Jiyoti Verma-Gaur

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all VH genes to be brought in proximity with DH-JH 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 VH 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 DH-JH-Eμ-CH domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the DH region and parts of the VH 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.

Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells.

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 germ-line 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 VH 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 VH genes close to the DJH 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 VH genes to undergo efficient rearrangement.

Deep Sequencing of the Murine Igh Repertoire Reveals Complex Regulation of Nonrandom V Gene Rearrangement Frequencies

A diverse Ab repertoire is formed through the rearrangement of V, D, and J segments at the IgH (Igh) loci. The C57BL/6 murine Igh locus has >100 functional VH gene segments that can recombine to a rearranged DJH. Although the nonrandom 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. Chromatin immunoprecipitation–sequencing results for several histone modifications and RNA polymerase II binding, RNA-sequencing for sense and antisense noncoding germline transcripts, and proximity to CCCTC-binding factor (CTCF) and Rad21 sites were compared with 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 noncoding RNA in general, but having a CTCF site near their recombination signal sequence 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 noncoding RNA, which may compensate for their poorer recombination signal sequences 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.

Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub

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 Vα-to-Jα 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 (Eα) interacts directly with Vα and Jα gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, Eα promotes interactions between these Vα and Jα segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to Eα and to many locus promoters, biases Eα to interact with these promoters, and is required for efficient Vα–Jα recombination. Our data indicate that Eα and CTCF cooperate to create a developmentally regulated chromatin hub that supports Vα–Jα synapsis and recombination.

Unifying model for molecular determinants of the preselection Vβ repertoire

Significance The assembly of immunoglobulin and T-cell receptor genes by V(D)J (variable, diversity, joining) recombination must strike a balance between maximum diversification of antigen receptors and favoring gene segments with specialized functions. We quantified the use of V gene segments in the primary T-cell receptor β repertoire, defining the relative contribution of 13 parameters in shaping their recombination efficiencies. Computational analysis of these data provides a unifying model, revealing a minimal set of five parameters that predict Vβ use. This model building approach will help predict how natural alterations of large V clusters impact immune receptor repertoires. 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.

Germline Deletion of Igh 3′ Regulatory Region Elements hs 5, 6, 7 (hs5–7) Affects B Cell-Specific Regulation, Rearrangement, and Insulation of the Igh Locus

Regulatory elements located within an ∼28-kb region 3′ of the Igh gene cluster (3′ regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5–7 region (hs5–7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the DH region. To test the function of hs5–7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5–7 knockout (KO) (hs5–7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5–7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal VH7183 gene families. Hs5–7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5–7, a high-density CTCF-binding region at the 3′ end of the Igh locus, impacts usage of VH regions as far as 500 kb away.

Initiation of Antigen Receptor-Dependent Differentiation into Plasma Cells by Calmodulin Inhibition of E2A

Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca2+ sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca2+ signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

CTCF-cohesin complex: architect of chromatin structure regulates V(D)J rearrangement

The CTCF/cohesin complex regulates higher order chromatin structure by creating long-range chromatin loops and by insulating neighboring genes from each other. The lymphocyte antigen receptor loci have large numbers of CTCF/cohesin binding sites, and recent studies demonstrate that the CTCF/cohesin complex plays several important roles in regulating the process of V(D)J recombination at these megabase-sized receptor loci.

Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A

Signaling from the BCR is used to judge Ag-binding strengths of the Abs of B cells. BCR signaling enables the selection for successive improvements in the Ag affinity over an extremely broad range of affinities during somatic hypermutation. We show that the mouse BCR is subject to general negative feedback regulation of the receptor proteins, as well as many coreceptors and proteins in signal pathways from the receptor. Thus, the BCR can downregulate itself, which can enable sensitive detection of successive improvements in the Ag affinity over a very large span of affinities. Furthermore, the feedback inhibition of the BCR signalosome and most of its proteins, as well as most other regulations of genes by BCR stimulation, is to a large extent through inhibition of the transcription factor E2A by Ca2+/calmodulin.

Broad feedback inhibition of pre-B-cell receptor signaling components

During B lymphocyte development, first immunoglobulin heavy chain gene segments and then immunoglobulin light chain gene segments are rearranged to create antibody diversity. Early in the development, expression of a pre-B-cell receptor (pre-BCR) that has membrane-bound Ig heavy chain protein associated with surrogate light chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces survival and clonal expansion to select cells with good heavy chains, but it also down-regulates transcription of the genes for the SLC proteins and CD19 and limits its own proliferative signaling. Here we have analyzed whether the down-regulation is limited to the SLC proteins and CD19, and we show that the pre-BCR of primary mouse pre-B-cells instead is subject to a broad feedback inhibition of pre-BCR signaling components. Activation of signaling leads to down-regulation of the receptor proteins, many co-receptors and proteins participating in signal pathways from the receptor. Thus the down-regulation of the pre-BCR is much broader than previously assumed. We also show that Ca(2+)/calmodulin inhibition of the transcription factor E2A is required for the feedback inhibition of the pre-BCR signaling proteins.