Search PubMed⌕ Search

Biomedical subjects

Xiaodong Cheng

Publications and source records attributed to Xiaodong Cheng.

49 records · Page 3Linked to original sources
PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗
PubMed2002

Structure and dynamics of the modular halves of Escherichia coli cyclic AMP receptor protein.

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains. To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides. The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets. The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices. The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209. The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP. Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP. In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific. Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding. The acquisition of this unique property is intimately associated with the dynamics of the molecule.

Amino Acid Motifs↗
PubMed2002

Mismatch repair in methylated DNA. Structure and activity of the mismatch-specific thymine glycosylase domain of methyl-CpG-binding protein MBD4.

MBD4 is a member of the methyl-CpG-binding protein family. It contains two DNA binding domains, an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain. Limited in vitro proteolysis of mouse MBD4 yields two stable fragments: a 139-residue fragment including the MBD, and the other 155-residue fragment including the glycosylase domain. Here we show that the latter fragment is active as a glycosylase on a DNA duplex containing a G:T mismatch within a CpG sequence context. The crystal structure confirmed the C-terminal domain is a member of the helix-hairpin-helix DNA glycosylase superfamily. The MBD4 active site is situated in a cleft that likely orients and binds DNA. Modeling studies suggest the mismatched target nucleotide will be flipped out into the active site where candidate residues for catalysis and substrate specificity are present.

Amino Acid Sequence↗
PubMed2002

Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.

Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21(Waf1/Cip1) (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha-helical conformation within the transactivation domain. Mutagenesis-derived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF(II)31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF(II)31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF(II)31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF(II)31 recruitment.

Amino Acid Sequence↗
PubMed2002

Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase.

AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.

Amino Acid Sequence↗
PubMed2002

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1.

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.

Amino Acid Substitution↗
PubMed2002

Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP.

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.

Amino Acid Motifs↗
PubMed2002

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.

Base Sequence↗
PubMed2002

Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p.

From transcription to translation, mRNA is complexed with heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that mediate mRNA processing, export from the nucleus, and delivery into the cytoplasm. Although the mechanism is unknown, export of mature mRNA from the nucleus is a critical regulatory step in gene expression. Analyses of hnRNP proteins have shown that many of these proteins are required for this essential cellular process. In this study, we characterize the Saccharomyces cerevisiae Nab2 protein, which was first identified as a poly(A) RNA-binding protein (Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol. 13, 2730-2741). Our work indicates that poly(A) RNA export from the nucleus is dependent upon a functional Nab2 protein; correspondingly, export of Nab2p from the nucleus is dependent upon ongoing RNA polymerase II transcription. Furthermore, we show that Nab2p is modified within its RGG domain by the type I protein-arginine methyltransferase, Hmt1p. Our experiments demonstrate that arginine methylation is required for the export of Nab2p from the nucleus and therefore establish an in vivo effect of this modification. Overall, these experiments provide evidence that Nab2p is an hnRNP protein that is required for poly(A) RNA export and whose export from the nucleus is regulated by Hmt1p.

ATP-Binding Cassette Transporters↗
PubMed2002

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds.

The PWWP domain is a weakly conserved sequence motif found in > 60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223--357) has been structurally characterized at 1.8 A resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Delta218 and Delta369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Delta218 protein, which includes the PWWP domain, binds DNA more strongly than Delta369, which lacks the PWWP domain.

Amino Acid Motifs↗
PubMed2002

Structure of the SET domain histone lysine methyltransferase Clr4.

Methylation of histone H3 lysine 9 is an important component of the 'histone code' for heterochromatic gene silencing. The SET domain-containing Clr4 protein, a close relative of Su(var)3-9 proteins in higher eukaryotes, specifically methylates lysine 9 of histone H3 and is essential for silencing in Schizosaccharomyces pombe. Here we report the 2.3 A resolution crystal structure of the catalytic domain of Clr4. The structure reveals an overall fold rich in beta-strands, a potential active site consisting of a SAM-binding pocket, and a connected groove that could accommodate the binding of the N-terminal tail of histone H3. The pre-SET motif contains a triangular zinc cluster coordinated by nine cysteines distant from the active site, whereas the post-SET region is largely flexible but proximal to the active site. The structure provides insights into the architecture of SET domain histone methyltransferases and establishes a paradigm for further characterization of the Clr4 family of epigenetic regulators.

Amino Acid Sequence↗
PubMed2002

Synergy among nuclear receptor coactivators: selective requirement for protein methyltransferase and acetyltransferase activities.

Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1, GRIP1, and ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates than CARM1, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300 or CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. The transfection assay described here is a valuable new tool for investigating the mechanism of coactivator function and demonstrates the importance of multiple coactivators, including CARM1 and its specific protein methyltransferase activity, in transcriptional activation.

Acetyltransferases↗