Sumy Mathew, Eduardo Mascareno and M.A.Q. Siddiqui *

From the Department of Anatomy and Cell Biology, Center for Cardiovascular and Muscle Research, State University of New York-Downstate Medical Center, Brooklyn, New York 11203

* To whom correspondence should be addressed at Department of Anatomy and Cell Biology, Box 5, State University of New York-Downstate Medical Center, 450 Clarkson Ave, Brooklyn, New York 11203.

Tel: 718-270-1014; Fax: 718-270-3732;
Email: maq.siddiqui@downstate.edu

The ventricular myosin light chain-2 (MLC-2v)1 gene serves as a paradigm for understanding the complexities of eukaryotic transcriptional regulation in development and disease, as it displays a strict cardiac tissue specificity (1, 2) and also serves as a marker for cardiac hypertrophy (3-6). Cardiac hypertrophy, an adaptive response, is characterized by an increase in cardiac cell size (7, 8), sarcomeric re-organization (3-6, 9), re-expression of embryonic genes (10) and induction of other hypertrophy-associated genes (11); among them, MLC-2v gene transcription is preferentially stimulated in response to agonist-induced hypertrophy (3, 12). To understand the mechanism(s) that dictates the transcriptional regulation of MLC-2v gene during development and disease, several regulatory cis-elements and their cognate proteins in both the avian (13-17) and murine (18) MLC-2v genes have been identified. Previous studies on chicken MLC-2v gene from our laboratory (1, 13-17) and on murine MLC-2v of others (18) have revealed that the basal promoter architecture of MLC-2v gene is conserved in both genomes and share the sequence motifs of the HF-1 box containing the CArG, MEF-2 and AP-2 elements (18). In chicken MLC-2v gene, there is an additional upstream sequence, CSS (Cardiac Specific Sequence) (17,   19), that represses the chicken MLC-2v gene expression in skeletal muscle cells. Recently, we have identified and partly characterized a transcription factor, Nishéd, as a putative repressor that interacts with CSS (17). In studies using transgenic mice, the HF-1 box in the 250-bp rat promoter region of rat MLC-2v gene was assigned a role in cardiac tissue-specificity (20).

Several inducible regulatory sequence elements have been identified in hypertrophy responsive genes. For example, the serum responsive element (SRE) and SP-1 confer a-adrenergic stimulation to the ANF promoter (21, 22). The AP-1 and/or cAMP-responsive elements (CRE) cause induction of ANF expression in pressure overload hypertrophy (22). Similarly, the GATA binding element and its interaction with GATA-4, a zinc finger protein, mediate the induction of b-myosin heavy chain ( b-MHC) in left ventricular hypertrophy resulting from aortic constriction (23-26). More recently, the Nuclear Factor of Activated T cells (NFAT) family of transcription factors were reported to induce the expression of target genes associated with hypertrophy via the formation of a protein complex involving GATA-4 (27), MEF-2 (28) and AP-1 (29). The HF-1 box implicated in tissue specificity above also confers the a-adrenergic inducible activation of rat MLC-2v gene (3), but the role of the regulatory proteins, HF-1b (30) and EF1_A /YB-1 (31), that interact with the HF-1b/MEF-2 site and the CArG box in HF-1 respectively, has not been defined. Thus, the mechanism of transcriptional regulation of MLC-2v gene in hypertrophy response remains elusive.

Here, we provide evidence of a new cardiac-specific cis-regulatory element, IRE (Intronic Regulatory Element), located in the first intron of the chicken MLC-2v gene. IRE shares a common core motif 5’ GAAG/CTTC 3’ with the upstream negative CSS element and plays a dual role i.e. it overrides the repressive effect of CSS via its binding to a common transcription factor, Nishéd, and in hypertrophy, IRE responds to signals triggered by the
hypertrophic ligand, angiotensin II (Ang II) to recruit activator proteins that induces MLC-2v gene transcription. Mutating the IRE sequence in the MLC-2v-luciferase reporter totally abolishes the Ang II-induced transcriptional up-regulation in transient transfection assays. In ovo treatment of chick embryos with Ang II results in a selective increase in IRE/Nishéd complex formation in which, Nishéd associates with NFATc4 and the co-activator p300, the major components of the endogenous IRE/protein binding activity in cardiac muscle cells. This ternary complex formation, is enhanced in response to Ang II, is, therefore, likely to play a key
role in the transcriptional up-regulation of MLC-2v gene in hypertrophy.

Transcription of genes is known to be regulated by distal sequence elements, illustrated by the fact that the proximal promoter alone rarely supports correct cell-type-specific expression of linked reporter genes in transgenic animals. Distal regulatory regions spanning large segments of DNA are often needed (44, 45). In transgenic mice studies (46, 47) the presence of rat MLC-2v 5’ flanking sequence alone does not display an expression profile similar to that of the endogenous gene. To date, only three cis-elements located at the proximal promoter have been characterized in the rat MLC-2v gene. They alone are insufficient to support the regulation of its expression in development and hypertrophy. Our present work utilizes the chicken MLC-2v
gene promoter, extensively characterized in our laboratory (13-17), and documents the role of an intronic element, IRE in the cardiac hypertrophic response resulting in its preferential up-regulation. IRE or IRE-like sequences are also present in other mammalian genes. The murine small heat shock protein/aB-crystallin gene that is expressed, in addition to the lens, in cardiac myocytes at high levels (48, 49) contains a sequence similar to IRE, the aBE-4 cis element (5’ GAAGATTC 3’), in its enhancer. Like IRE, aBE-4 is a heart specific regulatory element required for the maximal expression of this gene in myocardial cells (50) and has been implicated in the induction of the aB-crystallin gene in response to MAPK kinase induced stress in cardiac myocytes (51). However, there is no evidence on the identity of the aBE-4 binding protein raising the possibility that Nishéd or an analogous factor is functional at this site. IRE–like sequence (5’ GAAGCTCC 3’), identified by the web-based program, VISTA is also present at an analogous position (+155 bp) in the rat MLC-2v gene, although its potential in the regulation of transcription has not been investigated.

IRE shares the core sequence motif within the upstream CSS element identified previously as a repressor in the MLC-2v promoter (17). Although Nishéd binds both CSS and IRE, these two elements perform distinct functions: CSS is a repressor and IRE an activator. The functional activity of these elements may be dictated by the ability of Nishéd to recruit specific co-activators or co-repressors at IRE and CSS sites respectively. In a parallel study
(unpublished), we have shown that Nishéd recruits a co-repressor mSin3A at the CSS site to afford repression of cardiac MLC-2v gene in skeletal muscle cells. We demonstrate here, that the recruitment of the common transcription factor, Nishéd, and its association with NFAT and the co-activator p300 at IRE is likely to play a critical role in the regulation of MLC-2v gene transcription in cardiac cells.
...

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1. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells".

2. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming".

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