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 ...

Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complexcyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6⌬ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis.

Monoclonal antibodies (MAbs) provide scope for the development of better therapeutics and diagnostic tools. Herein, we describe the binding and neutralization profile(s) for a panel of murine MAbs generated against influenza A H1N1 viruses elicited by immunization with pandemic H1 recombinant hemagglutinin (rHA)/whole virus or seasonal H1 rHA. Neutralizing MAbs, MA-2070 and MA-M, were obtained after pandemic A/California/07/2009 (H1N1) virus/rHA immunization(s). Both MAbs reacted specifically with rHA from A/California/07/2009 and A/England/195/2009 in ELISA. MA-2070 bound rHA of A/California/07/2009 with high affinity (KD = 51.36 ± 9.20 nM) and exhibited potent in vitro neutralization (IC50 = 2.50 μg/mL). MA-2070 bound within the stem domain of HA. MA-M exhibited both hemagglutination inhibition (HI, 1.50 μg/mL) and in vitro neutralization (IC50 = 0.66 μg/mL) activity against the pandemic A/California/07/2009 virus and showed higher binding affinity (KD = 9.80 ± 0.67 nM) than MA-2070. MAb, MA-H generated against the seasonal A/Solomon Islands/03/2006 (H1N1) rHA binds within the head domain and bound the seasonal H1N1 (A/Solomon Islands/03/2006 and A/New Caledonia/20/1990) rHAs with high affinity (KD; 0.72-8.23 nM). MA-H showed high HI (2.50 μg/mL) and in vitro neutralization (IC50 = 2.61 μg/mL) activity against the A/Solomon Islands/03/2006 virus. All 3 MAbs failed to react in ELISA with rHA from various strains of H2N2, H3N2, H5N1, H7N9, and influenza virus B, suggesting their specificity for either pandemic or seasonal H1N1 influenza virus. The MAbs reported here may be useful in developing diagnostic assays.

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