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In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1–cohesin binding is maintained until the formation of microtubule–kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.

Genome regulation requires control of chromosome organization by SMC–kleisin complexes. The cohesin complex contains the Smc1 and Smc3 subunits that associate with the kleisin Scc1 to form a ring-shaped complex that can topologically engage chromatin to regulate chromatin structure. Release from chromatin involves opening of the ring at the Smc3–Scc1 interface in a reaction that is controlled by acetylation and engagement of the Smc ATPase head domains. To understand the underlying molecular mechanisms, we have determined the 3.2-A resolution cryo-electron microscopy structure of the ATPγS-bound, heterotrimeric cohesin ATPase head module and the 2.1-A resolution crystal structure of a nucleotide-free Smc1–Scc1 subcomplex from Saccharomyces cerevisiae and Chaetomium thermophilium. We found that ATP-binding and Smc1–Smc3 heterodimerization promote conformational changes within the ATPase that are transmitted to the Smc coiled-coil domains. Remodeling of the coiled-coil domain of Smc3 abrogates the binding surface for Scc1, thus leading to ring opening at the Smc3–Scc1 interface. Structural analysis reveals that ATP-binding and Smc1–Smc3 heterodimerization promotes conformational changes within the cohesin ATPase that are transmitted to the Smc coiled-coil domains, leading to ring opening at the Smc3–Scc1 interface.

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions., The histone acetyltransferase p300 mostly localizes to active chromatin; however, some repressed genes marked with H3K27me3 are also bound by p300. Here the authors show p300 is capable of phase separation, which relies on its catalytic core, and that p300 catalytic activity is decreased in phase-separated droplets that co-localize with H3K27me3-marked chromatin.

The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKe are noncanonical members of the inhibitor of the nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKKe inhibitor BAY-985. BAY-985 inhibits the cellular phosphorylation of interferon regulatory factor 3 and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2 but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model.

Histone acetylation plays an important role in transcriptional activation. Histones are also modified by chemically diverse acylations that are frequently deposited by p300, a transcriptional coactivator that uses a number of different acyl-CoA cofactors. Here we report that while p300 is a robust acetylase, its activity gets weaker with increasing acyl-CoA chain length. Crystal structures of p300 in complex with propionyl-, crotonyl-, or butyryl-CoA show that the aliphatic portions of these cofactors are bound in the lysine substrate-binding tunnel in a conformation that is incompatible with substrate transfer. Lysine substrate binding is predicted to remodel the acyl-CoA ligands into a conformation compatible with acyl-chain transfer. This remodeling requires that the aliphatic portion of acyl-CoA be accommodated in a hydrophobic pocket in the enzymes active site. The size of the pocket and its aliphatic nature exclude long-chain and charged acyl-CoA variants, presumably explaining the cofactor preference for p300.

Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features., Graphical Abstract, Highlights • Active gene TSSs are marked by competing H4 K5K8 acetylation and butyrylation • Histone butyrylation directly stimulates transcription • H4K5 butyrylation prevents binding of the testis specific gene expression-driver Brdt • H4K5K8 butyrylation is associated with delayed histone removal in spermatogenic cells, Histone butyrylation stimulates gene transcription while competing with acetylation at H4K5 to control Brdt bromodomain binding. Differential chromatin labeling with interchangeable H4 acylations is an important epigenetic regulatory mechanism controlling gene expression and chromatin reorganization.

The cohesin ring complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair and transcriptional regulation. How cohesin engages its chromatin substrate has remained an unresolved question. We show here, by determining a crystal structure of the budding yeast cohesin HEAT-repeat subunit Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.

Significance Cytokines interact with their receptors and activate JAK–STAT signaling pathways that lead to changes in gene expression. In mammals, there are seven STATs that have arisen due to gene duplication and genetic drift. STATs have similar DNA binding specificity, and how individual STATs have subfunctionalized to regulate very specific cytokine responses in cells is poorly understood. Here we describe X-ray structures that show how one STAT family member, STAT2, specifically pairs with a member of the IRF family of transcription factors, IRF9. Despite overall structural similarity among STAT and IRF family members, surface features in the interacting domains of IRF9 and STAT2 have diverged to enable specific interaction between these family members and to enable the antiviral response., Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9–IAD alone and in a complex with STAT2–CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9–IAD and STAT2–CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.

The transcriptional co-activator p300 is a histone acetyltransferase (HAT) that is typically recruited to transcriptional enhancers and regulates gene expression by acetylating chromatin. Here we show that the activation of p300 directly depends on the activation and oligomerization status of transcription factor ligands. Using two model transcription factors, IRF3 and STAT1, we demonstrate that transcription factor dimerization enables the trans-autoacetylation of p300 in a highly conserved and intrinsically disordered autoinhibitory lysine-rich loop, resulting in p300 activation. We describe a crystal structure of p300 in which the autoinhibitory loop invades the active site of a neighbouring HAT domain, revealing a snapshot of a trans-autoacetylation reaction intermediate. Substrate access to the active site involves the rearrangement of an autoinhibitory RING domain. Our data explain how cellular signalling and the activation and dimerization of transcription factors control the activation of p300, and therefore explain why gene transcription is associated with chromatin acetylation.

Nap1 is a histone chaperone involved in the nuclear import of H2A–H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A–H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1‐mediated H2A–H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A–H2B heterodimer. Oligomerization of the Nap1–H2A–H2B complex results in burial of surfaces required for deposition of H2A–H2B into nucleosomes. Chromatin immunoprecipitation‐exonuclease (ChIP‐exo) analysis shows that Nap1 is required for H2A–H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1‐mediated H2A–H2B deposition and nucleosome assembly. ![][1] A co‐crystal structure defines the stoichiometry of H2A–H2B binding to the Nap1 histone chaperone and shows that higher‐order oligomerization is needed for proper histone incorporation into nucleosomes. [1]: /embed/graphic-1.gif

The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a "crossover" binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

The interferon-beta (IFN-beta) enhanceosome is a paradigm for understanding the role of transcription factor complexes in eukaryotic signal integration. Recent structural studies provide a complete atomic model of the enhanceosome at the protein-DNA interface. The composite model shows how binding of eight transcription factors to enhancer DNA creates a continuous recognition surface. The extensive overlap of individual binding sites creates a composite element that ensures that the enhancer operates as a single unit of regulation. The absence of major protein-protein interfaces between the transcription factors suggests that cooperative binding occurs through a combination of binding-induced conformational changes in DNA structure and specific interactions with coactivator proteins such as CBP/p300. Contacts with virtually every nucleotide explain why the enhancer is evolutionary conserved in mammalian genomes.

Transcriptional activation of the interferon-beta (IFN-beta) gene requires assembly of an enhanceosome containing ATF-2/c-Jun, IRF-3/IRF-7, and NFkappaB. These factors bind cooperatively to the IFN-beta enhancer and recruit coactivators and chromatin-remodeling proteins to the IFN-beta promoter. We describe here a crystal structure of the DNA-binding domains of IRF-3, IRF-7, and NFkappaB, bound to one half of the enhancer, and use a previously described structure of the remaining half to assemble a complete picture of enhanceosome architecture in the vicinity of the DNA. Association of eight proteins with the enhancer creates a continuous surface for recognizing a composite DNA-binding element. Paucity of local protein-protein contacts suggests that cooperative occupancy of the enhancer comes from both binding-induced changes in DNA conformation and interactions with additional components such as CBP. Contacts with virtually every nucleotide pair account for the evolutionary invariance of the enhancer sequence.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.

McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis. DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart. The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites. DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved. However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site. These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping. A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart. This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis. We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA. Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA.

Antibodies contribute to the pathogenesis of many chronic inflammatory diseases, including autoimmune disorders and allergies. They are secreted by proliferating plasmablasts, short-lived plasma cells and non-proliferating, long-lived memory plasma cells. Memory plasma cells refractory to immunosuppression are critical for the maintenance of both protective and pathogenic antibody titers. Here, we studied the response of plasma cells in spleen, bone marrow and inflamed kidneys of lupus-prone NZB/W mice to high-dose dexamethasone and/or cyclophosphamide. BrdU+, dividing plasmablasts and short-lived plasma cells in the spleen were depleted while BrdU- memory plasma cells survived. In contrast, all bone marrow plasma cells including anti-DNA secreting cells were refractory to both drugs. Unlike bone marrow and spleen, which showed a predominance of IgM-secreting plasma cells, inflamed kidneys mainly accommodated IgG-secreting plasma cells, including anti-DNA secreting cells, some of which survived the treatments. These results indicate that the bone marrow is the major site of memory plasma cells resistant to treatment with glucocorticoids and anti-proliferative drugs, and that inflamed tissues and secondary lymphoid organs can contribute to the autoreactive plasma cell memory. Therefore, new strategies targeting autoreactive plasma cell memory should be considered. This could be the key to finding a curative approach to the treatment of chronic inflammatory autoantibody-mediated diseases.

Background Dendritic cells (DCs) have a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Reduced numbers of blood DCs and the accumulation of DCs at inflammatory sites have been observed in SLE. One crucial feature of DCs is their ability to migrate. Objective To analyse the maturation/activation state and the migratory capacity of different DC precursor subsets in SLE to further elucidate their role in autoimmunity. Methods Plasmacytoid DCs (pDCs), myeloid DCs (mDCs) and monocytes from patients with SLE, healthy volunteers and healthy volunteers immunised with tetanus/diphtheria were examined by flow cytometry for expression of subset-specific antigens (BDCA-2, CD11c, CD14, HLA-DR), activation/maturation markers (CD83, CD86, CD40, BLyS) and chemokine receptors (CCR1, CCR5, CCR7, ChemR23). Additionally, migratory capacity to chemokine receptors was investigated in vitro using the chemokines RANTES, CCL19 and chemerin. Results SLE monocytes and mDCs had higher CD86 and B-lymphocyte stimulatory factor (BLyS) expression levels. ChemR23 expression was lower in SLE pDCs and mDCs. Basal and CCL19-specific migration levels were higher in SLE pDCs. Altered DC function in SLE had no correlative changes in chemokine receptor expression, whereas immunisation-induced blood DC migration patterns in healthy donors were accompanied by changes in chemokine receptor expression. Conclusions The phenotypic and migratory disturbances observed in SLE blood DCs could result in altered distribution of DCs in peripheral tissues, contributing to dysregulated immune responses and autoimmunity.

The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKK epsilon and/or TBK1. Phosphorylation results in IRF-3 dimerization and removal of an autoinhibitory structure to allow interaction with the coactivators CBP/p300. The precise role of the different phosphorylation sites has remained controversial. Using purified proteins we show that TBK1 can directly phosphorylate full-length IRF-3 in vitro. Phosphorylation at residues in site 2 (Ser(396)-Ser(405)) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser(385) or Ser(386)). Phosphorylation at site 1 is, in turn, required for IRF-3 dimerization. The data support a two-step phosphorylation model for IRF-3 activation mediated by TBK1.

DNA in the cytosol activates immune responses by binding sensors such as cGAMP synthase (cGAS). A set of studies reveal the structural mechanism of DNA sensing and show that cGAS produces a cyclic 2′-5′-linked dinucleotide, a new cellular second messenger.

Transcriptional activation of the interferon-beta (IFN-beta) gene requires assembly of an enhanceosome containing the transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-kappaB and HMGI(Y). These factors cooperatively bind a composite DNA site and activate expression of the IFN-beta gene. The 3.0 A crystal structure of the DNA-binding domains of ATF-2/c-Jun and two IRF-3 molecules in a complex with 31 base pairs (bp) of the PRDIV-PRDIII region of the IFN-beta enhancer shows that association of the four proteins with DNA creates a continuous surface for the recognition of 24 bp. The structure, together with in vitro binding studies and protein mutagenesis, shows that protein-protein interactions are not critical for cooperative binding. Instead, cooperativity arises mainly through nucleotide sequence-dependent structural changes in the DNA that allow formation of complementary DNA conformations. Because the binding sites overlap on the enhancer, the unit of recognition is the entire nucleotide sequence, not the individual subsites.

Gene regulation in higher eukaryotes requires recruitment of the transcriptional co-activators CBP/p300 that associate with transcriptional regulators and integrate a large number of signal transduction pathways. Recruitment of CBP/p300 results in acetylation and remodeling of inhibitory chromatin. Recently we have determined the 2.8Å crystal structure of the catalytic core of p300 containing its Bromodomain, the CH2 region and HAT domain in complex with the bi-substrate inhibitor, Lys-CoA. Unexpectedly the structure reveals that the CH2 region contains a discontinuous PHD domain which is interrupted by a RING domain. The Bromodomain, PHD, RING and HAT domains adopt an assembled configuration in which the RING domain is positioned over the HAT substrate binding pocket. Disease mutations that disrupt RING attachment lead to upregulation of HAT activity, revealing an auto-inhibitory role for this domain. Detailed investigation of chromatin substrate recognition showed that the Bromodomain preferentially interacts with histones containing combinations of acetylations rather than singly modified sequences, whereas the p300 PHD domain did not interact with canonical substrates. Our results demonstrate that the Bromodomain substrate specificity is compatible with HAT substrate acetylation patterns suggesting that positive feedback is likely an important component in establishment of active chromatin states. We here present progress in our understanding of the regulation of p300 activity, chromatin modification, readout and how disease-related mutations result in dysregulation of these activities.

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA(+) proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg(2+) and GTP, GDP or GTP-gamma-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB(L) and McrB(S) form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5′ R m C 3′. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB L binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg 2+ . The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB L -binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPγS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.

McrBC is a methylation‐dependent endonuclease from Escherichia coli K‐12. The enzyme recognizes DNA with modified cytosines preceded by a purine. McrBC restricts DNA that contains at least two methylated recognition sites separated by 40–80 bp. Two gene products, McrB L and McrB s , are produced from the mcrB gene and one, McrC, from the mcrC gene. DNA cleavage in vitro requires McrB L , McrC, GTP and Mg 2+ . We found that DNA cleavage was optimal at a ratio of 3–5 McrB L per molecule of McrC, suggesting that formation of a multisubunit complex with several molecules of McrB L is required for cleavage. To understand the role of McrB s , we have purified the protein and analyzed its role in vitro . At the optimal ratio of 3–5 McrB L per molecule of McrC, McrB s acted as an inhibitor of DNA cleavage. Inhibition was due to sequestration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent. If McrC was in excess, a condition resulting in suboptimal DNA cleavage, addition of McrB s enhanced DNA cleavage, presumably due to sequestration of excess McrC. We suggest that the role of McrB s is to modulate McrBC activity by binding to McrC.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.

Nat. Chem. Biol. 9, 533–534 (2013); published online 19 August 2013; corrected after print 25 September 2013 In the version of this article initially published, the sentence “Three papers now report that cGAS produces a new type of second messenger molecule1,4,5” did not also attribute the finding to reference 6.

SummarySister chromatid cohesion is a fundamental prerequisite to faithful genome segregation. Cohesion is precisely regulated by accessory factors that modulate the stability with which the cohesin complex embraces chromosomes. One of these factors, Pds5, engages cohesin through Scc1 and is both a facilitator of cohesion, and, conversely also mediates the release of cohesin from chromatin. We present here the crystal structure of a complex between budding yeast Pds5 and Scc1, thus elucidating the molecular basis of Pds5 function. Pds5 forms an elongated HEAT repeat that binds to Scc1 via a conserved surface patch. We demonstrate that the integrity of the Pds5-Scc1 interface is indispensable for the recruitment of Pds5 to cohesin, and that its abrogation results in loss of sister chromatid cohesion and cell viability.

SummaryTank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 Å and 4.0 Å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation.

Cohesin catalyses the folding of the genome into loops that are anchored by CTCF1. The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N terminus interacts with the SA2–SCC1 subunits of human cohesin. We report a crystal structure of SA2–SCC1 in complex with CTCF at a resolution of 2.7 A, which reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF binding sites. A similar motif is present in a number of established and newly identified cohesin ligands, including the cohesin release factor WAPL2,3. Our data suggest that CTCF enables the formation of chromatin loops by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables the dynamic regulation of chromatin folding by cohesin and CTCF. The crystal structure of the SA2–SCC1 subunits of human cohesin in complex with CTCF reveals the molecular basis of the cohesin–CTCF interaction that enables the dynamic regulation of chromatin folding.

The cohesin complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair, transcriptional regulation and control of 3D chromatin architecture. How cohesin engages chromatin has remained a major question. The basic subunits of cohesin, Smc1, Smc3, Scc1 assemble a ring-shaped complex via connection of the heterodimeric SMC ‘hinge’ domains contributed by of Smc1 and Smc3, and through linkage of the SMC ATPase domains by Scc1. Additional accessory factors play important roles in different aspects of cohesin function, such as Scc3, which promotes the association of cohesin with DNA, the loading and unloading complexes, Scc2-Scc4 and Pds5-Wapl respectively, responsible for cohesin loading and its disassociation from chromatin. During S phase, an acetyltransferase called Eco1 acetylates the ATPase domain of Smc3 and triggers the stabilization, or establishment, of cohesion. To further augment cohesion, an additional metazoan factor, sororin forms a complex with Pds5 to prevent Wapl binding. During metaphase, centromeric cohesin is protected by the shugoshin-PP2A complex. In metazoans, cohesin is released from chromosomes in two major steps. The first requires cohesin phosphorylation and allows Wapl to bind Pds5 again to mediate cleavage-independent release of cohesin from chromosome arms. The second transpires upon fulfilment of spindle assembly and requires activation of a protease called separase, resulting in Scc1 cleavage, thus releasing sister chromatids to be segregated into daughter cells. Beyond cohesion, it is also becoming apparent that cohesin plays more diverse roles by interacting with a plethora of other factors, most notably CTCF, a zinc finger protein that is known as an insulator, which has been reported to collaborate with cohesin in determining 3D genome structure.To understand how cohesin engages DNA, I investigated the DNA binding properties of previously identified globular sub-complexes. By determining a crystal structure of the budding yeast Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, I could demonstrate that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.To investigate the molecular basis of the reported functional collaboration between cohesin and CTCF in defining 3D chromosome structure, I identified and determined the structure of a ternary complex composed of human SA2 (an orthologue of Scc3), Scc1 and CTCF. The structure revealed a wide-spread SA2-Scc1 binding motif which was found to be present not only in CTCF, but also other functionally related factors, including shugoshin and Wapl. Competition pulldown assays indicated that binding of these factors to SA2-Scc1 was mutually exclusive, which strongly suggested that they interact with cohesin via similar mechanisms. To demonstrate this principle, I was able to determine a structure of shugoshin in complex with SA2-Scc1, which confirmed that both shugoshin and CTCF bind the same conserved surface on cohesin., Le complexe de la cohésine est requis pour de nombreuses transactions chromosomiques, la cohésion des chromatides soeurs, la réparation des dommages à l'ADN, la régulation de la transcription et le contrôle de l'architecture de la chromatine en 3D. La manière dont la cohésine engage la chromatine est restée une question majeure. Les sous-unités de base de cohesin, Smc1, Smc3, Scc1 assemblent un complexe en forme d’anneau via la connexion des domaines SMC «charnières» hétérodimères fournis par Smc1 et Smc3, et par la liaison des domaines SMC ATPase par Scc1. D'autres facteurs jouent un rôle dans différents aspects de la fonction de la cohésine, tels que Scc3, qui favorise l'association de la cohésine à l'ADN, les complexes de chargement et de déchargement, Scc2-Scc4 et Pds5-Wapl, respectivement responsables du chargement de la cohésine et de sa dissociation de la chromatine. . Au cours de la phase S, une acétyltransférase appelée Eco1 acétyle le domaine ATPase de Smc3 et déclenche l’établissement de la cohésion. Pour augmenter davantage la cohésion, un facteur métazoaire supplémentaire, la sororine forme un complexe avec Pds5 pour empêcher la liaison de Wapl. Pendant la métaphase, la cohésine centromérique est protégée par shugoshin-PP2A. Chez les métazoaires, la cohésine est libérée des chromosomes en deux étapes. La première nécessite la phosphorylation de la cohésine et permet à Wapl de se lier à nouveau à Pds5 afin d'assurer la médiation de la libération de cohésine indépendante du clivage à partir des bras des chromosomes. La seconde se produit lors de la réalisation de l'assemblage de la broche et nécessite l'activation d'une protéase appelée séparase, ce qui entraîne le clivage Scc1, libérant ainsi des chromatides soeurs à séparer dans des cellules filles. Au-delà de la cohésion, il devient également évident que la cohésine joue des rôles plus divers en interagissant avec une multitude d'autres facteurs, notamment la CTCF, une protéine connue comme un isolant, dont il a été rapporté qu'elle collabore à la détermination du génome 3D. structure.Pour comprendre comment la cohesine engage l'ADN, j'ai étudié les propriétés de liaison à l'ADN de sous-complexes précédemment identifiés. En déterminant une structure cristalline de la levure Scc3 liée à un fragment de la sous-unité Scc1 kleisin et de l'ADN, j'ai pu démontrer que Scc3 et Scc1 forment un module d'interaction composite de l'ADN. Le sous-complexe Scc3-Scc1 engage un ADN double brin à travers une surface conservée, chargée positivement. Nous démontrons que ce domaine est requis pour la liaison à l'ADN par Scc3-Scc1 in vitro, ainsi que pour l'enrichissement de la cohésine sur des chromosomes et pour la viabilité cellulaire. Ces résultats suggèrent que l'interface de liaison à l'ADN Scc3-Scc1 joue un rôle central dans le recrutement des complexes de la cohésine sur les chromosomes et donc que cette dernière exécute fidèlement ses fonctions lors de la division cellulaire.Pour étudier les bases moléculaires de la collaboration fonctionnelle signalée entre la cohésine et le CTCF dans la définition de la structure chromosomique 3D, j'ai identifié et déterminé la structure d'un complexe ternaire composé de SA2 humain (un orthologue de Scc3), de Scc1 et de CTCF. La structure révélait un motif de liaison SA2-Scc1 très répandu qui était présent non seulement dans le CTCF, mais aussi dans d’autres facteurs connexes fonctionellement, tels que shugoshin et Wapl. Les tests de compétition déroulants ont indiqué que la liaison de ces facteurs à SA2-Scc1 était mutuellement exclusive, ce qui suggère fortement qu'ils interagissent avec la cohésine via des mécanismes similaires. Pour démontrer ce principe, j'ai pu déterminer une structure de shugoshin en complexe avec SA2-Scc1, ce qui a confirmé que tant le shugoshin que le CTCF se lient à la même surface conservée sur la cohésine.