Spatial organization and nuclear positioning of murine immunoglobulin loci in developing B cells

Background

Genomic DNA in eukaryotic cells is highly organized and shows multiple levels of compaction. Contraction and folding of DNA enables long-range interactions between widely dispersed genes to facilitate their expression. Furthermore, genes are positioned in the nucleus to either transcriptionally active or permissive compartments [1]. During B-cell development in the bone marrow, immunoglobulins (Ig) are assembled through stepwise recombination of V, (D) and J genes. Imaging studies have shown that Ig loci are organized into rosette-like clusters of loops and contract prior to rearrangement [2]. Moreover, during B cell development Ig loci change their nuclear positioning [3]. However, how the chromatin fiber is organized into higher-order structures and how this is regulated is still unknown.

Material and methods

To study 3D organization and nuclear compart-mentalization of Ig loci, we performed 3D DNA FISH on progenitor B cells derived from E2A-/-, RAG1-/- and RAG1-/-Vh81X mice. Three regions along the IGH and IGK loci were detected with BAC probes. Spatial distances between genetic markers and from each probe to the nuclear lamins were measured with a confocal microscopy and analyzed.

Results

Spatial distant measurements between IGH and IGK probes showed that both loci were contracted in RAG1-/- pro-B cells as compared with E2A-/- pre-pro-B cells. Interestingly, both loci remained contracted in RAG1-/-Vh81X pre-B cells. The nuclear gene positioning analysis revealed that IGH moved from the nuclear periphery to the central domains in pro-B cells, while IGK remained peripherally located. Similarly, IGK relocated in pre-B cells to nuclear center, whereas IGH repositioned peripherally. However, in other cell stages both loci were kept in the peripheral compartments.

Conclusions

Our studies confirm that spatial chromatin organization of murine IGH and IGK changes during development. Both loci undergo contraction prior to gene rearrangement which allows juxtaposing of genomically distant gene segments and creates equal opportunities for recombination. Specifically, IGH and IGK were contracted in pro-B cells, although IGK is assembled later during the development. This observation can be explained by changes in the nuclear gene positioning. IGH and IGK were tightly associated with the nuclear lamins in early pre-pro-B cells. However, in committed pro-B and pre-B cells, IGH and IGK were consecutively positioned to the active central compartments. Hence, we conclude that spatial chromatin organization and nuclear positioning orchestrate stepwise antigen receptor formation. Chromosome conformation capture experiments are ongoing to further dissect the DNA folding mechanism.