Structural basis of centromeric cohesion protection.

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.

Structural insights into p300 regulation and acetylation-dependent genome organisation.

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity.

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 structure of the cohesin ATPase elucidates the mechanism of SMC-kleisin ring opening.

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-Šresolution cryo-electron microscopy structure of the ATPγS-bound, heterotrimeric cohesin ATPase head module and the 2.1-Šresolution 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.

The structural basis for cohesin-CTCF-anchored loops.

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 Å, 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.

Discovery of BAY-985, a Highly Selective TBK1/IKKε Inhibitor.

The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε 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/IKKε 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.

Transcription factor dimerization activates the p300 acetyltransferase.

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.

Mechanistic insights into histone deposition and nucleosome assembly by the chromatin assembly factor-1.

Eukaryotic chromatin is a highly dynamic structure with essential roles in virtually all DNA-dependent cellular processes. Nucleosomes are a barrier to DNA access, and during DNA replication, they are disassembled ahead of the replication machinery (the replisome) and reassembled following its passage. The Histone chaperone Chromatin Assembly Factor-1 (CAF-1) interacts with the replisome and deposits H3-H4 directly onto newly synthesized DNA. Therefore, CAF-1 is important for the establishment and propagation of chromatin structure. The molecular mechanism by which CAF-1 mediates H3-H4 deposition has remained unclear. However, recent studies have revealed new insights into the architecture and stoichiometry of the trimeric CAF-1 complex and how it interacts with and deposits H3-H4 onto substrate DNA. The CAF-1 trimer binds to a single H3-H4 dimer, which induces a conformational rearrangement in CAF-1 promoting its interaction with substrate DNA. Two CAF-1•H3-H4 complexes co-associate on nucleosome-free DNA depositing (H3-H4)2 tetramers in the first step of nucleosome assembly. Here, we review the progress made in our understanding of CAF-1 structure, mechanism of action, and how CAF-1 contributes to chromatin dynamics during DNA replication.

Nut Directs p300-Dependent, Genome-Wide H4 Hyperacetylation in Male Germ Cells.

Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome.

Structural basis for Scc3-dependent cohesin recruitment to chromatin.

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.

Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function.

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.

Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1.

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3-H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

Structure of p300 in complex with acyl-CoA variants.

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.

Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly.

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.

Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters.

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.

Structure of the Pds5-Scc1 Complex and Implications for Cohesin Function.

Sister 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.

Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation.

CBP and p300 are histone acetyltransferases (HATs) that associate with and acetylate transcriptional regulators and chromatin. Mutations in their catalytic 'cores' are linked to genetic disorders, including cancer. Here we present the 2.8-Å crystal structure of the catalytic core of human p300 containing its bromodomain, CH2 region and HAT domain. The structure reveals that the CH2 region contains a discontinuous PHD domain interrupted by a RING domain. The bromodomain, PHD, RING and HAT domains adopt an assembled configuration with the RING domain positioned over the HAT substrate-binding pocket. Disease mutations that disrupt RING attachment led to upregulation of HAT activity, thus revealing an inhibitory role for this domain. The structure provides a starting point for understanding how chromatin-substrate targeting and HAT regulation are coupled and why mutations in the p300 core lead to dysregulation.

Crystal structure and mechanism of activation of TANK-binding kinase 1.

Tank-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.

Bone marrow of NZB/W mice is the major site for plasma cells resistant to dexamethasone and cyclophosphamide: implications for the treatment of autoimmunity.

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.