Chromatin Remodeling and Gene Activation

David J. Clark, PhD, Head, Section on Chromatin and Gene Expression
Peter Eriksson, PhD, Research Fellow
Neil McLaughlin, PhD, Postdoctoral Fellow
Nagarajavel Vivekananthan, PhD, Visiting Fellow

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 and 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., SWI/SNF complexes) 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 they have lacked a system to investigate the structural basis of the connections. 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, our work reveals 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

Kim,¹ McLaughlin; in collaboration with Tsukiyama

We chose budding yeast as a model organism because biochemical studies of chromatin structure can be combined with molecular genetics. 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, 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 different positions 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 to the function of domain remodeling, we speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. The aims of our current work are (1) to elucidate the structure of the remodeled nucleosome; there are at least two possibilities—unstable nucleosomes (remodeled such that they fall apart easily) and nucleosomes with a dramatically altered conformation—and (2) to map remodeled nucleosomes on a genome-wide scale and determine which domains are remodeled by the SWI/SNF complex.

Kim Y, McLaughlin N, Lindstrom K, Tsukiyama T, Clark DJ. Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilization of nucleosomes over the entire gene. Mol Cell Biol 2006;26:8607-8622.

The yeast Spt10 protein contains a DNA-binding domain fused to a putative histone acetylase domain

Mendiratta,² Eriksson, McLaughlin, Kotomura,³ Vivekananthan

We have shown previously that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter. 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. After confirming by expression microarray analysis that Spt10p has global effects on transcription, we addressed the mechanism of global regulation. Surprisingly, using the chromatin immunoprecipitation (ChIP) assay we were unable to detect Spt10p at any of the most strongly affected genes in vivo. However, we confirmed that Spt10p is present 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 observed in spt10Δ cells are the indirect effects 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 rather than to a classical activation domain. Spt10p is therefore a highly unusual transactivator in which the HAT domain, normally recruited as a co-activator to promoters through an activation domain, is attached directly to a sequence-specific DNA-binding 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 proposed that this integrase might also be a sequence-specific DNA-binding protein. To test our hypothesis, we initiated a new project that used a SELEX approach to determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein.

We addressed the mechanism through which Spt10p simultaneously recognizes two UAS elements (Mendiratta et al., 2007). We demonstrated that Spt10p is an elongated dimer and that its N-terminal domain is necessary for dimer formation (Mendiratta et al., 2007). Unlike the full-length protein dimer, which requires two UAS elements, the isolated DNA-binding domain is a monomer and binds tightly to a single UAS element. We propose that the Spt10p dimer undergoes a major conformational change in order to recognize two UAS elements at the same time.

Our current work aims to (1) demonstrate the putative histone/protein acetylase activity of Spt10p by using a proteomics approach; (2) understand the role of Spt10p in the cell cycle–dependent regulation of the core histone genes; and (3) identify negative regulatory proteins at the histone promoters, which counteract activation by Spt10p.

Mendiratta G, Eriksson PR, Clark DJ. Cooperative binding of the yeast Spt10p activator to the histone UAS elements is mediated through an N-terminal dimerization domain. Nucleic Acids Res 2007;35:812-821.

¹Yeonjung Kim, PhD, former Visiting Fellow
²Geetu Mendiratta, PhD, former Visiting Fellow
³Naoe Kotomura, PhD, former Volunteer

Collaborator

Toshio Tsukiyama, PhD, Fred Hutchinson Cancer Research Center, Seattle, WA

For further information, contact clarkda@mail.nih.gov or visit http://clarklab.nichd.nih.gov/.