A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.

Abstract

Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

“…The HFla site has been reported to bind two ubiquitous proteins, USF (Navankasattusas et al, 1994) and the Y-box protein EFRA/YB-1 (Zhou and Chien, 1995). The HFlb site can bind MEF2/RSRF proteins Navankasattusas et al, 1992) but also a novel, tissue-restricted zinc finger protein (Zhu et al, 1993). It is thought that gene expression from this DNA element is regulated by controlling the binding of these various proteins.…”

“…However, this experiment must be interpreted cautiously. Mutation of the HF1 sequence results in considerably reduced expression from this promoter (Zhu et al, , 1993Navankasattusas et al, 1992). Thus it was possible that we observed less difference between the wild-type MKKand MKK (AN3/S218E/S222D)-transfected cells with the HFla mutant simply because this molecule was much less active to begin with.…”

“…Several sequence elements that are important in conferring cardiac specificity and induction have been identified (Zhu et al, , 1993Navankasattusas et al, 1992Navankasattusas et al, , 1994Lee et al, 1994). These include a negative regulatory sequence (HF3) that seems to prevent noncardiac expression (Lee et al, 1994), an E box (MLE) that binds the helix-loop-helix transcription factor USF (Navankasattusas et al, 1994), and a sequence (HF1) that is sufficient and required for cardiac-specific expression Lee et al, 1994 1993 ;Navankasattusas et al, 1992;Zhou and Chien, 1995). To screen for possible target sequences that could mediate inhibition of the MLC-2 promoter by active MKK, we performed transient transfection experiments with mutated versions of the 250-bp promoter containing inactivating point mutations in each of these various DNA elements Navankasattusas et al, 1992).…”

“…These include a negative regulatory sequence (HF3) that seems to prevent noncardiac expression (Lee et al, 1994), an E box (MLE) that binds the helix-loop-helix transcription factor USF (Navankasattusas et al, 1994), and a sequence (HF1) that is sufficient and required for cardiac-specific expression Lee et al, 1994 1993 ;Navankasattusas et al, 1992;Zhou and Chien, 1995). To screen for possible target sequences that could mediate inhibition of the MLC-2 promoter by active MKK, we performed transient transfection experiments with mutated versions of the 250-bp promoter containing inactivating point mutations in each of these various DNA elements Navankasattusas et al, 1992). Cells were transfected with the wild-type or mutant promoters along with constitutively active MKK (AN3/S218E/S222D) or the wildtype MKK expression construct and stimulated with phenylephrine.…”

The RSRF/MEF2 protein SL1 regulates cardiac muscle-specific transcription of a myosin light-chain gene in Xenopus embryos.

“…Putative RSRF-binding sites have been identified in the control regions of several growth factor-induced genes (Pollock and Treisman 1991), raising the possibility that RSRF proteins may play a role in regulating cell proliferation. Among vertebrate tissues, RSRF transcripts are relatively prevalent in cardiac as well as skeletal muscle (Chambers et al 1992;Navankasattusas et al 1992;Molkentin and Markham 1993), and MEF2-1ike motifs are essential for cardiocyte-restricted expression of several genes (Iannello et al 1991;Nakatsuji et al 1992;Navankasattusas et al 1992;Parmacek et al 1992;Molkentin and Markham 1993). Elevated levels of RSRF transcripts are also found in neural tissue, and expression of one RSRF gene, MEF2C, is largely restricted to cortical neurons within the mammalian brain (Leifer et al 1993;Martin et al 1993).…”