The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

Permanganate/S1 Nuclease Footprinting Reveals Non-B DNA Structures with Regulatory Potential across a Mammalian Genome.

DNA in cells is predominantly B-form double helix. Though certain DNA sequences in vitro may fold into other structures, such as triplex, left-handed Z form, or quadruplex DNA, the stability and prevalence of these structures in vivo are not known. Here, using computational analysis of sequence motifs, RNA polymerase II binding data, and genome-wide potassium permanganate-dependent nuclease footprinting data, we map thousands of putative non-B DNA sites at high resolution in mouse B cells. Computational analysis associates these non-B DNAs with particular structures and indicates that they form at locations compatible with an involvement in gene regulation. Further analyses support the notion that non-B DNA structure formation influences the occupancy and positioning of nucleosomes in chromatin. These results suggest that non-B DNAs contribute to the control of a variety of critical cellular and organismal processes.

Ups and Downs of Poised RNA Polymerase II in B-Cells.

Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II) at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively) attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG), which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression regulation.

B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity.

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

Global regulation of promoter melting in naive lymphocytes.

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.

A genome-wide map of CTCF multivalency redefines the CTCF code.

The "CTCF code" hypothesis posits that CTCF pleiotropic functions are driven by recognition of diverse sequences through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is supported by in vitro binding studies of a limited number of sequences. To study CTCF multivalency in vivo, we define ZF binding requirements at ∼50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs 4-7 anchor CTCF to ∼80% of targets containing the core motif. Nonconserved flanking sequences are recognized by ZFs 1-2 and ZFs 8-11 clusters, which also stabilize CTCF broadly. Alternatively, ZFs 9-11 associate with a second phylogenetically conserved upstream motif at ∼15% of its sites. Individually, ZFs increase overall binding and chromatin residence time. Unexpectedly, we also uncovered a conserved downstream DNA motif that destabilizes CTCF occupancy. Thus, CTCF associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.

A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus.

Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.

A West Nile virus DNA vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase I clinical trial.

BACKGROUND: West Nile virus (WNV) is a flavivirus that causes meningitis and encephalitis. There are no licensed vaccines to prevent WNV in humans. The safety and immunogenicity of a first-generation WNV DNA vaccine was demonstrated in a clinical trial and a similar DNA vaccine has been licensed for use in horses. METHODS: A DNA vaccine encoding the protein premembrane and the E glycoproteins of the NY99 strain of WNV under the transcriptional control of the CMV/R promoter was evaluated in an open-label study in 30 healthy adults. Half of the subjects were age 18-50 years and half were age 51-65 years. Immune responses were assessed by enzyme-linked immunosorbent assay, neutralization assays, intracellular cytokine staining, and ELISpot. RESULTS: The 3-dose vaccine regimen was safe and well tolerated. Vaccine-induced T cell and neutralizing antibody responses were detected in the majority of subjects. The antibody responses seen in the older age group were of similar frequency, magnitude, and duration as those seen in the younger cohort. CONCLUSIONS: Neutralizing antibody responses to WNV were elicited by DNA vaccination in humans, including in older individuals, where responses to traditional vaccine approaches are often diminished. This DNA vaccine elicited T cell responses of greater magnitude when compared with an earlier-generation construct utilizing a CMV promoter. CLINICAL TRIALS REGISTRATION: NCT00300417.

Complement protein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of West Nile virus.

Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remain uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of cells expressing Fc-gamma receptors (CD32) and explains how C1q restricts the ADE of flavivirus infection.

Protonation of individual histidine residues is not required for the pH-dependent entry of west nile virus: evaluation of the "histidine switch" hypothesis.

Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a requirement for any single histidine as a pH-sensing "switch," and they suggest that additional features of the E protein are involved in triggering pH-dependent steps in the flavivirus life cycle.

Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.

Helical virus particles formed from morphological subunits of a membrane containing icosahedral virus.

The classic publication by Caspar and Klug in 1962 [Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27:1-24.] has formed the basis of much research on virus assembly. Caspar and Klug predicted that a single virus morphological unit could form a two dimensional lattice composed of 6-fold arrays (primitive plane), a family of icosahedra of increasing triangulation numbers (T) and helical arrays of varying length. We have shown that icosahedral viruses of varying T numbers can be produced using Sindbis virus [Ferreira, D. F. et al. 2003. Morphological variants of Sindbis virus produced by a mutation in the capsid protein. Virology 307:54-66]. Other studies have shown that Sindbis glycoproteins can also form a 2-dimensional lattice confirming Caspar and Klug's prediction of the primitive plane as a biologically relevant structure [VonBonsdorff, C. H., and S. C. Harrison. 1978. Sindbis virus glycoproteins form a regular icosahedral surface lattice. J. Virol. 28:578]. In this study we have used mutations in the glycoproteins of membrane containing Sindbis virus to create helical-virus-like particles from the morphological subunits of a virus of icosahedral geometry. The resulting virus particles were examined for subunit organization and were determined to be constructed of only 6-fold rotational arrays of the virus glycoproteins. A model of the tubular virus particles created from the 6-fold rotational arrays of Sindbis virus confirmed the observed structure. These experiments show that a common morphological unit (the Sindbis E1-E2 heterodimer) can produce three different morphological entities of varying dimensions in a membrane-containing virus system.

Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50 to 100 million human infections each year, highlighting the need for a safe and effective vaccine. In this study, we describe the production of pseudoinfectious DENV reporter virus particles (RVPs) using two different genetic complementation approaches, including the creation of cell lines that release reporter viruses in an inducible fashion. In contrast to studies with West Nile virus (WNV), production of infectious DENV RVPs was temperature-dependent; the yield of infectious DENV RVPs at 37 degrees C is significantly reduced in comparison to experiments conducted at lower temperatures or with WNV. This reflects both a significant reduction in the rate of infectious DENV RVP release over time, and the more rapid decay of infectious DENV RVPs at 37 degrees C. Optimized production approaches allow the production of DENV RVPs with titers suitable for the study of DENV entry, assembly, and the analysis of the humoral immune response of infected and vaccinated individuals.

Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization.

West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV.

The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection.

Antibody binding to the icosahedral arrangement of envelope proteins on the surface of flaviviruses can result in neutralization or enhancement of infection. We evaluated how many antibodies must bind to a given epitope on West Nile virus (WNV) to achieve neutralization. The most potent monoclonal antibodies (mAbs) block infection at concentrations that result in low occupancy of accessible sites on the virion, with neutralization occurring when as few as 30 of 180 envelope proteins are bound. In contrast, weakly neutralizing mAbs recognize fewer sites on the virion and require almost complete occupancy to inhibit WNV infection. For all mAbs studied, enhancement of infection is possible in cells bearing activating Fc-gamma receptors when the number of mAbs docked to the virion is not sufficient for neutralization. Thus, neutralization is best described by a model requiring "multiple hits" with the cumulative functional outcome determined by interplay between antibody affinity and epitope accessibility.

Structural characterization of the E2 glycoprotein from Sindbis by lysine biotinylation and LC-MS/MS.

Sindbis is an Alphavirus capable of infecting and replicating in both vertebrate and invertebrate hosts. Mature Sindbis virus particles consist of an inner capsid surrounded by a host-derived lipid bilayer, which in turn is surrounded by a protein shell consisting of the E1 and E2 glycoproteins. While a homolog of the E1 glycoprotein has been structurally characterized, the amount of structural data on the E2 glycoprotein is considerably less. In this study, the organization of the E2 glycoprotein was probed by surface biotinylation of intact virions. The virus remained fully infectious, demonstrating that the biotinylation did not alter the topology of the proteins involved in infection. Seven sites of modification were identified in the E2 glycoprotein (K70, K76, K97, K131, K149, K202, and K235), while one site of modification in the E1 glycoprotein (K16) was identified, confirming that the E1 protein is almost completely buried in the virus structure.

In vivo processing and isolation of furin protease-sensitive alphavirus glycoproteins: a new technique for producing mutations in virus assembly.

Sindbis virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding, and maturation.