Alexander Kagansky

Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin

Summary The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3Sp, the ortholog of budding yeast Scm3Sc. Scm3Sp depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3Sp coaffinity purifies with CENP-ACnp1 and associates with CENP-ACnp1 in vitro, yet localizes independently of intact CENP-ACnp1 chromatin and is differentially released from chromatin. While Scm3Sc has been proposed to form a unique hexameric nucleosome with CENP-ACse4 and histone H4 at budding yeast point centromeres, we favor a model in which Scm3Sp acts as a CENP-ACnp1 receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-ACnp1 from the Sim3 escort and mediate assembly of CENP-ACnp1 into subkinetochore chromatin.

Stc1: A Critical Link between RNAi and Chromatin Modification Required for Heterochromatin Integrity

Summary In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

Synthetic heterochromatin bypasses RNAi and centromeric repeats to establish functional centromeres.

Synthetic Centromere Every eukaryotic chromosome must have a centromere where the cell division machinery latches onto each chromosome pair to ensure an even apportioning of the genetic material between daughter cells. The characteristic (but not conserved) repeat sequences associated with most centromeres are thought to be required to induce an RNA interference (RNAi) response and thereby promote the formation of heterochromatin, needed for centromere function. Kagansky et al. (p. 1716) now show in fission yeast that these outer repeat sequences can be replaced in their entirety by very short sequences that recruit an enzyme, Clr4, which promotes the formation of heterochromatin in the absence of RNAi. Thus, flanking heterochromatin, regardless of its derivation, is all that is required for the formation of a functional centromere. A tethered methyltransferase induces the tight packing of DNA, the formation of a kinetochore, and chromosome segregation. In the central domain of fission yeast centromeres, the kinetochore is assembled on CENP-ACnp1 nucleosomes. Normally, small interfering RNAs generated from flanking outer repeat transcripts direct histone H3 lysine 9 methyltransferase Clr4 to homologous loci to form heterochromatin. Outer repeats, RNA interference (RNAi), and centromeric heterochromatin are required to establish CENP-ACnp1 chromatin. We demonstrated that tethering Clr4 via DNA-binding sites at euchromatic loci induces heterochromatin assembly, with or without active RNAi. This synthetic heterochromatin completely substitutes for outer repeats on plasmid-based minichromosomes, promoting de novo CENP-ACnp1 and kinetochore assembly, to allow their mitotic segregation, even with RNAi inactive. Thus, the role of outer repeats in centromere establishment is simply the provision of RNAi substrates to direct heterochromatin formation; H3K9 methylation-dependent heterochromatin is alone sufficient to form functional centromeres.

Splicing Factors Facilitate RNAi-Directed Silencing in Fission Yeast

Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.

Restricted epigenetic inheritance of H3K9 methylation

Inheritance of a covalent histone modification Genomic DNA is the repository of all genetic information and is packaged into chromatin. Chromatin is also a repository of regulatory information in the form of covalent marks added to the histones that package the DNA. These marks can determine tissue- and organ-specific gene expression patterns, which must be transmitted to daughter cells to maintain their identity. Ragunathan et al. and Audergon et al. show that in fission yeast, a chromatin mark, like genetic information, can be inherited across many cell generations. The mark can be inherited independently of DNA sequence, DNA methylation, or RNA interference. Thus, histone marks constitute true epigenetic information. Science, this issue 10.1126/science.1258699; see also p. 132 Covalent histone modifications are inherited epigenetically across many generations. Posttranslational histone modifications are believed to allow the epigenetic transmission of distinct chromatin states, independently of associated DNA sequences. Histone H3 lysine 9 (H3K9) methylation is essential for heterochromatin formation; however, a demonstration of its epigenetic heritability is lacking. Fission yeast has a single H3K9 methyltransferase, Clr4, that directs all H3K9 methylation and heterochromatin. Using releasable tethered Clr4 reveals that an active process rapidly erases H3K9 methylation from tethering sites in wild-type cells. However, inactivation of the putative histone demethylase Epe1 allows H3K9 methylation and silent chromatin maintenance at the tethering site through many mitotic divisions, and transgenerationally through meiosis, after release of tethered Clr4. Thus, H3K9 methylation is a heritable epigenetic mark whose transmission is usually countered by its active removal, which prevents the unauthorized inheritance of heterochromatin.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated ‘centrochromatin’, despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity – kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere‐derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5′‐monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well‐conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non‐coding sequence in itself may be of importance. Consistent with this, secondary structure‐probing experiments indicate that this centromeric RNA is partially double‐stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Δ cells. Our data suggest a pathway for siRNA generation that is distinct from the well‐documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin‐like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post‐transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double‐stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of high levels of Argonaute‐associated small interfering RNAs (siRNAs), which can induce heterochromatin formation at a remote locus. This silencing does not require any manipulation apart from hairpin expression. In cells expressing a ura4+–GFP fusion gene, production of GFP siRNAs causes the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RNA‐dependent RNA polymerase Rdp1 (RDRPRdp1) function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRPRdp1 can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.

Silencing Mediated by the Schizosaccharomyces pombe HIRA Complex Is Dependent upon the Hpc2-Like Protein, Hip4

Background HIRA (or Hir) proteins are conserved histone chaperones that function in multi-subunit complexes to mediate replication-independent nucleosome assembly. We have previously demonstrated that the Schizosaccharomyces pombe HIRA proteins, Hip1 and Slm9, form a complex with a TPR repeat protein called Hip3. Here we have identified a new subunit of this complex. Methodology/Principal Findings To identify proteins that interact with the HIRA complex, rapid affinity purifications of Slm9 were performed. Multiple components of the chaperonin containing TCP-1 complex (CCT) and the 19S subunit of the proteasome reproducibly co-purified with Slm9, suggesting that HIRA interacts with these complexes. Slm9 was also found to interact with a previously uncharacterised protein (SPBC947.08c), that we called Hip4. Hip4 contains a HRD domain which is a characteristic of the budding yeast and human HIRA/Hir-binding proteins, Hpc2 and UBN1. Co-precipitation experiments revealed that Hip4 is stably associated with all of the other components of the HIRA complex and deletion of hip4+ resulted in the characteristic phenotypes of cells lacking HIRA function, such as temperature sensitivity, an elongated cell morphology and hypersensitivity to the spindle poison, thiabendazole. Moreover, loss of Hip4 function alleviated the heterochromatic silencing of reporter genes located in the mating type locus and centromeres and was associated with increased levels of non-coding transcripts derived from centromeric repeat sequences. Hip4 was also found to be required for the distinct form of silencing that controls the expression of Tf2 LTR retrotransposons. Conclusions/Significance Overall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci.

Antibacterial and antibiotic potentiating activities of tropical marine sponge extracts

Increasing prevalence of antibiotic resistance has led research to focus on discovering new antimicrobial agents derived from the marine biome. Although ample studies have investigated sponges for their bioactive metabolites with promising prospects in drug discovery, the potentiating effects of sponge extracts on antibiotics still remains to be expounded. The present study aimed to investigate the antibacterial capacity of seven tropical sponges collected from Mauritian waters and their modulatory effect in association with three conventional antibiotics namely chloramphenicol, ampicillin and tetracycline. Disc diffusion assay was used to determine the inhibition zone diameter (IZD) of the sponge total crude extracts (CE), hexane (HF), ethyl acetate (EAF) and aqueous (AF) fractions against nine standard bacterial isolates whereas broth microdilution method was used to determine their minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and antibiotic potentiating activity of the most active sponge extract. MIC values of the sponge extracts ranged from 0.039 to 1.25mg/mL. Extracts from Neopetrosia exigua rich in beta-sitosterol and cholesterol displayed the widest activity spectrum against the 9 tested bacterial isolates whilst the best antibacterial profile was observed by its EAF particularly against Staphylococcus aureus and Bacillus cereus with MIC and MBC values of 0.039mg/mL and 0.078mg/mL, respectively. The greatest antibiotic potentiating effect was obtained with the EAF of N. exigua (MIC/2) and ampicillin combination against S. aureus. These findings suggest that the antibacterial properties of the tested marine sponge extracts may provide an alternative and complementary strategy to manage bacterial infections.