Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly

The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a "histone code" essential for the epigenetic inheritance of heterochromatin assembly.     

JMJD6 is a histone arginine demethylase

Arginine methylation occurs on a number of proteins involved in a variety of cellular functions. Histone tails are known to be mono- and dimethylated on multiple arginine residues where they influence chromatin remodeling and gene expression. To date, no enzyme has been shown to reverse these regulatory modifications. We demonstrate that the Jumonji domain-containing 6 protein (JMJD6) is a JmjC-containing iron- and 2-oxoglutarate-dependent dioxygenase that demethylates histone H3 at arginine 2 (H3R2) and histone H4 at arginine 3 (H4R3) in both biochemical and cell-based assays. These findings may help explain the many developmental defects observed in the JMJD6(-/-) knockout mice.     

Crystal structure of Argonaute and its implications for RISC slicer activity

Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.     

Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail

The chromodomain of the HP1 family of proteins recognizes histone tails with specifically methylated lysines. Here, we present structural, energetic, and mutational analyses of the complex between the Drosophila HP1 chromodomain and the histone H3 tail with a methyllysine at residue 9, a modification associated with epigenetic silencing. The histone tail inserts as a beta strand, completing the beta-sandwich architecture of the chromodomain. The methylammonium group is caged by three aromatic side chains, whereas adjacent residues form discerning contacts with one face of the chromodomain. Comparison of dimethyl- and trimethyllysine-containing complexes suggests a role for cation-pi and van der Waals interactions, with trimethylation slightly improving the binding affinity.     

Role of histone H3 lysine 27 methylation in Polycomb-group silencing

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.     

Establishment and maintenance of a heterochromatin domain

The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails. Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation. We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself. We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery. Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous mat locus but are dispensable for its subsequent inheritance. This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains.     

Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries

Eukaryotic genomes are organized into discrete structural and functional chromatin domains. Here, we show that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast. H3 methylated at lysine 9 (H3 Lys9), and its interacting Swi6 protein, are strictly localized to a 20-kilobase silent heterochromatic interval. In contrast, H3 methylated at lysine 4 (H3 Lys4) is specific to the surrounding euchromatic regions. Two inverted repeats flanking the silent interval serve as boundary elements to mark the borders between heterochromatin and euchromatin. Deletions of these boundary elements lead to spreading of H3 Lys9 methylation and Swi6 into neighboring sequences. Furthermore, the H3 Lys9 methylation and corresponding heterochromatin-associated complexes prevent H3 Lys4 methylation in the silent domain.     

A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8

The T cell coreceptors CD4 and CD8 both associate via their cytoplasmic tails with the N-terminus of the Src-family tyrosine kinase Lck. These interactions require zinc and are critical for T cell development and activation. We examined the folding and solution structures of ternary CD4-Lck-Zn2+ and CD8alpha-Lck-Zn2+ complexes. The coreceptor tails and the Lck N-terminus are unstructured in isolation but assemble in the presence of zinc to form compactly folded heterodimeric domains. The cofolded complexes have similar "zinc clasp" cores that are augmented by distinct structural elements. A dileucine motif required for clathrin-mediated endocytosis of CD4 is masked by Lck.     

Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates

We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain to its substrates.     

Heterochromatin and epigenetic control of gene expression

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.     

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.     

Histone H4-K16 acetylation controls chromatin structure and protein interactions

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.     

Salmonella pathogenesis and processing of secreted effectors by caspase-3

The enteric pathogen Salmonella enterica serovar Typhimurium causes food poisoning resulting in gastroenteritis. The S. Typhimurium effector Salmonella invasion protein A (SipA) promotes gastroenteritis by functional motifs that trigger either mechanisms of inflammation or bacterial entry. During infection of intestinal epithelial cells, SipA was found to be responsible for the early activation of caspase-3, an enzyme that is required for SipA cleavage at a specific recognition motif that divided the protein into its two functional domains and activated SipA in a manner necessary for pathogenicity. Other caspase-3 cleavage sites identified in S. Typhimurium appeared to be restricted to secreted effector proteins, which indicates that this may be a general strategy used by this pathogen for processing of its secreted effectors.     

Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1

The Arabidopsis gene DDM1 is required to maintain DNA methylation levels and is responsible for transposon and transgene silencing. However, rather than encoding a DNA methyltransferase, DDM1 has similarity to the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes, suggesting an indirect role in DNA methylation. Here we show that DDM1 is also required to maintain histone H3 methylation patterns. In wild-type heterochromatin, transposons and silent genes are associated with histone H3 methylated at lysine 9, whereas known genes are preferentially associated with methylated lysine 4. In ddm1 heterochromatin, DNA methylation is lost, and methylation of lysine 9 is largely replaced by methylation of lysine 4. Because DNA methylation has recently been shown to depend on histone H3 lysine 9 methylation, our results suggest that transposon methylation may be guided by histone H3 methylation in plant genomes. This would account for the epigenetic inheritance of hypomethylated DNA once histone H3 methylation patterns are altered.     

Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication

Acetylation of histone H3 lysine 56 (H3-K56) occurs in S phase, and cells lacking H3-K56 acetylation are sensitive to DNA-damaging agents. However, the histone acetyltransferase (HAT) that catalyzes global H3-K56 acetylation has not been found. Here we show that regulation of Ty1 transposition gene product 109 (Rtt109) is an H3-K56 HAT. Cells lacking Rtt109 or expressing rtt109 mutants with alterations at a conserved aspartate residue lose H3-K56 acetylation and exhibit increased sensitivity toward genotoxic agents, as well as elevated levels of spontaneous chromosome breaks. Thus, Rtt109, which shares no sequence homology with any other known HATs, is a unique HAT that acetylates H3-K56.