Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

Abstract

Sin1p/Spt2p is a yeast chromatin protein that, when mutated or deleted, alters the transcription of a family of genes presumably by modulating local chromatin structure. In this study, we investigated the ability of different domains of this protein to bind four-way junction DNA (4WJDNA) since 4WJDNA can serve as a model for bent double helical DNA and for the crossed structure formed at the exit and entry of DNA to the nucleosomes. Sequence alignment of Sin1p/Spt2p homologues from 11 different yeast species showed conservation of several domains. We found that three domains of Sin1p/ Spt2p fused to glutathione S-transferase can each bind independently in a structure-specific manner to 4WJDNA as measured in a gel mobility shift assay. A feature common to these domains is a cluster of positively charged amino acids. Modification of this cluster resulted in either abolishment of binding or a change in the binding properties. One of the domains tested clearly bound superhelical DNA, although it failed to induce bending in a circularization assay. Poly-L-lysine, which may be viewed as a cluster of positively charged amino acids, bound 4WJDNA as well. Phenotypic analysis showed that disruption of any of these domains resulted in suppression of a his4-912␦ allele, indicating that each domain has functional significance. We propose that Sin1p/Spt2p is likely to modulate local chromatin structure by binding two strands of double-stranded DNA at their crossover point.Sin1p/Spt2p is a yeast chromatin non-histone protein. While the precise function of the protein is still unknown, it is known to function as a negative transcriptional regulator of a number of genes including SUC2 (1), INO1 (2), and SSA3 (3). Activity of the HO promoter, as measured from an HO promoter driving a lacZ gene (4), is also regulated by SIN1/SPT2. swi1, swi2, and swi3 mutants are unable to transcribe RNA from the HO promoter. However, in sin1/swi double mutants, transcription is restored. Mutants in SPT2 were first identified as suppressors of ty and ␦ insertions in the 5Ј non-coding region of the HIS4 gene (5). As the negative regulation of these genes is overcome by the SW1/SNF chromatin remodeling complex, it was suggested that a function of SIN1p/SPT2p is to somehow maintain chromatin compaction at specific locations in the chromatin.

Recruitment of mRNA cleavage/polyadenylation machinery by the yeast chromatin protein Sin1p/Spt2p

“…A fusion peptide containing only a part of this HMG1-like domain (amino acids 1-64) was able to bind Fir1p, but less well than those peptides containing the entire domain. In previous work, we showed that a Sin1p peptide containing this HMG1-like domain could bind four-way junction DNA (9). Taken together, these results demonstrate that the first HMG1-like domain of the protein can interact with both Fir1p and with DNA.…”

“…Sequence analysis of Sin1p showed sequence similarity in two domains to HMG1 (6, 7), a known chromatin protein. Work from our laboratory showed that Sin1p can bind four-way junction and crossing DNA structures (9), supporting the idea that Sin1p binds DNA as it enters and exits the nucleosome.…”

“…Previous genetic and biochemical evidence (6,9) showed that the C terminus of Sin1p has functional significance. We therefore tested a strain lacking the nine terminal amino acids of Sin1p (Fig.…”

“…Several hypothetical functional domains in Sin1p have been defined based on genetic, structural, and biochemical considerations (6,9,21,(23)(24)(25). These domains include two HMG1-like domains, (amino acids 26-88 and 98-159), an acidic domain (amino acids 224-304), and a basic C-terminal domain (amino acids 303-333).…”

“…Plasmids encoding the GST͞ Sin1p fusions were described in ref. 9. The in vitro-binding assay between the Sin1p and Fir1p peptides was carried out as in Novoseler et al (9).…”