CHROMATIN REMODELING AND GENE ACTIVATION
, Head, Section on Chromatin and Gene Expression
Peter Eriksson, PhD, Research Fellow
Neil McLaughlin, PhD, Postdoctoral Fellow
Naoe Kotomura, PhD, Volunteer
Gene activation involves the regulated recruitment of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II as well as in transcription. These events must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent the chromatin block, eukaryotic cells possess chromatin-remodeling and nucleosome-modifying complexes. The former (e.g., the SWI/SNF complex) use ATP to drive conformational changes in nucleosomes and to slide nucleosomes along DNA. The latter contain enzymatic activities (e.g., histone acetylases) that modify the histones post-translationally to mark them for recognition by other complexes. Geneticists have described many interesting connections between chromatin components and transcription, but a model system in which to investigate these phenomena has been lacking. We have developed such a model system, involving native plasmid chromatin purified from the yeast Saccharomyces cerevisiae, to perform high-resolution studies of the chromatin structures of active and inactive genes. Remarkably, the studies reveal that activation correlates with large-scale movements of nucleosomes and conformational changes within nucleosomes over entire genes. Our current work focuses on the roles of chromatin remodeling and histone acetylation in gene regulation.
Transcriptional activation and SWI/SNF-dependent nucleosome mobilization
Recognizing that biochemical studies of chromatin structure could be combined with molecular genetics, we selected budding yeast as a model organism. Current models for the role of the SWI/SNF ATP-dependent chromatin-remodeling complex in gene regulation focus on promoters, which is where the most obvious changes in chromatin structure occur. However, by using our plasmid model system with HIS3, a SWI/SNF-regulated gene, we discovered that transcriptional activation creates a domain of remodeled chromatin structure that extends far beyond the promoter to include the entire gene. We addressed the effects of transcriptional activation on the chromatin structure of HIS3 by mapping the precise positions of nucleosomes in basal-expressing and transcriptionally activated chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a dominant nucleosomal array. In wild-type chromatin, the array is disrupted, and several alternative, overlapping nucleosomal arrays form. Disruption of the dominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring positions different from those preferred by the SWI/SNF complex. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure (Kim et al., 2006). Our work on HIS3 and our earlier work on CUP1 indicate that, at least for these two genes, the target of remodeling complexes is a domain rather than just the promoter. This important finding suggests that remodeling complexes act on chromatin domains. As regards the function of domain remodeling, we speculate that the remodeling entire of genes might facilitate elongation through nucleosomes by RNA polymerase II. Our current work aims to elucidate the structure of the remodeled nucleosome and focuses on at least two possibilities: unstable nucleosomes (remodeled such that they easily fall apart) and nucleosomes with a dramatically altered conformation.
In collaboration with Len Lutter, we compared the structures of yeast and mammalian chromatin, leading us to suggest that yeast chromatin is composed of relatively ordered arrays of closely spaced nucleosomes that are separated by substantial gaps, possibly corresponding to regulatory regions (Tong et al., 2006).
Kim Y, McLaughlin N, Lindstrom K, Tsukiyama T, Clark DJ. Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilisation of nucleosomes over the entire gene. Mol Cell Biol 2006;26:8607-22.
Tong W, Kulaeva OI, Clark DJ, Lutter LC. Topological analysis of plasmid chromatin from yeast and mammalian cells. J Mol Biol 2006;361:813-22.
The yeast Spt10 protein contains a DNA-binding domain fused to a putative histone acetylase domain
We have shown that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter (Clark and Shen, 2006). The acetylation is dependent on SPT10, which encodes a putative histone acetylase (HAT). SPT10 has been implicated as a global regulator of core promoter activity. We confirmed such a role for SPT10 by expression microarray analysis and then addressed the mechanism of global regulation. Using the chromatin immunoprecipitation (ChIP) assay, we were unable to detect Spt10p at any of the most strongly affected genes in vivo, but we confirmed its presence at the core histone gene promoters, which it activates. We presented evidence that a defective chromatin structure forms in the absence of Spt10p, with consequent activation of basal promoters. Furthermore, we found that Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequences (UAS elements) in the core histone promoters [consensus: (G/A)TTCCN6TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Our observations are consistent with the idea that the global changes in gene expression in spt10Δ cells are the indirect effect of defective regulation of the core histone genes. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain. We have identified the DNA-binding domain of Spt10p: it contains an unusual zinc finger (His2-Cys2), with homology to the DNA integrase of foamy retroviruses. We propose that the integrase might also be a sequence-specific DNA-binding protein (Mendiratta et al., 2006). We also showed that Spt10p is a dimer and that the N-terminal domain is required for dimer formation (Mendiratta et al., 2007). Our current work aims (1) to demonstrate the putative histone/protein acetylase activity of Spt10p; (2) to identify proteins that interact with Spt10p; and (3) to identify the molecular mechanisms underpinning the cell cycle regulation of the core histone genes. We have also initiated a project to determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein.
Clark DJ, Shen CH. Mapping histone modifications by nucleosome immunoprecipitation. Methods Enzymol 2006;410:416-29.
Mendiratta G, Eriksson PR, Clark DJ. Cooperative binding of the yeast Spt10p activator to the histone UAS elements is mediated through an N-terminal dimerisation domain. Nucl Acids Res 2007;35:812-21.
Mendiratta G, Eriksson PR, Shen CH, Clark DJ. The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase. J Biol Chem 2006;281:7040-8.
1 Yeonjung Kim, PhD, former Visiting Fellow
2 Geetu Mendiratta, PhD, former Visiting Fellow
COLLABORATORS
Len Lutter, PhD, Henry Ford Hospital, Detroit, MI
Chang-Hui Shen, PhD, City University of New York, Staten Island, NY
Toshio Tsukiyama, PhD, Fred Hutchinson Cancer Research Center, Seattle, WA
For further information, contact clarkda@mail.nih.gov.
