Michael Bulger

ACF, an ISWI-Containing and ATP-Utilizing Chromatin Assembly and Remodeling Factor

We describe the purification and characterization of ACF, an ATP-utilizing chromatin assembly and remodeling factor. ACF is a multisubunit factor that contains ISWI protein and is distinct from NURF, another ISWI-containing factor. In chromatin assembly, purified ACF and a core histone chaperone (such as NAP-1 or CAF-1) are sufficient for the ATP-dependent formation of periodic nucleosome arrays. In chromatin remodeling, ACF is able to modulate the internucleosomal spacing of chromatin by an ATP-dependent mechanism. Moreover, ACF can mediate promoter-specific nucleosome reconfiguration by Gal4-VP16 in an ATP-dependent manner. These results suggest that ACF acts catalytically both in chromatin assembly and in the remodeling of nucleosomes that occurs during transcriptional activation.

Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.

β-globin Gene Switching and DNase I Sensitivity of the Endogenous β-globin Locus in Mice Do Not Require the Locus Control Region

We have generated mice with a targeted deletion of the beta-globin locus control region (LCR). Mice homozygous for the deletion die early in embryogenesis but can be rescued with a YAC containing the human beta-globin locus. After germline passage, deletion of the LCR leads to a severe reduction in expression of all mouse beta-like globin genes, but no alteration in the developmental specificity of expression. Furthermore, a DNase I-sensitive open chromatin conformation of the locus is established and maintained. Thus, the dominant role of the LCR in the native locus is to confer high-level transcription, and elements elsewhere in the locus are sufficient to establish and maintain an open conformation and to confer developmentally regulated globin gene expression.

A Complex Chromatin Landscape Revealed by Patterns of Nuclease Sensitivity and Histone Modification within the Mouse β-Globin Locus

ABSTRACT In order to create an extended map of chromatin features within a mammalian multigene locus, we have determined the extent of nuclease sensitivity and the pattern of histone modifications associated with the mouse β-globin genes in adult erythroid tissue. We show that the nuclease-sensitive domain encompasses the β-globin genes along with several flanking olfactory receptor genes that are inactive in erythroid cells. We describe enhancer-blocking or boundary elements on either side of the locus that are bound in vivo by the transcription factor CTCF, but we found that they do not coincide with transitions in nuclease sensitivity flanking the locus or with patterns of histone modifications within it. In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse β-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active β-globin locus and illustrate the complexity of broad structural changes that accompany gene activation.

Enhancers: the abundance and function of regulatory sequences beyond promoters.

Transcriptional control in mammals and Drosophila is often mediated by regulatory sequences located far from gene promoters. Different classes of such elements - particularly enhancers, but also locus control regions and insulators - have been defined by specific functional assays, although it is not always clear how these assays relate to the function of these elements within their native loci. Recent advances in genomics suggest, however, that such elements are highly abundant within the genome and may represent the primary mechanism by which cell- and developmental-specific gene expression is accomplished. In this review, we discuss the functional parameters of enhancers as defined by specific assays, along with the frequency with which they occur in the genome. In addition, we examine the available evidence for the mechanism by which such elements communicate or interact with the promoters they regulate.

The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.

ChIPs of the β-globin locus: unraveling gene regulation within an active domain

Recent studies of β-globin gene expression have concentrated on the analysis of factor binding and chromatin structure within the endogenous locus. These studies have more precisely defined the extent and nature of the active chromosomal domain and the elements that organize it. Surprisingly, the β-globin locus control region (LCR), although critical for high-level gene expression, plays little role in the overall architecture of the active locus. Analysis of the effects of targeted deletion of the β-globin LCR, along with emerging knowledge of the behavior of the erythroid transcription factor NF-E2, leads to a new perspective on factor binding and LCR function.

ATP-facilitated Chromatin Assembly with a Nucleoplasmin-like Protein from Drosophila melanogaster

To gain a better understanding of the factors that can mediate chromatin assembly, we have purified and cloned a core histone-binding protein from Drosophila melanogaster embryos. This protein resembles Xenopus laevis nucleoplasmin, and it has therefore been termed dNLP, for Drosophila nucleoplasmin-like protein. dNLP is a nuclear protein that is present throughout development. Both purified native and recombinant dNLP bind to core histones and can function in the assembly of approximately regularly spaced nucleosomal arrays in a reaction that additionally requires DNA, purified core histones, ATP, and a partially purified fraction (containing at least one other assembly activity). We also analyzed the properties of an N-terminally truncated version of dNLP, termed dNLP-S, and found that the deletion of the N-terminal 31 residues of dNLP results in a loss of the specificity of the interaction of dNLP with core histones. We then compared the abilities of dNLP and Drosophila nucleosome assembly protein-1 (dNAP-1) to promote the decondensation of Xenopus sperm chromatin, a process that can be mediated by nucleoplasmin. We observed that dNAP-1, but not dNLP, was able to promote the decondensation of sperm chromatin. These and other data collectively suggest that dNLP may participate in parallel with other histone-binding proteins such as dNAP-1 in the assembly of chromatin.

Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1.

To study the relationship between DNA replication and chromatin assembly, we have purified a factor termed Drosophila chromatin assembly factor 1 (dCAF-1) to approximately 50% homogeneity from a nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA replication-coupled chromatin assembly revealed that both dCAF-1 and human CAF-1 mediate chromatin assembly preferentially with previously yet newly replicated DNA relative to unreplicated DNA. Moreover, the preferential assembly of the postreplicative DNA was observed at 30 min after inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it was no longer apparent at 120 min after inhibition of replication. These findings suggest that the coupling between DNA replication and chromatin assembly may not necessarily involve a direct interaction between the replication and assembly factors at a replication fork.

TRAPping enhancer function.

Enhancer elements act from distal regions to mediate the transcription of genes. A novel combination of FISH and ChIP techniques may provide new insight into the mechanism by which enhancer elements exert their control. Application of the technique to the Hbb (encoding β-globin) locus control region (LCR) suggests that elements of the LCR may be in close proximity to the transcribed β-globin genes.