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.
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•Genome-wide maps of 11 CTCF zinc finger mutants in B lymphocytes•Zinc finger mutations differentially affect CTCF binding and nuclear mobility•CTCF uses zinc finger clusters to recognize DNA sequence diversity•DNA sequences flanking the core motif modulate CTCF binding
CTCF is a nuclear architectural protein that binds to thousands of highly diverse sequences in eukaryotes. The current hypothesis, known as the “CTCF code,” proposes that CTCF binds DNA targets through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is mostly supported by in vitro binding studies. By expressing ZF mutants in B lymphocytes, Resch, Casellas, and colleagues now present genome-wide maps of CTCF multivalency. They show that CTCF reads sequence diversity by relying on well-defined ZF clusters.
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.
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•Lymphocyte activation induces a proportional amplification of the transcriptome•ssDNA-seq detects promoter melting and non-B DNA in living cells•Promoters in G0 lymphocytes are PolII loaded but unmelted•TFIIH expression and activity are limited in G0 lymphocytes
In addition to preinitiation complex assembly and polymerase pausing, DNA melting can also regulate transcription in eukaryotes. In lymphocytes, immune promoters loaded with polymerase are restrained until the DNA is melted, possibly via changes in TFIIH levels.
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.
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.
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.
Ups and Downs of Poised RNA Polymerase II in B-Cells Dao, Phuong; Wojtowicz, Damian; Nelson, Steevenson ...
PLOS computational biology/PLoS computational biology,
04/2016, Letnik:
12, Številka:
4
Journal Article
Recenzirano
Odprti dostop
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.
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