Professor, Department of Cell Biology
Chanin Bldg., Room 417
Regulation of Antibody Heavy Chain Gene Rearrangements and Expression
The immune system is our spacesuit for life in an environment containing enormous numbers of infectious agents. An essential part of the immune system is the B cell, which is the only cell type that produces antibodies. Antibody (Ig) genes are constructed via a series of DNA rearrangements. Occasionally, mistakes occur during antibody construction, which activate oncogenes and lead to cancers. My long term goal is to understand the mechanisms that initiate and control antibody gene rearrangements within the 3 megabase heavy chain gene (IgH) locus. Our experiments focus on a complex 3' IgH regulatory "ignition" region that lies immediately downstream of the antibody heavy chain gene cluster in mouse and humans. The ~50 kb 3' IgH regulatory region (3’ RR), containing multiple regulatory elements, and a 1 kb intronic enhancer, are the only two currently known regulatory regions for the Igh locus. We have recently identified an extension of the 3’ RR (hs5, 6, 7), which like the other 3' enhancers, has binding sites for Pax5, a transcription factor essential for B cell development. In addition, the 3' RR extension contains insulator activity, i.e. prevention of communication between an enhancer and its target promoter. This is associated with binding sites for CTCF, a protein identified in all mammalian insulators. Chromatin analysis shows that the 3' RR extension is likely to be active throughout B cell development while other 3' RR segments become progressively and stage-specific active.
Murine germline Igh locus, with variable (VH), diversity (DH), joining (JH) and constant region genes (µ,δ,γ,ε,α). Regulatory elements, Eµ and the 3' regulatory region (3'RR) are depicted
A major goal of our laboratory is to understand the mechanisms by which the 3' RR functions in both mouse and human, both by acting on the Igh locus and by insulating the locus from its non-Igh neighboring genes. We predict that the 3’ RR promotes Igh DNA rearrangement events throughout B cell development. In addition to its known essential contribution to the expression of different classes of antibodies (class switch recombination), the 3' RR is likely to regulate high levels of expression of antibodies in fully differentiated plasma cells. The 3’ RR is also implicated in the formation (VDJ joining) and subsequent hypermutation (SHM) of the variable region. CTCF binding sites are anticipated to play a major role in these activities by promoting interactions between distal DNA sequences through loop formation.
We are interested in identifying the extent of B cell-specific regulation of the IgH locus, by analyzing histone modifications in and the binding of CTCF and Pax5 to 3' RR sequences during B cell development and class switching. We have generated a knockout of the CTCF binding extension of the 3' RR. The influence of progressive changes in DNA methylation during B cell development on CTCF binding is under study. Furthermore, we are examining the interaction of the 3’ RR with target sequences within the IgH locus, as assessed by the chromosome capture conformation technique (3C). Together, this approach is designed to understand how molecular acrobatics can occur only within the IgH locus without intruding on downstream non-IgH genes.