Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

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.

Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1.

In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

Molecular Mechanism of Nucleosome Recognition by the Pioneer Transcription Factor Sox.

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.

Toward more potent imidazopyridine inhibitors of Candida albicans Bdf1: Modeling the role of structural waters in selective ligand binding.

Novel agents to treat invasive fungal infections are urgently needed because the small number of established targets in pathogenic fungi makes the existing drug repertoire particularly vulnerable to the emergence of resistant strains. Recently, we reported that Candida albicans Bdf1, a bromodomain and extra-terminal domain (BET) bromodomain with paired acetyl-lysine (AcK) binding sites (BD1 and BD2) is essential for fungal cell growth and that an imidazopyridine (1) binds to BD2 with selectivity versus both BD1 and human BET bromodomains. Bromodomain binding pockets contain a conserved array of structural waters. Molecular dynamics simulations now reveal that one water molecule is less tightly bound to BD2 than to BD1, explaining the site selectivity of 1. This insight is useful in the performance of ligand docking studies to guide design of more effective Bdf1 inhibitors, as illustrated by the design of 10 new imidazopyridine BD2 ligands 1a-j, for which experimental binding and site selectivity data are presented.

Enantioselective Approach for Expanding the Three-Dimensional Space of Tetrahydroquinoline to Develop BET Bromodomain Inhibitors.

The pharmaceutical industry has a pervasive need for chiral specific molecules with optimal affinity for their biological targets. However, the mass production of such compounds is currently limited by conventional chemical routes, that are costly and have an environmental impact. Here, we propose an easy access to obtain new tetrahydroquinolines, a motif found in many bioactive compounds, that is rapid and cost effective. Starting from simple raw materials, the procedure uses a proline-catalyzed Mannich reaction followed by the addition of BF3 ⋅ OEt2 , which generates a highly electrophilic aza-ortho-quinone methide intermediate capable of reacting with different nucleophiles to form the diversely functionalized tetrahydroquinoline. Moreover, this enantioselective one-pot process provides access for the first time to tetrahydroquinolines with a cis-2,3 and trans-3,4 configuration. As proof of concept, we demonstrate that a three-step reaction sequence, from simple and inexpensive starting compounds and catalysts, can generate a BD2-selective BET bromodomain inhibitor with anti-inflammatory effect.

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends.

The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.

Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B.

The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle, yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified, we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B reassembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant that plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg.

Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.

A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.

Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA.

Acetyl and other acyl groups from different short-chain fatty acids (SCFA) competitively modify histones at various lysine sites. To fully understand the functional significance of such histone acylation, a key epigenetic mechanism, it is crucial to characterize the cellular sources of the corresponding acyl-CoA molecules required for the lysine modification. Like acetate, SCFAs such as propionate, butyrate and crotonate are thought to be the substrates used to generate the corresponding acyl-CoAs by enzymes known as acyl-CoA synthetases. The acetyl-CoA synthetase, ACSS2, which produces acetyl-CoA from acetate in the nucleocytoplasmic compartment, has been proposed to also mediate the synthesis of acyl-CoAs such as butyryl- and crotonyl-CoA from the corresponding SCFAs. This idea is now widely accepted and is sparking new research projects. However, based on our direct in vitro experiments with purified or recombinant enzymes and structural considerations, we demonstrate that ACSS2 is unable to mediate the generation of non-acetyl acyl-CoAs like butyryl- and crotonyl-CoA. It is therefore essential to re-examine published data and corresponding discussions in the light of this new finding.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

Binding stoichiometry and structural model of the HIV-1 Rev/importin ß complex.

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.

Investigating a macromolecular complex: the toolkit of methods.

Structural biologists studying macromolecular complexes spend considerable effort doing strictly "non-structural" work: investigating the physiological relevance and biochemical properties of a complex, preparing homogeneous samples for structural analysis, and experimentally validating structure-based hypotheses regarding function or mechanism. Familiarity with the diverse perspectives and techniques available for studying complexes helps in the critical assessment of non-structural data, expedites the pre-structural characterization of a complex and facilitates the investigation of function. Here we survey the approaches and techniques used to study macromolecular complexes from various viewpoints, including genetics, cell and molecular biology, biochemistry/biophysics, structural biology, and systems biology/bioinformatics. The aim of this overview is to heighten awareness of the diversity of perspectives and experimental tools available for investigating complexes and of their usefulness for the structural biologist.

ATAD2 controls chromatin-bound HIRA turnover.

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.

Interaction of Epstein-Barr virus BZLF1 C-terminal tail structure and core zipper is required for DNA replication but not for promoter transactivation.

The Epstein-Barr virus (EBV) protein BZLF1 contains a bZIP DNA binding domain in which C-terminal tail residues fold back against a zipper region that forms a coiled coil and mediates dimerization. Point mutagenesis in the zipper region reveals the importance of individual residues within the (208)SSENDRLR(215) sequence that is conserved in C/EBP for transactivation and EBV DNA replication. The restoration of BZLF1 DNA replication activity by the complementation of two deleterious mutations (S208E and D236K) indicates that the interaction of the C-terminal tail and the core zipper is required for DNA replication, identifying a functional role for this structural feature unique to BZLF1.

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Exploring the structure and dynamics of macromolecular complexes by native mass spectrometry.

Mass spectrometry (MS) is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity and speed. Over the past 25 years, MS performed under non-denaturing conditions ("native MS") has been successfully exploited to investigate non-covalently associated biomolecules. Here we illustrate native MS applications aimed at studying protein-ligand interactions and structures of biomolecular assemblies, including both soluble and membrane protein complexes. Moreover, we review how the partial dissociation of holo-complexes can be used to determine the stoichiometry of subunits and their topology. We also describe "native top-down MS", an approach based on Fourier Transform MS (FT MS), whereby non-covalent interactions are preserved while covalent bonds are selectively fragmented. Overall, native MS plays an increasingly important role in integrative structural biology, helping researchers to elucidate the three dimensional architecture of intricate macromolecular complexes.

Cryo-electron microscopy of the chromatin fiber.

The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.