Chuanbing Bian

Tet3 CXXC Domain and Dioxygenase Activity Cooperatively Regulate Key Genes for Xenopus Eye and Neural Development

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3s biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

Binding of Different Histone Marks Differentially Regulates the Activity and Specificity of Polycomb Repressive Complex 2 (PRC2)

The polycomb repressive complex 2 (PRC2) is the major methyltransferase for H3K27 methylation, a modification critical for maintaining repressed gene expression programs throughout development. It has been previously shown that PRC2 maintains histone methylation patterns during DNA replication in part through its ability to bind to H3K27me3. However, the mechanism by which PRC2 recognizes H3K27me3 is unclear. Here we show that the WD40 domain of EED, a PRC2 component, is a methyllysine histone-binding domain. The crystal structures of apo-EED and EED in complex respectively with five different trimethyllysine histone peptides reveal that EED binds these peptides via the top face of its β-propeller architecture. The ammonium group of the trimethyllysine is accommodated by an aromatic cage formed by three aromatic residues, while its aliphatic chain is flanked by a fourth aromatic residue. Our structural data provide an explanation for the preferential recognition of the Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and H1K26 marks by EED over lower methylation states and other histone methyllysine marks. More importantly, we found that binding of different histone marks by EED differentially regulates the activity and specificity of PRC2. Whereas the H3K27me3 mark stimulates the histone methyltransferase activity of PRC2, the H1K26me3 mark inhibits PRC2 methyltransferase activity on the nucleosome. Moreover, H1K26me3 binding switches the specificity of PRC2 from methylating H3K27 to EED. In addition to determining the molecular basis of EED-methyllysine recognition, our work provides the biochemical characterization of how the activity of a histone methyltransferase is oppositely regulated by two histone marks.

Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

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.

Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain.

Arginine methylation modulates diverse cellular processes and represents a molecular signature of germ-line-specific Piwi family proteins. A subset of Tudor domains recognize arginine methylation modifications, but the binding mechanism has been lacking. Here we establish that, like other germ-line Tudor proteins, the ancestral staphylococcal nuclease domain-containing 1 (SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells. We find that human SND1 binds PIWIL1 in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. The entire Tudor domain and a bifurcated SN domain are required for this binding activity, whereas the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Crystal structures show that the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations. This analysis explains how SND1 preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine-containing peptides by extended Tudor domains.

The structural basis for selective binding of non-methylated CpG islands by the CFP1 CXXC domain

CFP1 is a CXXC domain-containing protein and an essential component of the SETD1 histone H3K4 methyltransferase complex. CXXC domain proteins direct different chromatin-modifying activities to various chromatin regions. Here, we report crystal structures of the CFP1 CXXC domain in complex with six different CpG DNA sequences. The crescent-shaped CFP1 CXXC domain is wedged into the major groove of the CpG DNA, distorting the B-form DNA, and interacts extensively with the major groove of the DNA. The structures elucidate the molecular mechanism of the non-methylated CpG-binding specificity of the CFP1 CXXC domain. The CpG motif is confined by a tripeptide located in a rigid loop, which only allows the accommodation of the non-methylated CpG dinucleotide. Furthermore, we demonstrate that CFP1 has a preference for a guanosine nucleotide following the CpG motif.

Crystal structure of TDRD3 and methyl-arginine binding characterization of TDRD3, SMN and SPF30.

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3.

Tudor domains of the PRC2 components PHF1 and PHF19 selectively bind to histone H3K36me3

PRC2 is the major H3K27 methyltransferase and is responsible for maintaining repressed gene expression patterns throughout development. It contains four core components: EZH2, EED, SUZ12 and RbAp46/48 and some cell-type specific components. In this study, we focused on characterizing the histone binding domains of PHF1 and PHF19, and found that the Tudor domains of PHF1 and PHF19 selectively bind to histone H3K36me3. Structural analysis of these Tudor domains also shed light on how these Tudor domains selectively bind to histone H3K36me3. The histone H3K36me3 binding by the Tudor domains of PHF1, PHF19 and likely MTF2 provide another recruitment and regulatory mechanism for the PRC2 complex. In addition, we found that the first PHD domains of PHF1 and PHF19 do not exhibit histone H3K4 binding ability, nor do they affect the Tudor domain binding to histones.

Sequence-Specific Recognition of a PxLPxI/L Motif by an Ankyrin Repeat Tumbler Lock

Phosphorylation of a motif that binds to ankyrin repeat domains switches its binding preference to 14-3-3 proteins. Fitting Protein Keys to Multiple Protein Locks The ankyrin repeat motif is a 33-residue sequence that is important for mediating protein-protein interactions. ANKRA2 contains five such motifs, and it binds to histone deacetylase 4 (HDAC4), among other targets. Through peptide-binding arrays, mutagenesis studies, and x-ray crystallography, Xu et al. showed that the middle three ankyrin repeat domains of ANKRA2 form hydrophobic pockets that accommodate the linear arrangement of specific residues in the binding motif of HDAC4, similar to the way in which the tumblers of a lock accept a key. Phosphorylation of a serine residue within the ANKRA2-binding motif of HDAC4 attenuated the ANKRA2-HDAC4 interaction but enabled HDAC4 to bind to 14-3-3 proteins, which inhibit HDAC activity by sequestering HDACs in the cytoplasm. Proteome-wide screening identified other proteins with similar serine-containing, ankyrin domain–binding motifs, which suggests that phosphorylation acts as a switch that determines the binding preference of this motif in a signal-dependent manner. Ankyrin repeat family A protein 2 (ANKRA2) interacts with the plasma membrane receptor megalin and the class IIa histone deacetylases HDAC4 and HDAC5. We report that the ankyrin repeat domains of ANKRA2 and its close paralog regulatory factor X–associated ankyrin-containing protein (RFXANK) recognize a PxLPxI/L motif found in diverse binding proteins, including HDAC4, HDAC5, HDAC9, megalin, and regulatory factor X, 5 (RFX5). Crystal structures of the ankyrin repeat domain of ANKRA2 in complex with its binding peptides revealed that each of the middle three ankyrin repeats of ANKRA2 recognizes a residue from the PxLPxI/L motif in a tumbler-lock binding mode, with ANKRA2 acting as the lock and the linear binding motif serving as the key. Structural analysis showed that three disease-causing mutations in RFXANK affect residues that are critical for binding to RFX5. These results suggest a fundamental principle of longitudinal recognition of linear sequences by a repeat-type domain. In addition, phosphorylation of serine 350, a residue embedded within the PxLPxI/L motif of HDAC4, impaired the binding of ANKRA2 but generated a high-affinity docking site for 14-3-3 proteins, which may help sequester this HDAC in the cytoplasm. Thus, the binding preference of the PxLPxI/L motif is signal-dependent. Furthermore, proteome-wide screening suggested that a similar phosphorylation-dependent switch may operate in other pathways. Together, our findings uncover a previously uncharacterized sequence- and signal-dependent peptide recognition mode for a repeat-type protein domain.

Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2

Background Expansion of the CGG trinucleotide repeat in the 5′-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs are locally translated in response to stimuli. Methodology/Principal Findings Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 Å, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9. Conclusions The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Crystal structures of the Tudor domains of human PHF20 reveal novel structural variations on the Royal Family of proteins.

The human PHD finger protein 20 (PHF20) is a putative transcription factor. While little is known about its cognate cellular role, antibodies against PHF20 are present in sera from patients with hepatocellular carcinoma, glioblastoma and childhood medulloblastula. PHF20 comprises two N‐terminal Tudor domains, a central C2H2‐link zinc finger domain and a C‐terminal zinc‐binding PHD domain, and is a component of some MLL methyltransferase complexes. Here, we report the crystal structures of the N‐terminal Tudor domains of PHF20 and highlight the novel structural features of each domain. We also confirm previous studies suggesting that the second Tudor domain of PHF20 exhibits preference for dimethylated histone substrates.