ABSTRACT
xNAP1 (Xenopus nucleosome assembly protein) belongs to the family of nucleosome assembly proteins (NAPs) and shares 92% identity with human and mouse NAP1. NAPs have been reported to have a role in nucleosome assembly, cell cycle regulation, cell proliferation and transcriptional control, although the precise function of NAP1 is still not clear. Here we report the identification of a putative domain of xNAP1 by limited proteolysis. This domain has been mapped in the xNAP1 protein sequence to residues 38-282 and thus lacks the acidic sequences at the N- and C-termini. We have studied this domain and related fragments in vitro and by a functional assay involving over-expression of the protein in Xenopus laevis embryos. Analytical ultracentrifugation shows that removal of the acidic N- and C-terminal regions does not prevent the formation of larger multimers, which are predominantly hexadecamers. Injection of mRNA encoding the full-length xNAP1 or the putative domain and other related constructs into Xenopus embryos gave identical phenotypes. These results are discussed in relation to protein-protein interactions between NAP1 octamers and a possible 'squelching' mechanism.
Subject(s)
Nuclear Proteins/chemistry , Xenopus Proteins/chemistry , Xenopus laevis/embryology , Amino Acid Sequence , Animals , Body Patterning , Embryo, Nonmammalian/anatomy & histology , Embryo, Nonmammalian/metabolism , Molecular Sequence Data , Nuclear Proteins/metabolism , Nucleosome Assembly Protein 1 , Peptide Hydrolases/metabolism , Peptide Mapping , Phenotype , Protein Structure, Tertiary , Ultracentrifugation , Xenopus Proteins/metabolism , Xenopus laevis/anatomy & histology , Xenopus laevis/metabolismABSTRACT
Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates alpha-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non-cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues alpha-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.
