Functional Role of a Negative Regulatory Element in DevelopmentStage Specific Expression of Rat Cardiac Myosin Heavy Chain Gene. •259

Abstract

The αmyosin heavy chain (αMHC) gene encodes a major cardiac contractile protein. Steadystate level of its mRNA are decreased during embryonic heart development and hypertrophy of the adult rat heart, mainly due to transcriptional regulation. While several ciselements and cognate binding factors involved in its positive regulation are described, the mechanism of its negative regulation remains unknown. We have identified a strong negative regulatory element (NRE) in the 5′ untranslated region of this gene. To study if NRE sequences play a role in the negative regulation of αMHC gene transcription in embryonic heart, we transfected the αMHC promoter/ chloramphenicol acetyl transferase (CAT) reporter constructs into primary cultures of 18 day fetal rat heart. The wild type (WT) construct included 600/420 bp sequence (in relation to transcription start site) of αMHC promoter. The deletion mutants comprised 600/63 promoter sequences and internal deletion of NRE within WT construct. The percent increase in CAT expression due to NRE deletion was compared with that obtained by direct in vivo injections of these constructs in adult heart. NRE deletion resulted in 15fold induction of CAT expression in fetal heart, but 8fold increase in adult heart. Gelshift assay with NRE sequence and nuclear extracts prepared from fetal and adult heart showed two complexes with distinct mobilities in fetal heart but one complex in adult heart. The adult heart complex corresponded to the slowermobility complex of the fetal heart. These data suggests that differential binding of nuclear factors to NRE sequence may be responsible for its stronger effect in fetal heart that may contribute to the in vivo suppression of αMHC gene transcription during embryonic heart development (Supported by a grant from Christ Hospital Med Fund).