Role of Swi6/HP1 Self-association-mediated Recruitment of Clr4/Suv39 in Establishment and Maintenance of Heterochromatin in Fission Yeast

Abstract

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.Distinct sets of histone modifications organize chromatin into transcriptionally expressed or repressed structures. As in higher eukaryotes, fission yeast euchromatic regions are associated with hyperacetylated histones, particularly Lys-9 and Lys-14 acetylated and Lys-4 methylated histone H3. Conversely, heterochromatin assembly (silent mating type, centromere, rDNA, and telomere loci) involves deacetylation of histone H3 at Lys-9 and Lys-14 by Clr3, Clr6, and Sir2, followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/ Suv39H1. H3-Lys-9-Me2 is recognized by the chromodomain in Swi6/HP1 (1, 2). The property of Swi6 to form multimers is thought to cause folding of chromatin into a transcriptionally inactive structure (3). The RNAi pathway is also involved in heterochromatin assembly. The dcr1⌬, rdp1⌬, and ago1⌬ mutants are defective in H3-Lys-9 methylation and Swi6 recruitment at heterochromatin loci (4).Structural studies show that the chromoshadow domain (CSD) 3 in Swi6/HP1 contains a dimerization motif that creates a cleft for binding a pentapeptide (5, 6). Mutations in the conserved residues in the cleft inhibit interaction with the pentapeptide motif in proteins (6, 7). We found that mutation of one such residue (L315E) severely compromises the self-association property of Swi6. is not only defective in complementing the silencing defect in the swi6⌬ mutant but also exerts a dominant negative effect on silencing, accompanied by loss of heterochromatic localization not only of itself but also that of native Swi6 and H3-Lys-9-Me2 and, by implication, of Clr4. These results support a concerted rather than a sequential action of Clr4 and Swi6 in establishment and maintenance of heterochromatin. EXPERIMENTAL PROCEDURESStrains and Plasmids Used-All strains and plasmids used are listed in supplemental Tables 1 and 2, respectively.Media Compositions-All fission yeast media were prepared according to Moreno et al. (8). The serial dilution plate assay has been described earlier (9). Iodine staining assays for switching and silencing have ...

Pericentric Heterochromatin Generated by HP1 Protein Interaction-defective Histone Methyltransferase Suv39h1

“…Furthermore, it has been proposed that the physical interaction of Clr4 with Swi6 is important for the establishment/spreading of H3K9 methylation (30). A similar functional concept for the Suv39h-HP1 complex on the H3K9me-mediated heterochromatin establishment/spreading has been proposed for other species, including mammals (11,12).…”

Biochemical Basis for Distinct Roles of the Heterochromatin Proteins Swi6 and Chp2

“…HP1 proteins have been proposed to act as platform molecules that recruit diverse activities to heterochromatic regions 1,17,19–22 . The best-characterized HP1 domain that is known to recruit non-nucleosomal ligands is the CSD.…”

LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

“…CSDs are dimerization domains of HP1 proteins that serve as interaction interfaces that stabilize heterochromatic structures (60). Mutations that interfere with HP1 dimerization disrupt the protein's localization to heterochromatin (6,61). Similarly, we recently showed that mutation of the Pdd1 CSD severely reduces production of viable progeny and inhibits formation of DNA elimination foci; however, some IES excision still occurred, which suggests that other proteins, possibly including Lia4, contribute to the overall stability of DNA elimination complexes (37).…”