Organization and Expression of Mouse Vh Genes

A fundamental question in immunogenetics involves the mechanism by which the antigen receptor genes (immunoglobulin and T cell receptor) become targeted for rearrangement in a tissue and stage appropriate manner during lymphocyte development. Work from numerous investigators has shown that locus “accessibility” is the result of a dynamic process that includes alterations in both nuclear positioning and chromatin structure. Recent studies in our lab have focused on the regulation of variable region gene accessibility within the mouse immunoglobulin heavy chain locus (Igh). We are particularly interested in how usage of the many different Vh gene segments is controlled during rearrangement.

The illustration depicts the different Vh gene segment families. The D and J gene segments that are recombined with a Vh segment to create the coding region for the variable portion of an Ig molecule are represented by the small black boxes. The sequences coding for the constant region (C) are also shown
 


Accessibility of D-distal Vh genes is uniquely dependent on IL-7 signaling. We have recently explored the biochemical basis of this pathway using a cell line model developed in our lab. In this system, IL-7 induces accessible chromatin, as measured by germline transcription and histone H4 acetylation, at the D-distal VhA1 gene but not at a D-proximal Vh gene (V11). We used dominant negative and constitutively active Stat5 retroviruses to show that active Stat5 is both necessary and sufficient for the induction of VhA1 germline transcripts. Chromatin immunoprecipitation assays demonstrated the recruitment of Stat5 to the VhA1 gene. These results provide a molecular mechanism by which individual Vh genes of the Igh locus can be independently regulated during B cell development (Stanton and Brodeur, J. Immunology , 2005)

Other recent work in our lab has searched for novel regulatory elements within the Igh locus. We hypothesize that the 5' flanking region of Igh contains regulatory sequences important to the regulation of Vh gene rearrangement and expression. Analysis of the region immediately 5' of the most D-distal Vh gene segment revealed a cluster of DNase I hypersensitivity sites. One of these sites, named HS1, is found exclusively in pro-B cells, the stage of B cell development at which the Igh locus undergoes rearrangement.

DNase I hypersensitivity can indicate the presence of sites within the DNA that may be involved in controlling the accessibility of gene segments to the VDJ recombinase. The experiment shown to the right helped us identify such a site near the 5' border of the Igh locus. 22D6 is a B lineage cell line while KBALB is a non-lymphoid cell line. The HS1 site is detected in 22D6 and not in KBALB, an early indication that it is B lineage specific.
We are working to understand how HS1 and other hypersensitive sites located within the Igh locus function to regulate VDJ recombination. Our hypothesis predicts that transcription factors known to play a central role in controlling B cell development interact with such sites and influence the rearrangement process. To date we have confirmed the presence of two such factors, PU.1 and Pax5, at HS1, as diagrammed below.
The diagram depicts one way PU.1 and Pax5 might influence HS1. The unlabeled pink oval represents additional factors that may be required in the HS1 complex.