Critical regulatory domains in intron 2 of a porcine sarcomeric myosin heavy chain gene.

Abstract

The porcine sarcomeric fast 2a myosin heavy chain (MyHC) gene was previously found to require a region extending 3' from the transcriptional start site for high levels of expression. Here we established the existence of two novel opposing regulatory domains in intron 2. A positive regulatory element, defined to a 75bp region, resembles a TATA-less intronic promoter, with a consensus transcription initiation element. It can up-regulate its endogenous or a heterologous muscle promoter in a position specific manner, and on its own drive a reporter gene. In tandem with it is a dominant negative regulatory element, localised to a 81bp region, which can down-regulate its native gene and a heterologous muscle promoter. Bandshift and DNase I footprinting assays demonstrated that specific nuclear factors bound to both regulatory elements are distinctly different. Both elements appear to have no counterpart in intron 2 of the porcine fast 2x and 2b MyHC genes. Taken together, we demonstrate for the first time that a 5'-end terminal intron of a sarcomeric MyHC gene contains two critical regulatory domains, which may be involved in the complexity of temporo-spatial expression.