Insertion of an imprinted insulator into the IgH locus reveals developmentally regulated, transcription-dependent control of V(D)J recombination.

The assembly of antigen receptor loci requires a developmentally regulated and lineage-specific recombination between variable (V), diversity (D), and joining (J) segments through V(D)J recombination. The process is regulated by accessibility control elements, including promoters, insulators, and enhancers. The IgH locus undergoes two recombination steps, D-J(H) and then V(H)-DJ(H), but it is unclear how the availability of the DJ(H) substrate could influence the subsequent V(H)-DJ(H) recombination step. The Eµ enhancer plays a critical role in V(D)J recombination and controls a set of sense and antisense transcripts. We epigenetically perturbed the early events at the IgH locus by inserting the imprinting control region (ICR) of the Igf2/H19 locus or a transcriptional insulator devoid of the imprinting function upstream of the Eµ enhancer. The insertions recapitulated the main epigenetic features of their endogenous counterparts, including differential DNA methylation and binding of CTCF/cohesins. Whereas the D-J(H) recombination step was unaffected, both the insulator insertions led to a severe impairment of V(H)-DJ(H) recombination. Strikingly, the inhibition of V(H)-DJ(H) recombination correlated consistently with a strong reduction of DJ(H) transcription and incomplete demethylation. Thus, developmentally regulated transcription following D-J(H) recombination emerges as an important mechanism through which the Eµ enhancer controls V(H)-DJ(H) recombination.

Seeking sense of antisense switch transcripts.

In B lymphocytes, class switch recombination (CSR) machinery targets highly repetitive sequences, called switch (S) sequences, in the constant domain of the immunoglobulin heavy chain (IgH) locus. Cotranscriptional generation of R loops at S sequences provides the substrate for the mutagenic enzyme AID (Activation-Induced cytidine Deaminase), which initiates the DNA breaks at the transcribed sequences. Both sense and antisense transcripts across the S regions have been reported. Our recent work shows that, unlike its sense counterpart, antisense transcription of S sequences is dispensable for CSR in vivo.

Sense transcription through the S region is essential for immunoglobulin class switch recombination.

Class switch recombination (CSR) occurs between highly repetitive sequences called switch (S) regions and is initiated by activation-induced cytidine deaminase (AID). CSR is preceded by a bidirectional transcription of S regions but the relative importance of sense and antisense transcription for CSR in vivo is unknown. We generated three mouse lines in which we attempted a premature termination of transcriptional elongation by inserting bidirectional transcription terminators upstream of Sµ, upstream of Sγ3 or downstream of Sγ3 sequences. The data show, at least for Sγ3, that sense transcriptional elongation across S region is absolutely required for CSR whereas its antisense counterpart is largely dispensable, strongly suggesting that sense transcription is sufficient for AID targeting to both DNA strands.

Combined deficiency of MSH2 and Sµ region abolishes class switch recombination.

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sµ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sµ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iµ-Cµ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iµ exon that may decrease with transcription elongation across the Sµ region.

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis.

Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck(-/-) mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck(-/-) BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.