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Histone post-translational modifications (PTMs) are associated with epigenetic states that form the basis for cell-type-specific gene expression.sup.1,2. Once established, histone PTMs can be maintained by positive feedback involving enzymes that recognize a pre-existing histone modification and catalyse the same modification on newly deposited histones. Recent studies suggest that in wild-type cells, histone PTM-based positive feedback is too weak to mediate epigenetic inheritance in the absence of other inputs.sup.3-7. RNA interference (RNAi)-mediated histone H3 lysine 9 methylation (H3K9me) and heterochromatin formation define a potential epigenetic inheritance mechanism in which positive feedback involving short interfering RNA (siRNA) amplification can be directly coupled to histone PTM positive feedback.sup.8-14. However, it is not known whether the coupling of these two feedback loops can maintain epigenetic silencing independently of DNA sequence and in the absence of enabling mutations that disrupt genome-wide chromatin structure or transcription.sup.15-17. Here, using the fission yeast Schizosaccharomyces pombe, we show that siRNA-induced H3K9me and silencing of a euchromatic gene can be epigenetically inherited in cis during multiple mitotic and meiotic cell divisions in wild-type cells. This inheritance involves the spreading of secondary siRNAs and H3K9me3 to the targeted gene and surrounding areas, and requires both RNAi and H3K9me, suggesting that the siRNA and H3K9me positive-feedback loops act synergistically to maintain silencing. By contrast, when maintained solely by histone PTM positive feedback, silencing is erased by H3K9 demethylation promoted by Epe1, or by interallelic interactions that occur after mating to cells containing an expressed allele even in the absence of Epe1. These findings demonstrate that the RNAi machinery can mediate transgenerational epigenetic inheritance independently of DNA sequence or enabling mutations, and reveal a role for the coupling of the siRNA and H3K9me positive-feedback loops in the protection of epigenetic alleles from erasure. In Schizosaccharomyces pombe, histone H3K9 methylation acts synergistically with short interfering RNA to perpetuate gene silencing during multiple mitotic and meiotic cell divisions., Author(s): Ruby Yu [sup.1] , Xiaoyi Wang [sup.1] , Danesh Moazed [sup.1] Author Affiliations: (1) Department of Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, Boston, USA Main [...]

Author(s): Gloria Jih [1, 2]; Nahid Iglesias [1, 2]; Mark A. Currie [1, 2]; Natarajan V. Bhanu [3]; Joao A. Paulo [1]; Steven P. Gygi [1]; Benjamin A. Garcia [3]; [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.01.019 Byline: Mario Halic (1), Danesh Moazed (1)(2) Keywords: RNA; DNA Abstract: Assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here, we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associates with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA (2) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, 02115, USA Article History: Received 1 September 2009; Revised 16 November 2009; Accepted 6 January 2010 Article Note: (miscellaneous) Published: February 18, 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.07.013 Byline: Manisha Sinha (1)(2), Shinya Watanabe (1), Aaron Johnson (3), Danesh Moazed (3)(4), Craig L. Peterson (1) Keywords: DNA; PROTEINS Abstract: Heterochromatin plays a key role in protection of chromosome integrity by suppressing homologous recombination. In Saccharomyces cerevisiae, Sir2p, Sir3p, and Sir4p are structural components of heterochromatin found at telomeres and the silent mating-type loci. Here we have investigated whether incorporation of Sir proteins into minichromosomes regulates early steps of recombinational repair in vitro. We find that addition of Sir3p to a nucleosomal substrate is sufficient to eliminate yRad51p-catalyzed formation of joints, and that this repression is enhanced by Sir2p/Sir4p. Importantly, Sir-mediated repression requires histone residues that are critical for silencing in vivo. Moreover, we demonstrate that the SWI/SNF chromatin-remodeling enzyme facilitates joint formation by evicting Sir3p, thereby promoting subsequent Rad54p-dependent formation of a strand invasion product. These results suggest that recombinational repair in the context of heterochromatin presents additional constraints that can be overcome by ATP-dependent chromatin-remodeling enzymes. Author Affiliation: (1) Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA (2) Interdisciplinary Graduate Program, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA (3) Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA (4) Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Article History: Received 5 January 2009; Revised 23 April 2009; Accepted 6 July 2009 Article Note: (miscellaneous) Published: September 17, 2009

Small RNA molecules of about 20-30 nucleotides have emerged as powerful regulators of gene expression and genome stability. Studies in fission yeast and multicellular organisms suggest that effector complexes, directed by small RNAs, target nascent chromatin-bound non-coding RNAs and recruit chromatin-modifying complexes. Interactions between small RNAs and nascent non-coding transcripts thus reveal a new mechanism for targeting chromatin-modifying complexes to specific chromosome regions and suggest possibilities for how the resultant chromatin states may be inherited during the process of chromosome duplication., RNA interference (RNAi) originally referred to the ability of exogenously introduced double-stranded RNA (dsRNA) molecules to silence the expression of homologous sequences in the nematode Caenorhabditis elegans (1). It has [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.03.038 Byline: Marc Buhler (1), Wilhelm Haas (1), Steven P. Gygi (1), Danesh Moazed (1) Keywords: DNA; RNA Abstract: In fission yeast, the RNAi pathway is required for heterochromatin-dependent silencing of transgene insertions at centromeric repeats and acts together with other pathways to silence transgenes at the silent mating-type locus. Here, we show that transgene transcripts at centromeric repeats are processed into siRNAs and are therefore direct targets of RNAi. Furthermore, we show that Cid14, a member of the Trf4/5 family of poly(A) polymerases, has poly(A) polymerase activity that is required for heterochromatic gene silencing. Surprisingly, while siRNA levels in cid14[DELTA] cells are dramatically reduced, the structural integrity of heterochromatin appears to be preserved. Cid14 resides in a complex similar to the TRAMP complex found in budding yeast, which is part of a nuclear surveillance mechanism that degrades aberrant transcripts. Our findings indicate that polyadenylation by a TRAMP-like complex contributes to robust silencing of heterochromatic genes in fission yeast via the recruitment of the exosome and/or the RNAi machinery. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 20 December 2006; Revised 14 February 2007; Accepted 16 March 2007 Article Note: (miscellaneous) Published: May 17, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.02.002 Byline: Corey A. Morris (1), Danesh Moazed (1) Abstract: Centromere assembly provides a unique example of how elaborate protein structures can be assembled onto DNA, independent of sequence, and then stably propagated through numerous cell divisions. Here, we review the possible epigenetic strategies that organisms ranging from yeast to human use to assemble and propagate active centromeres. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

The protein interaction network involved in suppressing homologous recombination within the rDNA locus is analyzed using affinity purification combined with highly sensitive mixture mass spectrometry. A model is suggested in which regulator of nucleolar silencing and telophase exit [RENT], Tof2 and Lrs4/Csm1 physically clamp rDNA to the cohesin ring, thereby restricting the movement of rDNA sister chromatids relative to each other to inhibit unequal exchange.

The establishment of transcriptional silencing in yeast requires cell-cycle progression, but the nature of this requirement is unknown. Sir2 is a protein deacetylase that is required for gene silencing in yeast. We have used temperature-sensitive alleles of the SIR2 gene to assess Sir2's contribution to silencing as a function of the cell cycle. When examined in vivo, these conditional alleles fall into two classes: one class exhibits a loss of silencing when raised to the nonpermissive temperature regardless of cell-cycle position, while the second class exhibits a mitosis-specific silencing defect. Alleles of the first class have a primary defect in protein deacetylase activity, while the alleles of the second class are specifically defective in Sir2-Sir4 interactions at nonpermissive temperatures. Using a SIR2 temperature-sensitive allele, we show that silencing can be established at the HML locus during progression through the [G.sub.2]/M-[G.sub.1] interval. These results suggest that yeast heterochromatin undergoes structural transitions as a function of the cell cycle and support the existence of a critical assembly step for silent chromatin in mitosis.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.025 Byline: Marc Buhler (1), Andre Verdel (1), Danesh Moazed (1) Abstract: In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4.sup.+ gene silences ura4.sup.+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4.sup.+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4.sup.+ siRNAs to silence a second ura4.sup.+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4.sup.+ , or ura4.sup.+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 11 November 2005; Revised 24 February 2006; Accepted 20 April 2006 Article Note: (miscellaneous) Published: June 1, 2006

The association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails is examined. The results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and O-acetyl-ADP-ribose (AAR) synthesis.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.03.035 Byline: Gunn-Guang Liou (1), Jason C. Tanny (1), Ryan G. Kruger (2), Thomas Walz (1), Danesh Moazed (1) Abstract: Assembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 (2) Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Article Note: (miscellaneous) Published: May 19, 2005

Eukaryotic DNA is organized into structurally distinct domains that regulate gene expression and chromosome behavior. Epigenetically heritable domains of heterochromatin control the structure and expression of large chromosome domains and are required for proper chromosome segregation. Recent studies have identified many of the enzymes and structural proteins that work together to assemble heterochromatin. The assembly process appears to occur in a stepwise manner involving sequential rounds of histone modification by silencing complexes that spread along the chromatin fiber by self-oligomerization, as well as by association with specifically modified histone amino-terminal tails. Finally, an unexpected role for noncoding RNAs and RNA interference in the formation of epigenetic chromatin domains has been uncovered., Chromatin and higher order chromosome structures play a central role in nearly every aspect of DNA biology in eukaryotes. Processes ranging from gene expression to chromosome dynamics during cell division [...]