Three-dimensional chromatin reorganization regulates B cell development during ageing.

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.

Switch Tandem Repeats Influence the Choice of the Alternative End-Joining Pathway in Immunoglobulin Class Switch Recombination.

Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions. The DSBs ends are fused by the classical non-homologous end-joining (C-NHEJ) and the alternative-NHEJ (A-NHEJ) pathways. Of the two pathways, the A-NHEJ displays a bias towards longer junctional micro-homologies (MHs). The Sµ region displays features that distinguish it from other S regions, but the molecular basis of Sµ specificity is ill-understood. We used a mouse line in which the downstream Sγ3 region was put under the control of the Eµ enhancer, which regulates Sµ, and analyzed its recombination activity by CSR-HTGTS. Here, we show that provision of Eµ enhancer to Sγ3 is sufficient to confer the recombinational features of Sµ to Sγ3, including efficient AID recruitment, enhanced internal deletions and robust donor function in CSR. Moreover, junctions involving Sγ3 display a bias for longer MH irrespective of sequence homology with switch acceptor sites. The data suggest that the propensity for increased MH usage is an intrinsic property of Sγ3 sequence, and that the tandem repeats of the donor site influence the choice of the A-NHEJ.

Mechanism and regulation of class switch recombination by IgH transcriptional control elements.

Class switch recombination (CSR) plays an important role in humoral immunity by generating antibodies with different effector functions. CSR to a particular antibody isotype is induced by external stimuli, and occurs between highly repetitive switch (S) sequences. CSR requires transcription across S regions, which generates long non-coding RNAs and secondary structures that promote accessibility of S sequences to activation-induced cytidine deaminase (AID). AID initiates DNA double-strand breaks (DSBs) intermediates that are repaired by general DNA repair pathways. Switch transcription is controlled by various regulatory elements, including enhancers and insulators. The current paradigm posits that transcriptional control of CSR involves long-range chromatin interactions between regulatory elements and chromatin loops-stabilizing factors, which promote alignment of partner S regions in a CSR centre (CSRC) and initiation of CSR. In this review, we focus on the role of IgH transcriptional control elements in CSR and the chromatin-based mechanisms underlying this control.

Altered 3D chromatin structure permits inversional recombination at the IgH locus.

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombination activating gene products (RAG) 1 and 2. Diversity (DH) gene segments rearrange first, followed by variable (VH) gene rearrangements. Here, we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, which recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on wild-type IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by distinct mechanisms from those that apply to DH gene segments. These distinctions may underlie differential allelic choice associated with each step of IgH gene assembly.

Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre.

Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eµ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eµ enhancer affects both transcription and recombination, making it difficult to conclude if Eµ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eµ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eµ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eµ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.

Two modes of cis-activation of switch transcription by the IgH superenhancer.

B cell isotype switching plays an important role in modulating adaptive immune responses. It occurs in response to specific signals that often induce different isotype (I) promoters driving transcription of switch regions, located upstream of the Ig heavy chain (IgH) constant genes. The transcribed switch regions can recombine, leading to a change of the constant gene and, consequently, of antibody isotype. Switch transcription is controlled by the superenhancer 3' regulatory region (3'RR) that establishes long-range chromatin cis-interactions with I promoters. Most stimuli induce more than one I promoter, and switch transcription can occur on both chromosomes. Therefore, it is presently unknown whether induced I promoters compete for the 3'RR on the same chromosome. Here we performed single-chromosome RT-qPCR assays to examine switch transcription monoallelically in the endogenous context. We show that there are two modes of 3'RR-mediated activation of I promoters: coactivation and competition. The nature of the inducing signal plays a pivotal role in determining the mode of activation. Furthermore, we provide evidence that, in its endogenous setting, the 3'RR has a bidirectional activity. We propose that the coactivation and competition modes mediated by the 3'RR may have evolved to cope with the different kinetics of primary immune responses.

Developmental regulation of DNA cytosine methylation at the immunoglobulin heavy chain constant locus.

DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.

Duplication of a germline promoter downstream of the IgH 3' regulatory region impairs class switch recombination.

During an adaptive immune response, B cells can change their surface immunoglobulins from IgM to IgG, IgE or IgA through a process called class switch recombination (CSR). Switching is preceded by inducible non-coding germline transcription (GLT) of the selected constant gene(s), which is largely controlled by a super-enhancer called the 3' regulatory region (3'RR). Despite intense efforts, the precise mechanisms that regulate GLT are still elusive. In order to gain additional insights into these mechanisms, we analyzed GLT and CSR in mutant B cells carrying a duplication of the promoter of the α constant gene (Iα) downstream of 3'RR. Duplication of the Iα promoter affected differently GLT and CSR. While for most isotypes a drop in GLT was accompanied by a decrease in CSR, that was not the case for switching to IgA, which diminished despite unchanged GLT. Unexpectedly, there was no obvious effect on GLT and CSR to IgG3. Remarkably, specific stimuli that normally induce switching to IgG2b had contrasting effects in mutant B cells; Iγ2b was now preferentially responsive to the stimulus that induced Iα promoter. We propose that one mechanism underlying the induced 3'RR-mediated activation of GL promoters involves, at least in part, specific transcription factories.

Inducible CTCF insulator delays the IgH 3' regulatory region-mediated activation of germline promoters and alters class switching.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3' regulatory region (3'RR) in a stimulus-dependent manner. Why the 3'RR-mediated up-regulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3'RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3'RR in developing B cells.

Developmental Switch in the Transcriptional Activity of a Long-Range Regulatory Element.

Eukaryotic gene expression is often controlled by distant regulatory elements. In developing B lymphocytes, transcription is associated with V(D)J recombination at immunoglobulin loci. This process is regulated by remote cis-acting elements. At the immunoglobulin heavy chain (IgH) locus, the 3' regulatory region (3'RR) promotes transcription in mature B cells. This led to the notion that the 3'RR orchestrates the IgH locus activity at late stages of B cell maturation only. However, long-range interactions involving the 3'RR were detected in early B cells, but the functional consequences of these interactions were unknown. Here we show that not only does the 3'RR affect transcription at distant sites within the IgH variable region but also it conveys a transcriptional silencing activity on both sense and antisense transcription. The 3'RR-mediated silencing activity is switched off upon completion of VH-DJH recombination. Our findings reveal a developmentally controlled, stage-dependent shift in the transcriptional activity of a master regulatory element.

Quantification of V(D)J recombination by real-time quantitative PCR.

B and T lymphocytes have the unique capacity to somatically rearrange their antigen receptor loci through V(D)J recombination. D-JH and VH-DJH recombination events are usually visualized by semi-quantitative PCR followed by detection of end products, which is time consuming and requires the use of hazardous elements. Additionally, it necessitates relatively large amounts of genomic DNA which could be limiting when the cell populations of interest are rare. Here, we describe a real-time quantitative PCR assay for a fast quantification of V(D)J recombination events at the IgH locus.