B lymphocytes recognize and destroy viruses and bacteria though special receptors on their cell surface called antibodies. The affinity and specificity of these receptors for pathogens depends to a great extent on three genetic processes: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles the 5 end of the antibody gene by combining related DNA segments. The recombination is catalyzed by the RAG1 and RAG2 enzymes. Somatic hypermutation on the other hand introduces random point mutations to increase the binding affinity of the antibody for the pathogen in question. Lastly, CSR introduces further changes to facilitate the elimination of the invading pathogen. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced Cytidine Deaminase (AID). The importance of RAGs and AID in the immune response is highlighted in humans and animals deficient for these enzymes, which are highly susceptible to infection and exhibit gut flora-dependent hyperplasia of intestinal villi. Complex diseases such as autoimmunity have long been associated with RAG and AID-dependent activity and both enzyme complexes are promiscuous, in that they can also damage non-immunoglobulin genes, including oncogenes (tumor-inducing genes). This off-targeting activity can lead to DNA mutations and oncogene deregulation, resulting in malignant transformation. Predominant among these irregularities are chromosomal translocations, which drive the formation of B cell lymphomas (e.g. Burkitt lymphomas and multiple myeloma) in humans. Thus, unraveling how RAG and AID activities are regulated under normal conditions and deregulated during tumorigenesis is key. This fiscal year we have furthered our understanding of AID biology and B cell transformation in several ways: i) we have resolved the nature of RPA recruitment to AID-mediated DNA breaks. As mentioned above, AID promotes chromosomal translocations by inducing DNA breaks at immunoglobulin genes and oncogenes in the G1 phase of the cell cycle. RPA is a ssDNA-binding protein that associates with damaged DNA in the S phase and facilitates the assembly of factors involved in homologous repair such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage. Because of the discrepancy in cell cycle stages, scientists have suggested that RPA might have a role in immunoglobulin gene recombination outside its homologous repair one. In a manuscript published in the January issue of Cell Reports we have demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1. However, recruitment in S-G2/M is extensive and associated with Rad51 accumulation as expected if RPA functions mainly in homologous recombination. Thus, most RPA recruitment are antibody genes represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. ii) AID is expressed in activated B lymphocytes. This process is initiated by a global increase in mRNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring ssDNA genome-wide, we have recently shown in the journal Cell that the genome of nave cells is poised for rapid activation. In G0, 90% of promoters from genes to be expressed in cycling lymphocytes are loaded with polymerases but unmelted and thus they support only basal transcription. Furthermore, we have found that the transition from abortive to productive elongation is kinetically limiting causing polymerases to accumulate nearer transcription start sites. Resting lymphocytes also limit expression of the TFIIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our publication identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens. iii) The Myc protein, which is deregulated by chromosomal translocations mediated by AID, has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and cell differentiation. No principle yet unifies Myc action due partly to an incomplete inventory of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, we collaborated with David Levens laboratory from NCI and created a mouse model that helps visualize Myc expression in vivo. In a publication in Cell we used these mice to analyze the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications in activated B cells and ES cells. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action thereore explains the vast majority of Myc biology observed in literature and provides a rationale as to how this protein transforms B lymphocytes.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Li, Guideng; So, Alex Yick-Lun; Sookram, Reeshelle et al. (2018) Epigenetic silencing of miR-125b is required for normal B-cell development. Blood 131:1920-1930
Kieffer-Kwon, Kyong-Rim; Nimura, Keisuke; Rao, Suhas S P et al. (2017) Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation. Mol Cell 67:566-578.e10
Wang, Qiao; Kieffer-Kwon, Kyong-Rim; Oliveira, Thiago Y et al. (2017) The cell cycle restricts activation-induced cytidine deaminase activity to early G1. J Exp Med 214:49-58
Canela, Andres; Maman, Yaakov; Jung, Seolkyoung et al. (2017) Genome Organization Drives Chromosome Fragility. Cell 170:507-521.e18
Juan, Aster H; Wang, Stan; Ko, Kyung Dae et al. (2017) Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells. Cell Rep 18:297
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Kouzine, Fedor; Wojtowicz, Damian; Baranello, Laura et al. (2017) Permanganate/S1 Nuclease Footprinting Reveals Non-B DNA Structures with Regulatory Potential across a Mammalian Genome. Cell Syst 4:344-356.e7
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Casellas, Rafael; Basu, Uttiya; Yewdell, William T et al. (2016) Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity. Nat Rev Immunol 16:164-76
Aiden, Erez Lieberman; Casellas, Rafael (2015) Somatic Rearrangement in B Cells: It's (Mostly) Nuclear Physics. Cell 162:708-11

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