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Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA–protein complexes is required for robust pairing. Moreover, the lncRNA–protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA–protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes., During meiosis, pairing of homologous chromosomes is critical for sexual reproduction. Here the authors reveal in S. pombe the role of lncRNA–protein complexes during the pairing of homologues chromosomes that assemble at specific chromosomal loci to mediate recognition of the pairs.

In eukaryotic cells, DNA is tightly compacted as chromatin. Chromatin states must be dynamically changed to increase the accessibility of transcription factors (TFs) to chromatin or to stably silence genes by higher-order chromatin structures known as heterochromatin. The regulation of chromatin needs cooperative action performed by a variety of proteins. Specific binding of TFs to target DNA is the initial step of chromatin regulation and promotes changes in the post-translational modifications of histone tails, which themselves are recognized by a set of histone reader proteins. Recent biochemical studies have revealed that some TFs that recognize specific DNA sequences can also interact with histones. Furthermore, histone reader proteins that recognize specific histone tail modifications have been shown to have the ability to directly bind to DNA. In this commentary, we introduce recent advances in the elucidation of how chromatin regulating factors recognize nucleosomal targets.

Triple-negative breast cancer (TNBC) is very aggressive and lacks specific therapeutic targets. Ribosome RNAs (rRNAs) are central components of ribosomes and transcribed in nucleoli, and the level of rRNA transcription greatly affects ribosome production and cell proliferation. We have reported that an epigenetic protein, KDM2A, exists in nucleoli and reduces rRNA transcription on glucose starvation. However, the molecular mechanism is still unclear. The purpose of this study is to examine the KDM2A-dependent regulation mechanism of rRNA transcription. In this study, we turned our attention to the nucleolar accumulation of KDM2A. We found that KDM2A had multiple regions for its nucleolar localization, and one of the regions was directly bound by heterochromatin protein 1γ (HP1γ) using valine 801 in the LxVxL motif of KDM2A. A knockdown of HP1γ or a point mutation of valine 801 in KDM2A decreased the nucleolar accumulation of KDM2A, and suppressed the reduction of rRNA transcription on glucose starvation. These results uncovered a novel function of HP1γ: the regulation of rRNA transcription, and suggested that HP1γ stimulates the nucleolar accumulation of KDM2A to support the KDM2A-dependent regulation of rRNA transcription. HP1γ was expressed in cancer cells in all breast carcinoma tissues examined, including TNBC tissues. A knockdown of HP1γ in a TNBC cell line, MDA-MB-231 cells, reduced the nucleolar accumulation of KDM2A, and suppressed the reductions of rRNA transcription and cell proliferation on glucose starvation. These results suggest that the KDM2A-dependent regulation of rRNA transcription requires HP1γ, and thus may be applicable to the treatment of TNBC.

The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing theHoxgenes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

Chromatin serves as a platform for a multitude of biological processes, including transcription and co-transcriptional RNA processing. Consequently, chromatin is likely to be covered with many RNA molecules.Here we describe a simple, reliable, and cross-link-free method for the systematic identification of chromatin-associated RBPs that exhibit RNA-dependent chromatin association.

The PRC2 (Polycomb repressive complex 2) complex is a multi-component histone H3K27 methyltransferase, best known for silencingHoxgenes during embryonic development. The Polycomb-like proteins PHF1, MTF2 and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem (ES) cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark, but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3tK27 in testis, and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

Chromatin serves as a platform for a multitude of biological processes, including transcription and co-transcriptional RNA processing. Consequently, chromatin is likely to be covered with many RNA molecules.Here we describe a simple, reliable, and cross-link-free method for the systematic identification of chromatin-associated RBPs that exhibit RNA-dependent chromatin association.

NAC1 is a cancer-related transcription regulator protein that is overexpressed in various carcinomas, including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation of intranuclear NAC1 in ovarian cancer cells remain poorly understood. In this study, analysis of ovarian cancer cell lysates by fast protein liquid chromatography on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Liquid chromatography-tandem mass spectrometry analysis identified CARM1 as interacting with NAC1 in the protein complex. Furthermore, tissue microarray analysis revealed a significant correlation between CARM1 and NAC1 expression levels. Ovarian cancer patients expressing high levels of NAC1 and CARM1 exhibited poor prognosis after adjuvant chemotherapy. Collectively, our results demonstrate that high expression levels of NAC1 and its novel binding partner CARM1 may serve as an informative prognostic biomarker for predicting resistance to chemotherapy for ovarian cancer.

The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.

Additional file 2. Supplementary data Figures S2â S9.

In eukaryotic cells, there are two chromatin states, silenced and active, and the formation of a so-called boundary plays a critical role in demarcating these regions; however, the mechanisms underlying boundary formation are not well understood. In this study, we focused on S. cerevisiae ADA1, a gene previously shown to encode a protein with a robust boundary function. Ada1 is a component of the histone modification complex Spt-Ada-Gcn5-acetyltransferase (SAGA) and the SAGA-like (SLIK) complex, and it helps to maintain the integrity of these complexes. Domain analysis showed that four relatively small regions of Ada1 (Region I; 66-75 aa, II; 232-282 aa, III; 416-436 aa and IV; 476-488 aa) have a boundary function. Among these, Region II could form an intact SAGA complex, whereas the other regions could not. Investigation of cellular factors that interact with these small regions identified a number of proteasome-associated proteins. Interestingly, the boundary functions of Region II and Region III were affected by depletion of Ump1, a maturation and assembly factor of the 20S proteasome. These results suggest that the boundary function of Ada1 is functionally linked to proteasome processes and that the four relatively small regions in ADA1 form a boundary via different mechanisms.

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

The chromodomain (CD) is a member of the Royal family of conserved chromatin-binding motifs with methylated substrate binding ability, and is often found in ‘readers’ or ‘writers’ of repressive histone marks. The regions upstream or downstream of the CD are generally highly charged. Several previous studies suggested that these charged regions modulate the CD’s chromatin-binding activity. Considering the relatively weak interaction between the CD and a modified histone tail, it is puzzling how the highly charged CD-flanking regions are ‘balanced’ on the highly charged nucleosomes to mediate a modification-dependent interaction. Interestingly, the charge distributions along the CD and surrounding regions appear to be distinct among different types of readers and writers, indicating their functional relevance. Here, we describe and discuss the current understanding of the highly charged CD-flanking regions and the potential experimental concerns caused by the regions.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that plays a crucial role in heterochromatin-mediated gene silencing. We previously showed that mammalian HP1α is constitutively phosphorylated at its N-terminal serine residues by casein kinase II (CK2), and that this phosphorylation enhances HP1α's binding specificity for nucleosomes containing lysine 9-methylated histone H3 (H3K9me). Although the presence of additional HP1α phosphorylation during mitosis was reported more than a decade ago, its biological significance remains largely elusive. Here we found that mitosis-specific HP1α phosphorylation affected HP1α's ability to bind chromatin. Using biochemical and mutational analyses, we showed that HP1α's mitotic phosphorylation was located in its hinge region and was reversibly regulated by Aurora B kinase and serine/threonine phosphatases. In addition, chromatin fractionation and electrophoretic mobility shift assays revealed that hinge region-phosphorylated HP1α was preferentially dissociated from mitotic chromatin and exhibited a reduced DNA-binding activity. Although HP1's mitotic behaviour was previously linked to H3 serine 10 phosphorylation, which blocks the binding of HP1's chromodomain to H3K9me3, our findings suggest that mitotic phosphorylation in HP1α's hinge region also contributes to changes in HP1α's association with mitotic chromatin.

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Various coding and non-coding transcripts are known to associate with chromatin and now there is accumulating evidence that interaction between RNA-binding proteins (RBPs) and RNA molecules regulate not only co-transcriptional mRNA processing, but also other biological processes within the nucleus. Although over a thousand of RBPs have been identified by several mass spectrometry-based methods, it is still unclear which of these RBPs actually associate with chromatin, especially through interaction with RNAs. In addition, biological outcomes of such RBP-RNA-chromatin interactions are yet to be elucidated.Here we describe a simple proteomics-based method for systematic screening of RBPs that are anchored to chromatin and/or insoluble nuclear substructures by RNA molecules. We used RNase A to release such RBPs from chromatin fraction and analyzed ‘RNase A-solubilized’ proteins by mass spectrometry. Using this method, we were able to identify 156 RNase A-solubilized proteins of which 144 were known RBPs/RBP candidates. Interestingly, several key players of the non-homologous end-joining (NHEJ) pathway were enriched in RNase A-solubilized fraction and the RNA-mediated chromatin association of these factors appeared to be dependent on transcriptional elongation. Furthermore, some enzymes involved in metabolic pathways were also released from chromatin and/or an insoluble nuclear structure by RNase A treatment. In summary, our methodology is highly versatile and is potentially a useful tool to unravel new biological functions for RBP-RNA-chromatin interactions.

Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis-specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis-specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis-specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis-specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.

Additional file 1: Figure S1. Fission yeast Hsp70 proteins and their homologs. (A) Phylogenic tree of Hsp70 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are associated with two-letter abbreviations and color-coded to indicate the species: “sp” for Schizosaccharomyces pombe (red), “sc” for Saccharomyces cerevisiae (black), and “dm” for Drosophila melanogaster (blue). (B) Domain structure of Hsp70 proteins. Protein names are depicted as in (A) and their amino acid lengths are shown. Protein domains are drawn as boxes according to the CATH-Gene3D classification. The N-terminal ATPase domains (ATPase) are composed of three internal domains that belong to the CATH superfamilies 3.30.420.40, 3.30.30.30, and 3.90.640.10. Substrate-binding domains (SB) belong to the 2.60.34.10 superfamily. The C-terminal lid domains (Lid) belong to the 1.20.1270.10 superfamily. Predicted localization signals for mitochondria (TargetP-M) and for the endoplasmic reticulum or beyond (TargetP-S) are shown as shaded boxes. Locations of the proteins in the cell are indicated. For clarity, D. melanogaster Hsp70 proteins other than Hsc70-4 are omitted. Figure S2. Hsp40 family proteins in fission yeast. Domain structure of all 26 fission yeast Hsp40 proteins are shown. Protein names and amino acid lengths are indicated. Protein domains are drawn as boxes according to the CATH-Gene3D, Pfam, and Prosite classifications. Predicted localization signals are shown as in Additional file 1: Figure S1. Predicted trans-membrane helix regions (TMhelix) are indicated as gray boxes. The DnaJ domains belong to the CATH superfamily 1.10.287.110. Type-I Hsp40 proteins are characterized by C-terminal (purple) and central (pink) domains that belong to the CATH superfamilies 2.60.260.20 and 2.10.230.10, respectively. The type-II Hsp40 protein Psi1 also contains the C-terminal domain but lacks the central domain. The other proteins are classified as the type-III Hsp40 proteins. Figure S3. Fission yeast type-I and -II Hsp40 proteins and their homologs. (A) Phylogenic tree of Hsp40 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are depicted as in Additional file 1: Figure S1. (B) Domain structure of Hsp40 proteins. Protein names are depicted as in (A) and amino acid lengths are shown. Protein domains and predicted localization signals are drawn as in Additional file 1: Figure S2. Locations of proteins in the cell are indicated. For clarity, D. melanogaster Hsp40 proteins other than Droj2 are omitted. Figure S4. Schematic representation of marker integration sites. The ade6+ and ura4+ marker genes were located in the SphI site of otr1R (otr1R(SphI)::ade6+) and in the NcoI site of imr1L (imr1L(NcoI)::ura4+), respectively. Figure S5. Hsp90 and Mas5 are dispensable for red pigment formation. Cells were spotted on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for six days. Figure S6. Derepression of marker genes inserted in the pericentromere. (A) Cells were serially diluted, spotted on normal EMMS plates and EMMS lacking adenine (EMMS - adenine) or uracil (EMMS - uracil), and incubated at 30°C for 6 days. (B) Strand-specific RT-qPCR for the ura4 marker gene. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). (C) Strand-specific RT-qPCR for the ade6 and ura4 genes located in the endogenous loci (ade6+ and ura4+) and in the pericentromere (otr1R::ade6+ and imr1L::ura4+). Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). **P

Chromobox 2 (CBX2), a component of polycomb repressive complex 1 (PRC1), binds lysine 27-methylated histone H3 (H3K27me3) via its chromodomain (CD) and plays a critical role in repressing developmentally regulated genes. The phosphorylation of CBX2 has been described in several studies, but the biological implications of this modification remain largely elusive. Here, we show that CBX2's phosphorylation plays an important role in its nucleosome binding. CBX2 is stably phosphorylated in vivo, and domain analysis showed that residues in CBX2's serine-rich (SR) region are the predominant phosphorylation sites. The serine residues in an SR region followed by an acidic-residue (AR) cluster coincide with the consensus target of casein kinase II (CK2), and CK2 efficiently phosphorylated the SR region in vitro. A nucleosome pull-down assay revealed that CK2-phosphorylated CBX2 had a high specificity for H3K27me3-modified nucleosomes. An electrophoretic mobility-shift assay showed that CK2-mediated phosphorylation diminished CBX2's AT-hook-associated DNA-binding activity. Mutant CBX2 lacking the SR region or its neighboring AR cluster failed to repress the transcription of p21, a gene targeted by PRC1. These results suggest that CBX2's phosphorylation is critical for its transcriptional repression of target genes.

In fission yeast, the formation of centromeric heterochromatin is induced through the RNA interference (RNAi)-mediated pathway. Some pre-mRNA splicing mutants (prp) exhibit defective formation of centromeric heterochromatin, suggesting that splicing factors play roles in the formation of heterochromatin, or alternatively that the defect is caused by impaired splicing of pre-mRNAs encoding RNAi factors. Herein, we demonstrate that the splicing factor spPrp16p is enriched at the centromere, and associates with Cid12p (a factor in the RNAi pathway) and the intron-containing dg ncRNA. Interestingly, removal of the dg intron, mutations of its splice sites, or replacement of the dg intron with an euchromatic intron significantly decreased H3K9 dimethylation. We also revealed that splicing of dg ncRNA is repressed in cells and its repression depends on the distance from the transcription start site to the intron. Inefficient splicing was also observed in other intron-containing centromeric ncRNAs, dh and antisense dg, and splicing of antisense dg ncRNA was repressed in the presence of the RNAi factors. Our results suggest that the introns retained in centromeric ncRNAs work as facilitators, co-operating with splicing factors assembled on the intron and serving as a platform for the recruitment of RNAi factors, in the formation of centromeric heterochromatin., Author summary Formation of centromeric heterochromatin is required for correct segregation of sister chromatids during mitosis. In fission yeast, formation of heterochromatin at centromeres is performed through the RNA interference (RNAi) system, which involves processing of noncoding RNAs transcribed from the centromeres. We found that the intron in the centromeric dg ncRNAs facilitates formation of centromeric heterochromatin in fission yeast. We showed that the splicing factor spPrp16p associates with the RNAi factor and intron-containing dg ncRNA. Removal of or mutations in the dg intron significantly decreased H3K9 dimethylation, suggesting that the intron and associated splicing factors serve as a platform for recruitment of RNAi factors. Inefficient splicing is a hallmark of intron-containing centromeric ncRNAs. Such repression of splicing seems to be important for facilitation of heterochromatin formation. Introns in euchromatic regions are removed by splicing to generate functional RNAs, whereas centromeric introns are retained in ncRNAs by splicing repression and play roles in gene silencing. Our findings shed light on the novel roles of introns in epigenetic regulation of gene expression and heterochromatin formation.

Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here, we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing histone deacetylase complex and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two histone deacetylase complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the Caenorhabditis elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1's phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α's intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin, and plays a crucial role in forming higher-order chromatin structures. HP1 has an N-terminal chromodomain and a C-terminal chromo shadow domain, linked by an unstructured hinge region. Although biochemical and structural studies have revealed each domain's properties, little is known about the mechanisms by which these domains cooperate to carry out HP1's function in forming higher-order chromatin structures. In this review, we summarize HP1's biochemical and structural properties and highlight the latest findings regarding HP1's interactions with nucleosomes.

The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α’s N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α’s chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α’s N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α’s N-terminal tail underlies the enhancement in H3 binding due to phosphorylation.

Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.

In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1 + mutant allele ( ers1-C62 ) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6 + reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.

Post-translational modifications of histones are critical not only for local regulation of gene expression, but also for higher-order structure of the chromosome and genome organization in general. These modifications enable a preset state to be maintained over subsequent generations and thus provide an epigenetic level of regulation. Heterochromatic regions of the genome are epigenetically regulated to maintain a ‘‘silent state’’ and protein coding genes inserted into these regions are subject to the same epigenetic silencing. The fission yeast Schizosaccharomyces pombe has well characterized regions of heterochromatin and has proven to be a powerful model for elucidation of epigenetic silencing mechanisms. Research in S. pombe led to the breakthrough discovery that epigenetic silencing is not solely a chromatin-driven transcriptional repression and that RNA interference of nascent transcripts can guide epigenetic silencing and associated histone modifications. Over the last 10 years, an eloquent integration of genetic and biochemical studies have greatly propelled our understanding of major players and effector complexes for regulation of RNAi-mediated epigenetic silencing in S. pombe. Here, we review recent research related to regulation of the epigenetic state in S. pombe heterochromatin, focusing specifically on the mechanisms by which transcription and RNA processing interact with the chromatin modification machinery to maintain the epigenetically silent state.

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.

The binding of heterochromatin protein 1 (HP1) to lysine 9-methylated histone H3 (H3K9me) is an essential step in heterochromatin assembly. Chp2, an HP1-family protein in the fission yeast Schizosaccharomyces pombe, is required for heterochromatic silencing. Chp2 recruits SHREC, a multifunctional protein complex containing the nucleosome remodeler Mit1 and the histone deacetylase Clr3. Although the targeting of SHREC to chromatin is thought to occur via two distinct modules regulated by the SHREC components Chp2 and Clr2, it is not clear how Chp2's chromatin binding regulates SHREC function. Here, we show that H3K9me binding by Chp2's chromodomain (CD) is essential for Chp2's silencing function and for SHREC's targeting to chromatin. Cells expressing a Chp2 mutant with defective H3K9me binding (Chp2-W199A) have a silencing defect, with a phenotype similar to that of chp2-null cells. Genetic analysis using a synthetic silencing system revealed that a Chp2 mutant and SHREC-component mutants had similar phenotypes, suggesting that Chp2's function also affects SHREC's chromatin binding. Size-exclusion chromatography of native protein complexes showed that Chp2-CD's binding of H3K9me3 ensures Clr3's chromatin binding, and suggested that SHREC's chromatin binding is mediated by separable functional modules. Interestingly, we found that the stability of the Chp2 protein depended on the Clr3 protein's histone deacetylase activity. Our findings demonstrate that Chp2's H3K9me binding is critical for SHREC function and that the two modules within the SHREC complex are interdependent.

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Delta set13 mutant cells. Delta set13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.

PALB2 physically and functionally connects the proteins encoded by the BRCA1 and BRCA2 breast and ovarian cancer genes into a DNA-damage-response network. However, it remains unclear how these proteins associate with chromatin that contains damaged DNA. We show here that PALB2 binds directly to a conserved chromodomain protein, MRG15, which is a component of histone acetyltransferase-deacetylase complexes. This interaction was identified by analysis of purified MRG15- and PALB2-containing protein complexes. Furthermore, MRG15 interacts with the entire BRCA complex, which contains BRCA1, PALB2, BRCA2 and RAD51. Interestingly, MRG15-deficient cells, similarly to cells deficient in PALB2 or BRCA2, showed reduced efficiency for homology-directed DNA repair and hypersensitivity to DNA interstrand crosslinking agents. Additionally, knockdown of MRG15 diminished the recruitment of PALB2, BRCA2 and RAD51 to sites of DNA damage and reduced chromatin loading of PALB2 and BRCA2. These results suggest that MRG15 mediates DNA-damage-response functions of the BRCA complex in chromatin.

Summary Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1β, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.