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Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression.sup.1-4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3.sup.5-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me.sup.12-15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me--which would lead to aberrant gene silencing and epigenetic instability--is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue.sup.16 suggest that the mechanism described here is broadly conserved.An autoinhibitory conformation of the histone H3K9 methyltransferase Clr4 of Schizosaccharomyces pombe helps to prevent aberrant heterochromatin formation and maintains epigenetic stability., Author(s): Nahid Iglesias [sup.1] [sup.2] , Mark A. Currie [sup.1] [sup.2] , Gloria Jih [sup.1] [sup.2] , Joao A. Paulo [sup.1] , Nertila Siuti [sup.1] [sup.2] , Marian Kalocsay [sup.1] [...]

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.2007.09.014 Byline: Jurgi Camblong (1), Nahid Iglesias (1), Celine Fickentscher (1), Guennaelle Dieppois (1), Francoise Stutz (1) Keywords: RNA Abstract: Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications. Author Affiliation: (1) Department of Cell Biology, Sciences III, University of Geneva, 30 Quai E. Ansermet, 1211 Geneva 4, Switzerland Article History: Received 2 February 2007; Revised 11 July 2007; Accepted 6 September 2007 Article Note: (miscellaneous) Published: November 15, 2007

Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein-DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i.e., ribosomal DNA (rDNA).