EFI(A) YB-1 IS A COMPONENT OF CARDIAC HF-1A BINDING-ACTIVITY AND POSITIVELY REGULATES TRANSCRIPTION OF THE MYOSIN LIGHT-CHAIN 2V GENE/

Transient assays in cultured ventricular muscle cells and studies in t ransgenic mice have identified two adjacent regulatory elements (HF-1a and HF-1b/MEF-2) as required to maintain ventricular chamber-specific expression of the myosin light-chain 2v (MLC-2V) gene, A rat neonatal heart cDNA library was screened with an HF-la binding site, resulting in the isolation of EFI(A), the rat homolog of human YB-1, Purified r ecombinant EFI(A)/YB-1 protein binds to the HF-1a site in a sequence-s pecific manner and contacts a subset of the HF-1a contact points made by the cardiac nuclear factor(s), The HF-1a sequence contains AGTGG, w hich is highly homologous to the inverted CCAAT core of the EFI(A)/YB- 1 binding sites and is found to be essential for binding of the recomb inant EFI(A)/YB-1. Antiserum against Xenopus YB-3 (100% identical in t he DNA binding domain and 89% identical in overall amino acid sequence to rat EFI(A)) can specifically abolish a component of the endogenous HF-la complex in the rat cardiac myocyte nuclear extracts, In cotrans fection assays, EFI(A)/YB-1 increased 250-bp MEC-2V promoter activity by 3.4-fold specifically in the cardiac cell context and in an HF-la s ite-dependent manner, EFI(A)/YB-1 complexes with an unknown protein in cardiac myocyte nuclear extracts to form the endogenous HF-1a binding activity. Immunocoprecipitation revealed that EFI(A)/YB-1 has a major associated protein of similar to 30 kDa (p30) in cardiac muscle cells , This study suggests that EFI(A)/YB-1, together with the partner p30, binds to the HF-la site and, in conjunction with HF-1b/MEF-2, mediate s ventricular chamber-specific expression of the MLC-2V gene.