Identification of a novel DNA-binding protein that interacts with the strong positive element of the human beta-myosin heavy chain gene.

Abstract

The human β-myosin heavy chain (βMHC) gene contains a strong positive element (SPE) at positions -298/-277 that is required for high-level expression in cultured fetal rat heart cells. This element was used to screen a fetal cardiac expression library and a single positive isolate was obtained (designated FβMBF). The nucleotide sequence of the 1.6 kb cDNA insert contained an open reading frame encoding a protein of 398 amino acids (44.1 kDa). Northern analysis to determine temporal expression of the FβMBF transcript showed expression in 15-, 17-, and 20-day rat placenta, but not in fetal rat heart or embryo at any of these developmental stages. Western blot detected an immunoreactive band of 44 kDa in 13-day fetal rat heart, and three immunoreactive bands in placenta. Coupled transcription/translation in rabbit reticulocyte lysate produced two proteins of 44 and 38 kDa. When cotransfected in an expression vector with a β-MHC reporter construct into fetal rat heart cell cultures, FβMBF caused a decrease in activity. The sequence of the 1.6 kb cDNA was not similar to any trans-acting factors previously identified in muscle, but is closely homologous to the NH₂-terminal region of two larger DNA-binding proteins PO-GA, a 128 kDa protein which may be involved in the regulation of the pituitary-specific pro-opiomelanocortin gene, and the 145 kDa subunit of Activator 1 (A1-p145), a DNA polymerase accessory protein. In the region of overlap, all three proteins contain helix-turn-turn helix (HTH) motifs which presumably represents the DNA-binding domain. FβMBF may represent an additional member of a newly described subgroup of eukaryotic HTH proteins that may play diverse roles in transcriptional regulation and DNA replication.