Nucleosome Remodeling and Transcriptional Repression Are Distinct Functions of Isw1 in Saccharomyces cerevisiae

Abstract

The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.In eukaryotic organisms, DNA is folded into a highly compacted chromatin structure. This packing restricts the accessibility of DNA to various nuclear factors involved in DNAtemplated processes, including transcription. There are several mechanisms that regulate chromatin structure, among them covalent modifications of histones provided by histone-modifying enzymes and disruption of histone-DNA interactions by ATP-dependent chromatin-remodeling enzymes. Although these two classes of chromatin-modifying factors are functionally distinct, their subunits share conserved motifs, such as the chromodomain, bromodomain, plant homeodomain finger, and SANT domain (56). These domains are involved in histone tail recognition and binding, thus providing recruitment of these enzymes to targeted regions within the chromatin. The bromodomain shows highly selective binding to acetylated lysines (24,25,47). The chromodomain and plant homeodomain finger bind to methylated lysines (17, 27, 28, 34-36, 39, 49). In each case, the specific binding is defined by a hydrophobic pocket, formed by aromatic residues and surrounded by residues determining the specificity of binding.In contrast, our understanding of the function of the SANT domain is still restricted to examples that often prove to be protein specific. Historically, the SANT domain was identified based on its homology to the DNA-binding domain of c-Myb (1). It consists of three alpha-helices arranged in a helix-turnhelix motif; each helix contains a conserved, bulky aromatic residue that plays a key role in helix packing. Although structurally similar, the SANT domain and the canonical Myb DNA-binding domain are functionally divergent (21,26,48).The DNA-contacting residues in Myb are not conserved in SANT and often prove to be incompatible with DNA ...

Nucleosome mobilization by ISW2 requires the concerted action of the ATPase and SLIDE domains

“…1a and 1b, lane 1 and 2, and data not shown). The SLIDE domain in ISW1a and ISW1b has a similar role in complex assembly 16 . The SANT domain is also required for complex assembly in ISW1b, but not in any other ISWI remodeler investigated so far.…”

The SNF2-Family Member Fun30 Promotes Gene Silencing in Heterochromatic Loci

“…Likewise, the deletion or mutation of critical domains flanking the ATPase, such as the bromodomain in SWI/SNF, has been shown to affect the function of the protein in many instances [42], [43], [44]. We tested the role of Fun30's ATP-binding site and putative CUE motif for silencing in the HMR .…”

Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

“…Isw1 associates with Ioc3 ( I sw O ne C omplex protein 3) or Ioc2 and Ioc4 to form two distinct remodeling complexes, Isw1a and Isw1b respectively 13 . Although Isw1 can interact with nucleosomes through its SANT and SLIDE domains 14,15 , it is thought that the associated Ioc proteins are involved in targeting the remodeling complexes to different genomic locations. Indeed previous experiments found Ioc3 associated with the promoter of MET16 , while Ioc2 and Ioc4 localized to the MET16 ORF 16 .…”