Non-canonical bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex.

Bivalent chromatin with active H3K4me3 and repressive H3K27me3 was initially identified in embryonic stem cells (ESCs) to poise expression of developmental genes upon lineage commitment. Since then, many more different bivalent modifications have been demonstrated in both ESCs and fully differentiated cells. Bivalency not only spatiotemporally controls gene transcription but also acts to fine-tune the level of transcription during development. Although increasing number of studies demonstrated the functional significance of bivalent chromatin, the molecular connection of bivalent chromatin and transcriptional regulation remains largely elusive. Recently, we showed Spindlin1/C11orf84 complex prefers to recognize the non-canonical histone H3K4me3K9me3 bivalent mark, which is required for timely ribosomal RNA transcription. Here, we hypothesize the recognition of K4me3 and K9me3 at the same histone tail by Spindlin1/C11orf84 complex may serve as a general mechanism of conversion from a repressed to an active chromatin structure for transcriptional activation.

Timeless Interacts with PARP-1 to Promote Homologous Recombination Repair.

Human Timeless helps stabilize replication forks during normal DNA replication and plays a critical role in activation of the S phase checkpoint and proper establishment of sister chromatid cohesion. However, it remains elusive whether Timeless is involved in the repair of damaged DNA. Here, we identify that Timeless physically interacts with PARP-1 independent of poly(ADP-ribosyl)ation. We present high-resolution crystal structures of Timeless PAB (PARP-1-binding domain) in free form and in complex with PARP-1 catalytic domain. Interestingly, Timeless PAB domain specifically recognizes PARP-1, but not PARP-2 or PARP-3. Timeless-PARP-1 interaction does not interfere with PARP-1 enzymatic activity. We demonstrate that rapid and transient accumulation of Timeless at laser-induced DNA damage sites requires PARP-1, but not poly(ADP-ribosyl)ation and that Timeless is co-trapped with PARP-1 at DNA lesions upon PARP inhibition. Furthermore, we show that Timeless and PARP-1 interaction is required for efficient homologous recombination repair.

Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation.

UHRF1 plays multiple roles in regulating DNMT1-mediated DNA methylation maintenance during DNA replication. The UHRF1 C-terminal RING finger functions as an ubiquitin E3 ligase to establish histone H3 ubiquitination at Lys18 and/or Lys23, which is subsequently recognized by DNMT1 to promote its localization onto replication foci. Here, we present the crystal structure of DNMT1 RFTS domain in complex with ubiquitin and highlight a unique ubiquitin binding mode for the RFTS domain. We provide evidence that UHRF1 N-terminal ubiquitin-like domain (UBL) also binds directly to DNMT1. Despite sharing a high degree of structural similarity, UHRF1 UBL and ubiquitin bind to DNMT1 in a very distinct fashion and exert different impacts on DNMT1 enzymatic activity. We further show that the UHRF1 UBL-mediated interaction between UHRF1 and DNMT1, and the binding of DNMT1 to ubiquitinated histone H3 that is catalyzed by UHRF1 RING domain are critical for the proper subnuclear localization of DNMT1 and maintenance of DNA methylation. Collectively, our study adds another layer of complexity to the regulatory mechanism of DNMT1 activation by UHRF1 and supports that individual domains of UHRF1 participate and act in concert to maintain DNA methylation patterns.

Structural characterization of the redefined DNA-binding domain of human XPA.

XPA (xeroderma pigmentosum complementation group A), a key scaffold protein in nucleotide excision repair (NER) pathway, is important in DNA damage verification and repair proteins recruitment. Earlier studies had mapped the minimal DNA-binding domain (MBD) of XPA to a region corresponding to residues 98-219. However, recent studies indicated that the region involving residues 98-239 is the redefined DNA-binding domain (DBD), which binds to DNA substrates with a much higher binding affinity than MBD and possesses a nearly identical binding affinity to the full-length XPA protein. However, the structure of the redefined DBD domain of XPA (XPA-DBD) remains to be investigated. Here, we present the crystal structure of XPA-DBD at 2.06 Šresolution. Structure of the C-terminal region of XPA has been extended by 21 residues (Arg211-Arg231) as compared with previously reported MBD structures. The structure reveals that the C-terminal extension (Arg211-Arg231) is folded as an α-helix with multiple basic residues. The positively charged surface formed in the last C-terminal helix suggests its critical role in DNA binding. Further structural analysis demonstrates that the last C-terminal region (Asp217-Thr239) of XPA-DBD might undergo a conformational change to directly bind to the DNA substrates. This study provides a structural basis for understanding the possible mechanism of enhanced DNA-binding affinity of XPA-DBD.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Identification of Shared and Asian-Specific Loci for Systemic Lupus Erythematosus and Evidence for Roles of Type III Interferon Signaling and Lysosomal Function in the Disease: A Multi-Ancestral Genome-Wide Association Study.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with differences in prevalence and severity among ancestral groups. This study was undertaken to identify novel genetic components, either shared by or distinct between Asian and European populations. METHODS: Both trans-ancestral and ancestry-specific meta-analyses of genome-wide association studies (GWAS) for SLE were performed, involving 30,604 participants of European, Chinese, or Thai origin. Using public epigenomic data and expression quantitative trait loci, fine-mapping analyses were conducted to identify putative causal variants and genes for the newly identified loci. Performance of polygenic risk scores for the Thai cohort was evaluated by comparing different training data. RESULTS: A 1-bp deletion upstream of IFNLR1 was found to be associated with SLE, with the risk allele correlated with increased expression of IFNLR1. This gene encodes interferon-λ (IFNλ) receptor 1, providing evidence of a role of type III IFN signaling in SLE. An intronic variant in SLC29A3 was found to be associated with SLE in Asians only. The putative risk variant may modulate SLC29A3 expression in a monocyte-specific manner. SLC29A3 encodes a lysosomal nucleoside transporter, and subsequent analyses suggested that reduced lysosomal function and phagocytosis might be the mechanism underlying this association. Ancestry-shared loci in or near TAOK3, CHD9, CAMK1D, ATXN1, and TARBP1 and Asian-specific loci close to PEX2, FCHSD2, and TMEM116 also reached the genome-wide significant association with SLE. In addition, trans-ancestral meta-analysis was shown to be valuable in risk prediction for individuals without ancestry-matched data. CONCLUSION: In this study both shared and Asian-specific loci for SLE were identified, and functional annotation provided evidence of the involvement of increased type III IFN signaling and reduced lysosomal function in SLE.

Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation.

Spindlin1 is a unique multivalent epigenetic reader that facilitates ribosomal RNA transcription. In this study, we provide molecular and structural basis by which Spindlin1 acts in complex with C11orf84 to preferentially recognize non-canonical bivalent mark of trimethylated lysine 4 and lysine 9 present on the same histone H3 tail (H3K4me3K9me3). We demonstrate that C11orf84 binding stabilizes Spindlin1 and enhances its association with bivalent H3K4me3K9me3 mark. The functional analysis suggests that Spindlin1/C11orf84 complex can displace HP1 proteins from H3K4me3K9me3-enriched rDNA loci, thereby facilitating the conversion of these poised rDNA repeats from the repressed state to the active conformation, and the consequent recruitment of RNA Polymerase I for rRNA transcription. Our study uncovers a previously unappreciated mechanism of bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex required for activation of rRNA transcription.

Structure and chromosomal DNA binding of the SWIRM domain.

The evolutionarily conserved Swi3p, Rsc8p and Moira (SWIRM) domain is found in many chromosomal proteins involved in chromatin modifications or remodeling. Here we report the three-dimensional solution structure of the SWIRM domain from the human transcriptional adaptor ADA2alpha. The structure reveals a five-helix bundle consisting of two helix-turn-helix motifs connected by a central long helix, reminiscent of the histone fold. Using structural and biochemical analyses, we showed that the SWIRM domains of human ADA2alpha and SMARC2 bind to double-stranded and nucleosomal DNA, and we identified amino acid residues required for this function. We demonstrated that the ADA2alpha SWIRM domain is colocalized with lysine-acetylated histone H3 in the cell nucleus and that it potentiates the ACF remodeling activity by enhancing accessibility of nucleosomal linker DNA bound to histone H1. These data suggest a functional role of the SWIRM domain in chromatin remodeling.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA.

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

The redefined DNA-binding domain of human xeroderma pigmentosum complementation group A: production, crystallization and structure solution.

Human xeroderma pigmentosum complementation group A (XPA) is a scaffold protein that plays significant roles in DNA-damage verification and in recruiting downstream endonucleases to facilitate the repair of DNA lesions in nucleotide-excision repair. XPA98-219 (residues 98-219) has been identified as a DNA-binding domain and has been extensively studied in the last two decades. However, the most recent studies have redefined the DNA-binding domain as XPA98-239 (residues 98-239); it exerts a remarkably higher DNA-binding affinity than XPA98-219 and has a binding affinity that is quite similar to that of the full-length protein. Here, the production, crystallization and structure solution of human XPA98-239 are described. Crystals were obtained using a precipitant composed of 1.8 M ammonium citrate tribasic pH 7.0. Native X-ray diffraction data and zinc single-wavelength anomalous diffraction (SAD) data were collected to 1.93 and 2.06 Šresolution, respectively. The crystals belonged to space group P3, with unit-cell parameters a = 67.1, b = 67.1, c = 35.6 Å, γ = 120.0°. Crystal-content analysis showed the presence of one molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.65 Å3 Da-1 and a solvent content of 53.6%. The initial phases were solved and the structure model was automatically built by zinc SAD using the AutoSol program. The initial structure model covered 119 of 142 residues in the asymmetric unit, with an Rwork of 22.15% and an Rfree of 25.82%. Compared with a previously obtained truncated solution NMR structure of XPA (residues 98-210), a 19-residue C-terminal extension (residues 211-229, corresponding to 10 of the 20 extra C-terminal residues in the redefined domain for enhanced DNA binding) was contained in this initial model. Refinement of the atomic coordinates of XPA is ongoing.

The Growing Complexity of UHRF1-Mediated Maintenance DNA Methylation.

Mammalian DNMT1 is mainly responsible for maintenance DNA methylation that is critical in maintaining stem cell pluripotency and controlling lineage specification during early embryonic development. A number of studies have demonstrated that DNMT1 is an auto-inhibited enzyme and its enzymatic activity is allosterically regulated by a number of interacting partners. UHRF1 has previously been reported to regulate DNMT1 in multiple ways, including control of substrate specificity and the proper genome targeting. In this review, we discuss the recent advances in our understanding of the regulation of DNMT1 enzymatic activity by UHRF1 and highlight a number of unresolved questions.

Structural insights of the specificity and catalysis of a viral histone H3 lysine 27 methyltransferase.

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.

Structural Insights into BAF47 and BAF155 Complex Formation.

Mammalian BAF complexes are a subfamily of SWI/SNF ATP-dependent chromatin remodelers that dynamically modulate chromatin structure to regulate fundamental cellular processes including gene transcription, cell cycle control, and DNA damage response. So far, many distinct BAF complexes have been identified with polymorphic assemblies of up to 15 subunits from 29 genes. The evolutionarily conserved BRG1/BRM, BAF47, and BAF155/BAF170 form a stable complex that carries out essential chromatin remodeling activity and therefore have been regarded as the core components of BAF complex. Here, we first confirmed that SWIRM domain of BAF155 is responsible for its interaction with BAF47 and then narrowed down the SWIRM-binding region in BAF47 to the Repeat 1 (RPT1) domain. We further presented the high-resolution crystal structure of SWIRM/RPT1 complex. Extensive mutagenesis experiments together with isothermal titration calorimetry and NMR titrations were performed to corroborate the interactions observed in crystal structure. Overall, we demonstrated that BAF155 SWIRM is a modular domain involved in BAF47 interaction, which is functionally distinct from other characterized SWIRM domains that possess DNA binding activity.

Structure of the adaptor protein p14 reveals a profilin-like fold with distinct function.

The adaptor protein p14 is associated with the cytoplasmic face of late endosomes that is involved in cell-surface receptor endocytosis and it also directly interacts with MP1, a scaffolding protein that binds the MAP kinase ERK1 and its upstream kinase activator MEK1. The interaction of p14 with MP1 recruits the latter to late endosomes and the endosomal localization of p14/MP1-MEK1-ERK1 scaffolding complex is required for signaling via ERK MAP kinase in an efficient and specific manner upon receptor stimulation. Here, we report the three-dimensional solution structure of the adaptor protein p14. The structure reveals a profilin-like fold with a central five-stranded beta-sheet sandwiched between alpha-helices. Unlike profilin, however, p14 exhibits weak interaction with selective phosphoinositides but no affinity towards proline-rich sequences. Structural comparison between profilin and p14 reveals the molecular basis for the differences in these functions. We further mapped the MP1 binding sites on p14 by NMR, and discuss the implications of these important findings on the possible function of p14.

Crystal structure and SUMO binding of Slx1-Slx4 complex.

The SLX1-SLX4 complex is a structure-specific endonuclease that cleaves branched DNA structures and plays significant roles in DNA recombination and repair in eukaryotic cells. The heterodimeric interaction between SLX1 and SLX4 is essential for the endonuclease activity of SLX1. Here, we present the crystal structure of Slx1 C-terminal zinc finger domain in complex with the C-terminal helix-turn-helix domain of Slx4 from Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals a conserved binding mechanism underling the Slx1-Slx4 interaction. Structural and sequence analyses indicate Slx1 C-terminal domain is actually an atypical C4HC3-type RING finger which normally possesses E3 ubiquitin ligase activity, but here is absolutely required for Slx1 interaction with Slx4. Furthermore, we found the C-terminal tail of S. pombe Slx1 contains a SUMO-interacting motif and can recognize Pmt3 (S. pombe SUMO), suggesting that Slx1-Slx4 complex could be recruited by SUMOylated protein targets to take part in replication associated DNA repair processes.

Deposition of 5-Methylcytosine on Enhancer RNAs Enables the Coactivator Function of PGC-1α.

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

NMR study of the conformational transition of cytochrome c upon the displacement of Met80 by exogenous ligand: structural and magnetic characterization of azidoferricytochrome c.

As the exogenous ligand-cytochrome c complexes were purported to represent models for the unfolding intermediate of cytochrome c, NMR spectroscopy has been utilized to study the azide adduct of horse heart cytochrome c. The structure of azidoferricytochrome c was modeled by restrained energy minimization using paramagenetic pseudocontact shifts as constraints. The bound azide moiety was found to be tilted approximately 15 degrees from the heme normal. The displacement of Met80 by the exogenous azide molecule causes large structural rearrangement in the distal cavity. Furthermore, the conformation transition associated with the swing out of the loop containing Met80 and the shift of the 50s-helix increases the solvent accessibility of the heme group. To elucidate the heme electronic structure of the complex, the paramagnetic 13C shifts were analyzed in terms of a model based on the pi molecular orbitals of the heme under perturbed D(4) symmetry. It turned out that the His-Fe bonding provides the protein constraint that orients the in-plane anisotropy in the complex. The electronic properties are in accordance with the calculated magnetic susceptibility anisotropy and the structural information.

Structure of RPA32 bound to the N-terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins.

Replication protein A subunit RPA32 contains a C-terminal domain that interacts with a variety of DNA damage response proteins including SMARCAL1, Tipin, UNG2 and XPA. We have solved the high-resolution crystal structure of RPA32 C-terminal domain (RPA32C) in complex with a 26-amino-acid peptide derived from the N-terminus of SMARCAL1 (SMARCAL1N). The RPA32C-SMARCAL1N structure reveals a 1 : 1 binding stoichiometry and displays a well-ordered binding interface. SMARCAL1N adopts a long α-helical conformation with the highly conserved 11 residues aligned on one face of the α-helix showing extensive interactions with the RPA32C domain. Extensive mutagenesis experiments were performed to corroborate the interactions observed in crystal structure. Moreover, the α1/α2 loop of the RPA32C domain undergoes a conformational rearrangement upon SMARCAL1N binding. NMR study has further confirmed that the RPA32C-SMARCAL1N interaction induces conformational changes in RPA32C. Isothermal titration calorimetry studies have also demonstrated that the conserved α-helical motif defined in the current study is required for sufficient binding of RPA32C. Taken together, our study has provided convincing structural information that redefines the common recognition pattern shared by RPA32C interacting proteins. DATABASE: The atomic coordinates of RPA32C in complex with 26-aa SMARCAL1 (SMARCAL1N) peptide have been deposited at the Protein Data Bank with accession code 4MQV. STRUCTURED DIGITAL ABSTRACT: RPA32 and SMARCAL1 bind by isothermal titration calorimetry(1, 2, 3, 4, 5, 6, 7, 8, 9) RPA32 and SMARCAL1 bind by molecular sieving (View interaction) RPA32 and SMARCAL1 bind by x-ray crystallography (View interaction) Tipin and RPA32 bind by isothermal titration calorimetry (1, 2) RPA32 and UNG2 bind by isothermal titration calorimetry (1, 2, 3) SMARCAL1 and RPA32 bind by nuclear magnetic resonance (View interaction) UNG2 and RPA32 bind by nuclear magnetic resonance (View interaction) Tipin and RPA32 bind by nuclear magnetic resonance (View interaction).

UHRF1 double tudor domain and the adjacent PHD finger act together to recognize K9me3-containing histone H3 tail.

Human multi-domain-containing protein UHRF1 has recently been extensively characterized as a key epigenetic regulator for maintaining DNA methylation patterns. UHRF1 SRA domain preferentially binds to hemimethylated CpG sites, and double Tudor domain has been implicated in recognizing H3K9me3 mark, but the role of the adjacent PHD finger remains unclear. Here, we report the high-resolution crystal structure of UHRF1 PHD finger in complex with N-terminal tail of histone H3. We found that the preceding zinc-Cys4 knuckle is indispensable for the PHD finger of UHRF1 to recognize the first four unmodified residues of histone H3 N-terminal tail. Quantitative binding studies indicated that UHRF1 PHD finger (including the preceding zinc-Cys4 knuckle) acts together with the adjacent double Tudor domain to specifically recognize the H3K9me3 mark. Combinatorial recognition of H3K9me3-containing histone H3 tail by UHRF1 PHD finger and double Tudor domain may play a role in establishing and maintaining histone H3K9 methylation patterns during the cell cycle.