Identification of a 68 kDa protein species as a specific DNA-binding component of the H3abp complex interacting with the histone H3.2 G1/S regulatory domain.
The hamster histone H3.2 promoter contains a protein binding site (referred to as site X) required for G1/S transcriptional activation. We report here that nuclear extracts prepared from serum synchronized cells at various stages of the cell cycle show a biphasic increase in the H3.2 specific complex, H3abp, binding to site X. An increase in binding activity occurs as cells first enter the cell cycle and later at the G1/S border. The H3.2 specific binding activity is enhanced by Mg2+ and Ca2+ in vitro, but is inhibited by Zn2+. Site X resembles a Jun/AP-1 site, but previously it has been shown that the H3abp complex is immunologically distinct from the characterized AP-1 proteins. Here, we identify the size of the hamster nuclear protein(s) that bind specifically to the H3abp site by ultra-violet crosslinking and renaturation of specific protein bands following gel electrophoresis. In addition, we purify H3abp by affinity chromatography and show that the purified H3abp has a different methylation interference profile from AP-1. Our results indicate that a protein species around 68 kDa is the major DNA binding component of the H3abp complex and it binds specifically to the histone promoter site required for G1/S regulation.
Naeve
G S
GS
Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles.
Zhou
Y
Y
Lee
A S
AS
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology*; D002457:Cell Extracts / Q000737:chemistry; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D018808:Transcription Factor AP-1; D014466:Ultraviolet Rays
D002413:Cations, Divalent; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D004247:DNA