Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases, members of a newly emerging transcription-factor family, remove methyl groups from the lysine residues of the histone tails and thereby regulate the transcriptional activity of target genes. JmjC-domain-containing proteins have been predicted to be demethylases. For example, the JmjC-containing protein JMJD2A has been characterized as a H3-K9me3- and H3-K36me3-specific demethylase. Here, structures of the catalytic-core domain of JMJD2A with and without alpha-ketoglutarate in the presence of Fe2+ have been determined by X-ray crystallography. The structure of the core domain, consisting of the JmjN domain, the JmjC domain, the C-terminal domain, and a zinc-finger motif, revealed the unique elements that form a potential substrate binding pocket. Sited-directed mutagenesis in conjunction with demethylase activity assays allowed us to propose a molecular model for substrate selection by the JMJD2 histone demethylase family.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

The Jumonji C domain is a catalytic motif that mediates histone lysine demethylation. The Jumonji C-containing oxygenase JMJD2A specifically demethylates tri-and dimethylated lysine-9 and lysine-36 of histone 3 (H3K9/36me3/2). Here we present structures of the JMJD2A catalytic core complexed with methylated H3K36 peptide substrates in the presence of Fe(II) and N-oxalylglycine. We found that the interaction between JMJD2A and peptides largely involves the main chains of the enzyme and the peptide. The peptide-binding specificity is primarily determined by the primary structure of the peptide, which explains the specificity of JMJD2A for methylated H3K9 and H3K36 instead of other methylated residues such as H3K27. The specificity for a particular methyl group, however, is affected by multiple factors, such as space and the electrostatic environment in the catalytic center of the enzyme. These results provide insights into the mechanisms and specificity of histone demethylation.demethylase ͉ oxygenase ͉ JmjC ͉ epigenetic ͉ chromatin

JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds α-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. Here, we describe the structures of JMJD6 with and without α-ketoglutarate, which revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, doublestranded RNA, or double-stranded DNA. These structural features and truncation analysis of JMJD6 suggest that JMJD6 may bind and modify single-stand RNA rather than the previously reported peptide substrates.RNA binding proteins | RNA modification | RNA splicing J MJD6 was first characterized as a receptor for phosphatidylserine (PSR), which facilitates the phagocytosis of dead and dying cells by macrophages and fibroblasts (1). Targeted deletion of gene encoding PSR in mice and morpholino knock-downs of PSR in zebrafish resulted in embryonic lethality, with severe defects in hematopoiesis and aberrant development of eye, brain, and heart (2-5). In contrast, knock-down of PSR expression in Caenorhabditis elegans produced only a mild phenotype (5). Somewhat surprisingly, sequence analysis suggested that JMJD6 contains a Jumonji C (JMJC) domain, which places it within a highly conserved, cupin fold-containing enzyme family (6-8). Further analysis demonstrated that the protein is localized specifically in the nucleus (7-9). Despite the significant effects of JMJD6 deficiency, knockout mice engulfed apoptotic cells normally (9). Based on these studies and additional sequence analysis, the protein was recategorized as an α-ketoglutarateand Fe 2þ -dependent hydroxylase and was named JMJD6 (10).Recent studies demonstrated that most JMJC domain-containing proteins function as histone demethylases by specifically acting on lysine residues in histone tails (11)(12)(13)(14). For example, the specific interactions between enzymes from the JMJD2 subfamily and methylated peptides have been structurally characterized (15-18). Interestingly, JMJD6 was reported to demethylate arginine residues in histone tails (10). Several laboratories including ours, however, have been unable to reproduce these results. In other studies, JMJD6 was identified as a lysine hydroxylase that specifically recognizes the protein tail of U2AF65, a mediator of RNA splicing (19).To resolve the disparate results and further elucidate the structure and functions of JMJD6, we determined X-ray crystallographic structures of the protein with and without α-ketoglutarate. To obtain these structures, JMJD6 was cocrystallized with a Fab fragment derived from a JMJD6-specific hamster monoclonal antibody. Intriguingly, the structure of JMJD6 is dramatically different from known structures of other JMJC domain superfamily proteins includ...

A major obstacle to developing small interfering RNAs (siRNAs) as cancer drugs is their intracellular delivery to disseminated cancer cells. Fusion proteins of single-chain fragmented antibodies (ScFvs) and positively charged peptides deliver siRNAs into specific target cells. However, the therapeutic potential of ScFv-mediated siRNA delivery has not been evaluated in cancer. Here, we tested whether Polo-like kinase 1 (PLK1) siRNAs complexed with a Her2-ScFv-protamine peptide fusion protein (F5-P) could suppress Her2(+) breast cancer cell lines and primary human cancers in orthotopic breast cancer models. PLK1-siRNAs transferred by F5-P inhibited target gene expression, reduced proliferation, and induced apoptosis of Her2(+) breast cancer cell lines and primary human cancer cells in vitro without triggering an interferon response. Intravenously injected F5-P/PLK1-siRNA complexes concentrated in orthotopic Her2(+) breast cancer xenografts and persisted for at least 72 hours, leading to suppressed PLK1 gene expression and tumor cell apoptosis. The intravenously injected siRNA complexes retarded Her2(+) breast tumor growth, reduced metastasis, and prolonged survival without evident toxicity. F5-P-mediated delivery of a cocktail of PLK1, CCND1, and AKT siRNAs was more effective than an equivalent dose of PLK1-siRNAs alone. These data suggest that F5-P could be used to deliver siRNAs to treat Her2(+) breast cancer.

Background: HJURP is a molecular chaperone essential for the deposition of the centromere marker CENP-A. Results: Mis18␤ binds with and specifies the centromere localization of HJURP. Conclusion: Mis18␤ governs centromere assembly via the Mis18␤-HJURP-CENP-A axis. Significance: Our finding reveals a novel mechanism underlying CENP-A incorporation into the centromere.

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Light-induced chemical reactions exist in nature in regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair, and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.