Research Interests

Assembling and Diversifying Antigen Receptor Genes During Lymphocyte Development

The B and T lymphocytes that constitute the adaptive immune system make use of antigen receptor molecules, known as immunoglobulins and T cell receptors, to combat viral and bacterial infections.  Each of the hundreds of millions of lymphocytes expresses a different antigen receptor on its surface, indicative of an extraordinary level of diversity in these receptors.  The fundamental interest of the lab is to understand the two major processes that generate this diversity:  V(D)J recombination and somatic hypermutation.

V(D)J recombination assembles immunoglobulin and T cell receptor genes from component V (variable), D (diversity), and J (joining) gene segments in developing B and T cells.  The reaction can be divided into two phases.  In the first, two DNA segments are bound by the recombination machinery, brought into close physical proximity, and the DNA is then cleaved adjacent to the two gene segments.  This generates four DNA ends, which in the second phase of the reaction are processed and joined by the cellular DNA repair machinery to form the reaction products.

One major interest of the lab is the enzymatic mechanism of the first phase of V(D)J recombination, which is catalyzed by the proteins encoded by the recombination activating genes, RAG1 and RAG2.  We have found that RAG1 is a multifunctional protein:  it binds RAG2 as well as the RSS, and it contains the crucial "active site" of the RAG1-RAG2 complex.  The functions of RAG2 remain more mysterious.  We are studying how the RAG proteins bend and twist the substrate DNA in order to execute DNA cleavage and have developed fluorescence resonance energy transfer (FRET) as a method to study the structure of protein-DNA complexes formed by the RAG proteins.  We are currently using FRET to characterize the dynamic conformational changes that accompany DNA binding by RAG1/RAG2.  We are also using X-ray crystallography to study RAG-DNA interactions, and have determined the structure of a major DNA binding domain of RAG1 in complex with DNA.

Recently, we have used chromatin immunoprecipitation (ChIP) to determine the pattern of binding of RAG1 and RAG2 in the genome of developing lymphocytes.  The RAG proteins associate with one small, focal region of each antigen receptor locus investigated.  We propose that these "recombination centers" are specialized sites within which the two recombining gene segments are brought together.  Interestingly, each RAG protein has the ability to be recruited into recombination centers in the absence of the other, suggesting several distinct levels of regulation of RAG binding.

Somatic hypermutation introduces point mutations into the variable regions of immunoglobulin genes (which encode antibodies) in B cells during an immune response.  These mutations allow for the generation of B cells expressing antibodies with high affinity for an invading microorganism, a process known as affinity maturation.  This process is important for the "memory" of the immune system, which helps protect individuals from recurrent infections with the same microorganism, and underlies the success of many vaccines.

Somatic hypermutation is initiated by an enzyme known as activation induced deaminase (AID), which deaminates cytosine to create uracil in immunoglobulin genes.  The uracil is then processed by the mismatch and base excision repair pathways to create mutations at the site of deamination and at nearby sites in the DNA.  We have demonstrated that mutations can spread approximately 30 base pairs in both directions from the site of deamination and that this spreading process exhibits a striking strand asymmetry:  a "top" (nontranscribed) strand uracil supports efficient spreading while a "bottom" strand uracil does not.

Somatic hypermutation has been linked to genomic instability and B cell lymphomas and a major interest of the lab is to understand how the reaction is targeted to immunoglobulin loci and how the rest of the genome is protected from its deleterious effects.  We have demonstrated that somatic hypermutation is associated with phosphorylation of serine 14 on histone H2B have demonstrated that the immunoglobulin promoter plays a previously unsuspected role in determining the efficiency of somatic hypermutation.  We are currently investigating the role of phosphorylated H2B and of promoters and other DNA elements in immunoglobulin loci in the targeting of the reaction. 

Recently, we have discovered that the genome is protected from damage due to somatic hypermutation by two distinct mechanisms:  specific targeting of AID and gene-specific high-fidelity DNA repair.  AID preferentially deaminates immunoglobulin genes and our data demonstrate that many genes avoid the action of AID altogether.  Surprisingly, however, a large number of non-immunoglobulin genes are hit by AID but fail to accumulate mutations because the uracils are repaired in an high-fidelity manner.  These genes include many, such as Myc, that have been implicated in the development of B cell malignancies.  It is clear that AID acts more widely in the genome than previously suspected.  An important implication of these findings is that anything that undermines high-fidelity repair would be expected to allow for widespread accumulation of mutations, with potentially disastrous consequences.  We are testing the hypothesis that cellular stress, viral infection, or the early steps in malignant transformation, perturb high-fidelity repair and thereby contribute to or hasten the development of cancer.