The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq.

For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies use RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per-cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here, we describe VDJ sequencing (VDJ-seq), which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer-extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMIs) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and nonproductive VDJ and DJ recombination events on a per-cell basis. Library preparation takes 3 d, with 2 d of sequencing and 1 d of data processing and analysis.

Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination.

Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.

The mouse immunoglobulin heavy chain V-D intergenic sequence contains insulators that may regulate ordered V(D)J recombination.

During immunoglobulin heavy chain (Igh) V(D)J recombination, D to J precedes V to DJ recombination in an ordered manner, controlled by differential chromatin accessibility of the V and DJ regions and essential for correct antibody assembly. However, with the exception of the intronic enhancer Emu, which regulates D to J recombination, cis-acting regulatory elements have not been identified. We have assembled the sequence of a strategically located 96-kb V-D intergenic region in the mouse Igh and analyzed its activity during lymphocyte development. We show that Emu-dependent D antisense transcription, proposed to open chromatin before D to J recombination, extends into the V-D region for more than 30 kb in B cells before, during, and after V(D)J recombination and in T cells but terminates 40 kb from the first V gene. Thus, subsequent V antisense transcription before V to DJ recombination is actively prevented and must be independently activated. To find cis-acting elements that regulate this differential chromatin opening, we identified six DNase I-hypersensitive sites (HSs) in the V-D region. One conserved HS upstream of the first D gene locally regulates D genes. Two further conserved HSs near the D region mark a sharp decrease in antisense transcription, and both HSs bind CTCF in vivo. Further, they both possess enhancer-blocking activity in vivo. Thus, we propose that they are enhancer-blocking insulators preventing Emu-dependent chromatin opening extending into the V region. Thus, they are the first elements identified that may control ordered V(D)J recombination and correct assembly of antibody genes.

Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.

Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.

Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.

Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Emu.

V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Emu and is abrogated in mice lacking this enhancer, indicating that Emu regulates DH antisense transcription. Emu was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Emu controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Emu can regulate chromatin accessibility throughout the DH region. In contrast, Emu deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.

Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region.

The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5' end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3' end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.

Antisense intergenic transcription in V(D)J recombination.

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJ(H) to VDJ(H) recombination and terminates concomitantly with VDJ(H) recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate V(H)-to-DJ(H) recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.