ABSTRACT
Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.
Subject(s)
Chromatin/metabolism , DNA/metabolism , Histone Chaperones/physiology , Histones/metabolism , Molecular Motor Proteins/physiology , Nucleosomes/metabolism , DNA/chemistry , Histone Chaperones/chemistry , Histone Chaperones/metabolism , Histones/chemistry , Molecular Motor Proteins/metabolismABSTRACT
CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. Here we show that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that yields regularly spaced nucleosomes. The comparative analysis of CHD1 and ACF revealed that CHD1 assembles chromatin with a shorter nucleosome repeat length than ACF. In addition, ACF, but not CHD1, can assemble chromatin containing histone H1, which is involved in the formation of higher-order chromatin structure and transcriptional repression. These results suggest a role for CHD1 in the assembly of active chromatin and a function of ACF in the assembly of repressive chromatin.
Subject(s)
Adenosine Triphosphate/metabolism , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Transcription Factors/metabolism , Animals , Catalysis , Cell Cycle Proteins , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Histones/metabolism , Nuclear Proteins , Nucleosome Assembly Protein 1 , Nucleosomes/chemistry , Nucleosomes/metabolism , Proteins/metabolism , Transcription Factors/chemistryABSTRACT
Eosinophil infiltration of the lung is a feature of both allergic and nonallergic asthma, and IL-5 is the key cytokine regulating the production and activation of these cells. Despite many studies focusing on the IL-5 promoter in both humans and mice there is as yet no clear picture of how the IL-5 gene is regulated. The aim of this study was to determine if distal regulatory elements contribute to appropriate regulation of the human IL-5 (hIL-5) gene. Activity of the -507/+44 hIL-5 promoter was compared to expression of the endogenous IL-5 gene in PER-117 T cells. The IL-5 promoter was not sufficient to reproduce a physiological pattern of IL-5 expression. Further, functional analysis of the 5' and 3' intergenic regions revealed a number of novel regulatory elements. We have identified a conserved enhancer located approximately 6.2 kb upstream of the hIL-5 gene. This region contains two potential GATA-3-binding sites and increases expression from the hIL-5 promoter by up to ninefold.
