Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.

Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells.centromeres ͉ RNA interference

Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin. Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.

Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1000 pediatric tumors to define the landscape of somatic mutations in epigenetic regulators in pediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different pediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukemia, medulloblastoma, and a paucity of mutations in low-grade glioma, and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A, and ZMYM3. Importantly, we identify novel loss-of-function mutations in the ubiquitin-specific-processing protease 7 (USP7) in pediatric leukemia, which result in a decrease in deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in pediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.

Transcription factor DRTF1/E2F coordinates events in the cell cycle with transcription by its cyclical interactions with important regulators of cellular proliferation like the retinoblastoma tumour-suppressor gene product (Rb) and the Rb-related protein, p107 (refs 1-8). DRTF1/E2F binding sites occur in the control regions of genes involved in proliferation, and both Rb and p107 repress the capacity of DRTF1/E2F to activate transcription (refs 11, 12; M. Zamanian and N.B.L.T., manuscript submitted). Mutant Rb proteins isolated from tumour cells are unable to bind DRTF1/E2F (refs 11-13), and certain viral oncoproteins, such as adenovirus E1A, sequester Rb and p107 in order to free active DRTF1/E2F (refs 5, 11, 12, 14, 15). Here we report the isolation of a complementary DNA encoding DRTF1-polypeptide-1 (DP-1), a major sequence-specific binding protein that is present in DRTF1/E2F, including Rb- and p107-associated DRTF1/E2F. The DNA-binding domain of DP-1 contains a region that resembles that of E2F-1 (refs 16, 17), and recognizes the same sequence. DRTF1/E2F thus appears to contain at least two sequence-specific DNA-binding proteins.

Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.

We have identified a novel promoter element that confers M/Gl-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G 1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcml binding site to which Mcml binds in vitro, and an Mcml-VP16 fusion, which places a constitutive activator on Mcml-binding sites in vivo, can deregulate ECB-containing promoters. Mcml is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcml mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication.

A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.