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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 modifications of histones are inherited epigenetically across many generations. Changes in histone posttranslational modifications are associated with epigenetic states that define distinct patterns of gene expression. It remains unclear whether epigenetic information can be transmitted through histone modifications independently of specific DNA sequence, DNA methylation, or RNA interference. Here we show that, in the fission yeast Schizosaccharomyces pombe , ectopically induced domains of histone H3 lysine 9 methylation (H3K9me), a conserved marker of heterochromatin, are inherited through several mitotic and meiotic cell divisions after removal of the sequence-specific initiator. The putative JmjC domain H3K9 demethylase, Epe1, and the chromodomain of the H3K9 methyltransferase, Clr4/Suv39h, play opposing roles in maintaining silent H3K9me domains. These results demonstrate how a direct “read-write” mechanism involving Clr4 propagates histone modifications and allows histones to act as carriers of epigenetic information.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression 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 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 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 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.

Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In Saccharomyces cerevisiae, heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading. Inside plant, fungi and animal cells, DNA wraps around disc-shaped histone proteins to form structures called nucleosomes. Chains of nucleosomes, each with a small stretch of DNA, help to package meters of genetic material into a compact form called chromatin in the cell’s nucleus. Changes to how chromatin is organized can affect how genes switch on and off. Critically, this allows cells to respond to changes in their environment and to develop into the many cell types required to build animals ranging from worms to humans. For example, specialized groups of proteins that bind to nucleosomes, spread along specific sites of chromatin and can change its structure into an inaccessible form called heterochromatin thereby switching off genes. Proteins that bind to specific nucleosomes control the spreading, gene activity, and even memory properties of heterochromatin. However, it is not clear how these proteins spread from their original binding point on the chromatin to other nucleosomes. Now, Behrouzi, Lu et al. show how heterochromatin spreads to form large, stable structures in budding yeast. Their experiments reveal that heterochromatin proteins attach to sites on neighbouring nucleosomes, forming bridges between them. These findings conflict a long-held view as they show that pairs of nucleosomes, rather than individual nucleosomes, are the natural binding partners for heterochromatin proteins. Also, because these proteins cannot bridge from one side of a nucleosome to the other, they are unlikely to form a continuous chain across multiple nucleosomes on the chromatin. Instead, Behrouzi, Lu et al. observed that a series of short bridges between nucleosomes helps heterochromatin to spread. To fully understand why bridging only happens between separate nucleosomes, the atomic structure of heterochromatin proteins bound to pairs of nucleosomes needs to be determined. In addition, it will be essential to develop more experimental methods to study the spreading of heterochromatin inside cells.

Heterochromatin formation involves histone H3 methylation, with H3K9me2 defining a distinct heterochromatin state that is transcriptionally permissive and can couple with RNAi, and the transition to non-permissive H3K9me3 required for the epigenetic heritability of heterochromatin. A transcriptionally permissive heterochromatin state Heterochromatin silences transcription of repetitive DNA elements and transposons, yet its formation involves a co-transcriptional mechanism that paradoxically promotes small RNA generation to initiate histone H3K9 methylation. Here, Danesh Moazed and colleagues show that, in fission yeast, H3K9me2 defines a distinct heterochromatin state that is transcriptionally permissive. The transition from H3K9me2 to the silent state marked by H3K9me3 seems to be required for the epigenetic heritability of heterochromatin. Given the conservation of H3K9 methylation in heterochromatin, a similar mechanism is likely to be used in other organisms. Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 . H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA 11 , 15 , 18 . Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.

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.

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.

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.