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Genomic Integrity and Immunity Group

Research Overview

DNA repair and immunity

The mechanisms that maintain the stability of the mammalian genome are a major focus of our laboratory. Mature B lymphocytes are an ideal system to address these questions since upon activation during the immune response, B cells undergo class switch recombination and somatic hypermutation at their immunoglobulin (Ig) genes. These genetic events require the molecular interplay between the transcription machinery, chromatin remodeling complexes, a plethora of DNA repair enzymes, and a B cell specific factor known as AID. Although much has been learned about these individual protein complexes, to date it has not been possible to unravel how their activity is orchestrated within the cell nucleus in a living cell. We are elucidating the interplay between these systems by using a combination of state of the art confocal microscopy, video imaging, and bioinformatic techniques.

Damage	Nbs1
PolI/Nbs1	Hoechst

Figure 1: A system to elucidate the interplay between gene transcription and DNA damage. RNA Polymerase I is labeled with GFP. This polymerase is exclusive for ribosomal gene transcription and thus localizes to the nucleoli (green). DNA damage can be specifically induced in boxed nucleoli and nuclear DNA with a 364nm laser. The repair enzyme Nbs1 (and many other repair factors) is recruited to DNA double-stranded breaks (red). Hoechst stains all nuclear DNA. Using photobleaching techniques the in vivo elongation time, recruitment and incorporation frequencies of PolI can be measured both in the presence or absence of DNA damage.

The second goal of our laboratory is to investigate how hypermutation and recombination of Ig genes are regulated in vivo to evade both tumorigenesis and autoimmunity. Both hypermutation and switch recombination require the activity of AID, a B cell specific DNA deaminase. AID-mediated DNA lesions downstream of hypermutation and recombination are believed to give rise to chromosomal translocations in B lymphocytes, and a direct link between these processes and tumorigenesis has been established. Furthermore, hypermutation has long been associated with autoimmunity in humans and animal models. In rheumatoid arthritis patients, for instance, extensive hypermutation activity occurs at the synovial membrane of affected joints, which exacerbates the progression of the disease. Using conventional gene targeting, bacterial artificial chromosome transgenesis, and lentiviral-mediated RNAi techniques we are investigating the regulation of AID expression and its activity in the animal.

Figure 2: AID genetic markers. (A) The green fluorescent protein (GFP) is fused to the AID gene by homologous recombination in bacteria. Immunized AID-GFP BAC transgenic mice show that nearly all activated B cells (CD95⁺ population) express AID upon immunization (panels 2, panel 1: profile of a non-transgenic immunized control). These animals provide a model to investigate AID expression in vivo.
(B) Mice expressing Cre under the AID promoter allow lox-mediated deletion of the neomycin (neo) cassette and subsequent expression of YFP in germinal center CD95^high) and post-germinal center (CD95^low) cells in Rosa-neo-EYFP knock-in animals. Because the Rosa promoter is constitutively expressed, post-germinal center cells are yellow-tagged permanently, as opposed to cells expressing AID-GFP which are green only during AID transcription. Left panel: lymph nodes of immunized animals, right panel: Peyer's patches. These double transgenic mice are useful to unravel the kinetics of the immune response and the memory B cell compartment both in health and disease.

Selected Publications

Teng G, Hakimpour P, Landgraf P, Rice A, Tuschl T, Casellas R, Papavasiliou FN. MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity. 2008 May;28(5):621-9. Epub 2008 May 1.

Takizawa M, Tolarová H, Li Z, Dubois W, Lim S, Callen E, Franco S, Mosaico M, Feigenbaum L, Alt FW, Nussenzweig A, Potter M, Casellas R. AID expression levels determine the extent of cMyc oncogenic translocations and the incidence of B cell tumor development. J Exp Med. 2008 Aug 4. [Epub ahead of print]

Kruhlak M, Crouch EE, Orlov M, Montaño C, Gorski SA, Nussenzweig A , Misteli T, Phair RD, Casellas R. The ATM Repair Pathway Inhibits RNA Polymerase I Transcription in Response to Chromosome Breaks. Nature 2007;447(7145):730-4.

Crouch EE, Li Z, Takizawa M, Fichtner-Feigl S, Gourzi P, Montaño C, Feigenbaum L, Wilson P, Janz S, Papavasiliou FN, Casellas R. Regulation of AID expression in the immune response. J Exp Med. 2007;204(5):1145-56.

Casellas R, Jankovic M, Meyer G, Gazumyan A, Luo Y, Roeder R, Nussenzweig M. OcaB is required for normal transcription and V(D)J recombination of a subset of immunoglobulin kappa genes. Cell. 2002; 110(5): 575-85.

See complete list of publications

 

Updated September 17, 2007