamma(3) M-CAT element, which is in agreement with reports showing that the M-CAT element binds the TEF-1 family of transcription factors. The 150 bp DPR contains three M-CAT elements, an INR element, an upstream stimulatory factor 1 element, and the transcription start site. We have shown that myocyte gamma(3) gene expression is regulated by myocyte-specific M-CAT and MEF-1 elements.