Summary HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9 methylated chromatin in vitro relies on a newly identified interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl-mark recognition is highly sensitive to inter-nucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled, and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

SUMMARY Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1Cdt2 ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1Cdt2 complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.

The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.

The universal bacterial transcription elongation factor NusA mediates elongation activities of RNA polymerase. By itself, NusA induces transcription pausing and facilitates intrinsic termination, but NusA also is a cofactor of antiterminators that antagonize pausing and prevent termination. We show that NusA is required for lambda-related phage 82 antiterminator Q(82) to construct a stable complex in which RNA-based termination mechanisms have restricted access to the emerging transcript; this result suggests a locale for both Q(82) and NusA near the beta flap domain of RNA polymerase. Furthermore, as NusA is not required for the antipausing activity of Q(82) in vitro, we distinguish two distinct activities of antiterminators, namely antipausing and RNA occlusion, and discuss their roles in Q(82) function.

Long noncoding RNAs (lncRNAs) can trigger repressive chromatin, but how they recruit silencing factors remains unclear. In Schizosaccharomyces pombe, heterochromatin assembly on transcribed noncoding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks a repressive chromatin mark (H3K9me [methylated Lys9 on histone H3]), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We show that the S. pombe ortholog Seb1 is associated with pericentromeric lncRNAs. Individual mutation of dcr1 + (Dicer) or seb1 + results in equivalent partial reductions of pericentromeric H3K9me levels, but a double mutation eliminates this mark. Seb1 functions independently of RNAi by recruiting the NuRD (nucleosome remodeling and deacetylase)-related chromatin-modifying complex SHREC (Snf2-HDAC [histone deacetylase] repressor complex).Supplemental material is available for this article.Received November 1, 2012; revised version accepted August 7, 2013. A major unsolved question in chromatin biology is how long intergenic noncoding RNAs (lincRNAs) trigger the formation of repressed chromatin. A large number of mammalian lincRNAs have been identified by systematic studies ). Many of these ncRNAs associate with chromatin-modifying complexes (Khalil et al. 2009). Multiple models have been proposed for how these ncRNAs are recognized and recruit chromatinmodifying factors, but little is understood mechanistically (Guttman and Rinn 2012).In Schizosaccharomyces pombe, pericentromeric heterochromatin assembly is promoted by transcription of the dg and dh repeat sequences by RNA polymerase II (Pol II) (Djupedal et al. 2005;Kato et al. 2005). The corresponding long ncRNAs (lncRNAs) are converted into dsRNAs and processed into siRNAs by the combined action of RNA-directed RNA polymerase complex (RDRC) and Dicer (Dcr1) (Verdel et al. 2009;Lejeune and Allshire 2011). siRNAs produced by Dicer are bound by Argonaute (Ago1), a component of the RNA-induced transcriptional silencing (RITS) complex, and together they promote both degradation of pericentromeric ncRNAs and transcriptional silencing via repressive histone methylation (Verdel et al. 2004). These complexes in turn recruit the Clr4 methyltransferase complex (CLRC), which methylates Lys9 on histone H3 (H3K9me) (Nakayama et al. 2001;Zhang et al. 2008). The methyl mark serves as a binding platform for the repressive HP1 proteins Swi6 and Chp2 (Thon and Verhein-Hansen 2000;Bannister et al. 2001;Fischer et al. 2009). Both proteins promote the recruitment of SHREC (Snf2-HDAC [histone deacetylase] repressor complex) to pericentromeric heterochromatin (Sugiyama et al. 2007;Sadaie et al. 2008). Moreover, Chp2 has been found to associate with SHREC to form the SHREC2 complex (SHREC complex associated with Chp2) (Motamedi et al. 2008). The core of SHREC consists of silencing factors Clr1 and Clr2, the HDAC Clr3, and the putative chromatin-remodeling enzyme Mit1 (Sugiyama et al. 2007). SHREC and SHREC2 resemble the mammalian nucle...