Purification and Functional Analysis of a Novel Leucine-Zipper/Nucleotide-Fold Protein, BZAP45, Stimulating Cell Cycle Regulated Histone H4 Gene Transcription

Abstract

Regulation of histone gene transcription at the G1/S phase transition via the Site II cell cycle control element is distinct from E2F-dependent mechanisms operative at the growth factor-related restriction point. E2F-independent activation of histone H4 gene expression combines contributions of several promoter factors, including HiNF-M/IRF2 and the HiNF-D/CDP-cut complex which contains pRB, CDK1, and cyclin A as non-DNA binding subunits. Mutational analyses suggest additional rate-limiting factors for Site II function. Using sequence-specific Site II DNA affinity chromatography, we identified a 45 kDa protein (KIAA0005 or BZAP45) that is embryonically expressed and phylogenetically conserved. Based on amino acid sequence analysis, BZAP45 contains a unique decapeptide that is part of a putative leucine-zipper protein with a nucleotide (ATP or GTP) binding fold. Bacterial expression of a full-length cDNA produces a 45 kDa protein. Binding studies reveal that highly purified BZAP45 does not interact with Site II, suggesting that BZAP45 function may require partner proteins. Forced expression of BZAP45 strongly stimulates H4 promoter (nt -215 to -1)/CAT reporter gene activity. Deletion analyses and point mutations indicate that BZAP45 enhances H4 gene transcription through Site II. Thus, BZAP45 is a novel regulatory factor that contributes to transcriptional control at the G1/S phase transition.

Transcriptional Activation of Histone Genes Requires NPAT-Dependent Recruitment of TRRAP-Tip60 Complex to Histone Promoters during the G1/S Phase Transition

“…The mechanism of HiNF-P action is also not fully understood. Additional protein factors, such as YY1, HIRA, FLASH, and BZAP45, have also been implicated in the regulation of histone gene transcription with unknown molecular mechanisms (3,12,19,31,41,61).We and others have demonstrated previously that the cyclin E-Cdk2 substrate NPAT associates with histone gene promoters in S phase (66,67). The overexpression of NPAT activates promoters of multiple histone genes through the SSREs within the promoters (66).…”

Expression analysis of nuclear W2-containing homologs of eukaryotic initiation factors in rice

“…Some well-defined W2 proteins also exhibit the ability for transcriptional regulation. The H sapiens basic leucine zipper and W2 domains 1 (BZW1 or BZAP45) is a conserved regulatory factor in transcriptional activation of histone H4 gene at the G1/S phase transition (Mitra et al 2001) for promoting cell growth (Li et al 2009). The W2 domain is a nucleotide-binding fold with unknown functions (Mitra et al 2001).…”

“…The H sapiens basic leucine zipper and W2 domains 1 (BZW1 or BZAP45) is a conserved regulatory factor in transcriptional activation of histone H4 gene at the G1/S phase transition (Mitra et al 2001) for promoting cell growth (Li et al 2009). The W2 domain is a nucleotide-binding fold with unknown functions (Mitra et al 2001). However, energy input through nucleoside triphosphate hydrolysis, such as SWI/SNF-dependent chromatin remodeling and covalent modifications of nucleosomal proteins (Peterson & Logie 2000;Spencer & Davie 1999) is required in many processes involving transcription factors and, moreover, nuclear import and correct protein folding of transcription factors are energy-dependent processes (Mitra et al 2001).…”

“…However, energy input through nucleoside triphosphate hydrolysis, such as SWI/SNF-dependent chromatin remodeling and covalent modifications of nucleosomal proteins (Peterson & Logie 2000;Spencer & Davie 1999) is required in many processes involving transcription factors and, moreover, nuclear import and correct protein folding of transcription factors are energy-dependent processes (Mitra et al 2001). Therefore, the nucleotide-binding fold may be a molecular adaptation for transport and nucleoside triphosphate hydrolysis to support a function in transcriptional activation (Mitra et al 2001). Our study focused on the three OsW2 genes also revealed their nuclear location and potential capacity for gene expression control, on the basis of their responses to phytohormones.…”

“…eIF-5, an eukaryotic initiation factor from Zea mays, containing a zinc-finger structure, binds RNA in a zinc-dependent manner (Lopez Ribera et al 1997), while the W2 domain of it has not been studied. BZW1 or BZAP45, a W2 protein in Homo sapiens containing a leucine-zipper, was found to be a conserved regulatory factor for transcriptional control of histone H4 gene at the G1/S transition (Mitra et al 2001), but not eIFs. However, the roles of the W2 domain within BZW1 involved in the transcriptional regulation have not been characterized.…”