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.

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.

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.

A major obstacle to a broadly neutralizing antibody (bnAb)-based HIV vaccine is the activation of appropriate B cell precursors. Germline-targeting immunogens must be capable of priming rare bnAb precursors in the physiological setting. We tested the ability of the VRC01-class bnAb germline-targeting immunogen eOD-GT8 60mer to activate appropriate precursors in mice transgenic for human immunoglobulin loci. Despite an average frequency of at most ~1 VRC01-class precursor per mouse, we found that at least 29% of singly-immunized mice produced a VRC01-class memory response, suggesting that priming generally succeeded when at least one precursor was present. The results demonstrate the feasibility of using germline targeting to prime specific and exceedingly rare bnAb precursor B cells within a human-like repertoire.

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.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

SummaryVariable (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.

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 V H region in pro-B cells, remodels chromatin for V H -to-DJ H recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh D H J H 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 E and is abrogated in mice lacking this enhancer, indicating that E regulates D H antisense transcription. E was recently demonstrated to regulate D H -to-J H recombination of the Igh locus. Together, these data suggest that E controls D H -to-J H recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which E can regulate chromatin accessibility throughout the D H region. In contrast, E deletion has no effect on V H antisense intergenic transcription, which is rarely associated with D H 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.In order to generate the primary repertoire of immunoglobulin (Ig) and T-cell receptor (TCR) molecules, antigen receptor loci undergo variable, diversity, and joining [V(D)J] recombination in B and T lymphocytes. Recombination is catalyzed by a recombinase complex containing the protein products of the recombinase-activating genes Rag1 and Rag2 (28). Within precursor lymphocytes, this process is strictly lineage specific, with heavy (Igh) and light (Ig and Ig) immunoglobulin loci fully recombining only in B lymphocytes and T-cell receptor loci (Tcra, Tcrb, Tcrg, and Tcrd) recombining only in T cells. Further, within lineages, loci are recombined in a precise order. Recombination of the Igh locus is the earliest step in the generation of the mature antibody repertoire in B lymphocytes. The Igh locus of the C57BL/6 mouse spans 3 Mb and comprises 195 V H genes spanning 2.5 Mb, 10 D H genes (ϳ60 kb), 4 J H genes (2 kb), and 8 constant (C H ) genes (200 kb) (31, 68). D H -to-J H recombination occurs on both Igh alleles before V H -to-DJ H recombination takes place (16).Lineage and stage specificity of V(D)J recombination are regulated by differential chromatin accessibility to the RAG proteins. Several mechanisms may contribute, but their relative importance is still unclear. The first process discovered was germ line transcription, which occurs in all antigen receptor loci across gene segments competent for recombination (34).This transcription was termed "sterile" or "germ line" to distinguish it from coding V(D)J transcription. In the Igh locus, the earliest germ line transc...