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.