Compaction and looping of the ∼2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V H genes to be brought in proximity with D H -J H segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ∼60 sites throughout the V H region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a D H -J H -Eμ-C H domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D H region and parts of the V H locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.A ntigen receptors are created through the highly regulated lineage-specific process of V(D)J recombination, creating a diverse repertoire of Ig and T-cell receptors. The generation of the mouse Ig heavy chain in pro-B cells begins with D H -to-J H rearrangement on both alleles, followed by V H -to-D H J H rearrangement. In order for the >100 functional murine V H genes spread across ∼2.5 Mb to gain access to the single D-J rearrangement on that allele, the Igh locus undergoes contraction and looping during the pro-B-cell stage of B-cell differentiation (1-5). By measuring spatial distances between 11 small probes spread throughout the Igh locus, Jhunjhunwala et al. (2) demonstrated that distal and proximal V H genes were approximately equidistant from the D genes specifically at the pro-B-cell stage when the V H genes are rearranging. Computational as well as geometrical approaches have suggested that the locus is organized into rosette-like clusters of loops that compact during rearrangement. Several proteins have been reported to influence Igh locus compaction, including Pax5, YY1, and Ikaros (5-7). These proteins and others, such as Ezh2 (8), are also necessary for the rearrangement of distal V H genes but not proximal V H genes. This is most likely a consequence of the lack of locus compaction in the absence of these proteins. How all these proteins funct...

The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4 − CD8 − double-negative thymocytes and Tcra gene segments in CD4 + CD8 + double-positive thymocytes. Initial V α -to-J α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E α ) interacts directly with V α and J α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E α promotes interactions between these V α and J α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E α and to many locus promoters, biases E α to interact with these promoters, and is required for efficient V α -J α recombination. Our data indicate that E α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V α -J α synapsis and recombination.T-cell development | T-cell receptor | thymus T and B cells produce diverse antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at the T-cell receptor (Tcra, Tcrb, Tcrg, and Tcrd) and Ig (Igh, Igκ, and Igλ) loci. This V(D)J recombination is initiated by the lymphoid-specific recombination-activating gene-1 (RAG-1) and RAG-2 proteins, which recognize the recombination signal sequences (RSSs) that flank all V, D, and J gene segments and then cleave the DNA between the RSSs and the adjacent coding gene segments (1). A critical feature of the reaction is the assembly of a synaptic complex composed of two RSSs before the generation of RAG-dependent DNA doublestrand breaks (DSBs). As such, lineage-and developmental stagespecific V(D)J recombination events can be regulated not only by changes in RAG protein expression and RSS accessibility to RAG proteins but also by the ability of those RSSs to undergo synapsis (2).Conformational changes of antigen receptor loci are believed to support V(D)J recombination events because they can bring distant RSSs into proximity and therefore increase the probability of RSS synapsis (2, 3). Studies using 3D-FISH have demonstrated that lineage-and development stage-specific locus contraction marks the recombination windows at antigen receptor loci (3). For example, the 3-Mb Igh locus contracts specifically in pro-B cells to support V H -to-D H J H recombination (4-7). This contracted conformation brings distal and proximal V H segments, which are separated by megabases in the linear DNA sequence, to the vicinity of the D H J H cluster, presumably allowing all V H segments a similar opportunity for recombination (8). In addition, the mapping of Igh locus DNA-DNA...

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage-and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1 −/− pro-B cells are greatly impaired in distal V H gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V H genes close to the DJ H rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V H genes to undergo efficient rearrangement.A ntigen receptors in lymphocytes are assembled in the highly regulated lineage-specific process of V(D)J recombination, which creates a diverse repertoire of Ig and T-cell receptors. In each precursor lymphocyte, one each of the many V, D, and J gene segments at the appropriate receptor loci are juxtaposed to create a V(D)J exon encoding the variable region of the antigen receptor. In B-lineage progenitors, rearrangement occurs first at the Ig heavy chain (Igh) locus, where D H to J H rearrangement occurs first on both alleles, followed by V H to DJ H rearrangement (1). After successful rearrangement of the Igh locus, rearrangement at the Igk light chain locus begins. These successive stages of rearrangement have been proposed to be regulated by differential accessibility of different portions of the loci at the appropriate time for rearrangement (2). Early indications of this stage-specific and lineage-specific accessibility came from the observation that unrearranged gene segments underwent noncoding transcription at the stage immediately preceding their rearrangement (3, 4).The murine Igh locus spans ∼2.8 Mb, of which ∼2.4 Mb contains the 195 V H gene segments (5). The V H genes are divided i...

A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5′-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that being positioned near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have higher levels of histone marks and non-coding RNA, which may compensate for their poorer RSSs and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for each of the four domains that comprise this locus.

The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vβ use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DβJβ targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vβ gene segments, all pseudo-Vβ segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vβ use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DβJβ clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.lymphocytes | T-cell receptor | gene regulation G ene activity is regulated at multiple levels to coordinate expression during development. At a most basic level, the collection of cis-acting elements for a genetic locus recruits transcription factors that alter its chromatin environment to either induce or repress gene activity. Emerging studies indicate that the 3D conformation of a locus also plays an important role in the regulation of its composite genes (1). At most genes, many levels of control are integrated to achieve the requisite gene expression state. For example, transcriptional promoters interact with their cognate enhancers over considerable distances in the linear genome to generate "hubs" where the two cis elements are in spatial proximity (1, 2).All of these regulatory strategies are used to generate functional Ig (Ig) and T-cell receptor (Tcr) genes during lymphocyte development (3). Each antigen receptor (AgR) locus is composed of multiple variable (V), joining (J), and sometimes diversity (D) gene segments that are assembled by the process of V (D)J recombination, creating a potential variable region exon (4). Recombination is mediated by the RAG-1/2 enzymatic complex, which is expressed in all developing lymphocytes and recognizes semiconserved recombination signal sequences (RSSs) flanking all AgR gene segments (5). On selection of two compatible gene segments by RAG-1/2, recombination proceeds via a DNA break/repair mechanism, ultimately fusing the two selected segments (4, 5).The assembly of AgR genes is strictly regulated despite a common collection of ge...

The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

Objectives: Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. Methods:We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms.Results: Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2DD mutant, we implicate matrix polysaccharide b-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy.Conclusions: Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.