Distinct regions of the chicken p46 polypeptide are required for its in vitro interaction with histones H2B and H4 and histone acetyltransferase-1

Abstract

We cloned cDNA encoding the chicken p46 polypeptide, chp46, homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48. It comprises 424 amino acids including a putative initiation Met, is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48 and 94.3% identity to the human and mouse p46 polypeptides (hup46 and mop46). The in vitro immunoprecipitation experiment established that chp46 interacts with histones H2B and H4 and chicken histone acetyltransferase-1, chHAT-1, whereas hup46 interacts with histones H2A and H4 and chHAT-1 and chCAF-1p48 with histone H4 and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a region comprising amino acids 359-404 (in fact, 375-404) binds to chHAT-1 in vitro. Taken together, these results indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with chHAT-1 and histones H2B and H4, but also that the proper propeller structure of chp46 is not necessary for its interaction with chHAT-1.