Progress during FY18: 1) Continued breeding mouse strains that carry point mutations in E. Current experimental standards suggest 5-6 backcrosses of mouse strains generated by CRISPR/Cas9 technology to minimize chances of confounding interpretations due to off-target mutations. We generated 4 such strains of mice, with 2-4 independent lines of each strain. Backcrossing to WT C57BL6 mice is almost complete for 2 strains. Preliminary developmental phenotyping shows a partial block at the pro- to pre-B transition in mice that carry E2, E5-double-mutated alleles. 2) Our working model is that inappropriate E activation directs the IgH rearrangement phenotype in DP thymocytes. To identify which enhancer motifs directed E activity in thymocytes we assayed DH recombination in D cells from E-mutated mice. We noted that the A/B-mutated enhancer conferred higher levels of recombination, suggesting protein binding to these sites may attenuate E activity in DP cells. 3) Two of the 4 enhancer-mutated strains were bred to a RAG2-deficient background to hold the IgH locus in unrearranged configuration. Bone marrow pro-B cells will be used to map the transcriptional and epigenetic state of the locus to identify the roles of individual DNA binding proteins in establishing the pre-rearrangement structure of the IgH locus. 4) continued collaboration with Stephen Desiderio's lab to study the function of the autoinhibitory domain of RAG2. these studies were published in Molecular and Cellular Biology (2018).

National Institute of Health (NIH)
National Institute on Aging (NIA)
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Qiu, Xiang; Kumari, Gita; Gerasimova, Tatiana et al. (2018) Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus. Mol Cell 70:21-33.e6
Ward, Alyssa; Kumari, Gita; Sen, Ranjan et al. (2018) The RAG-2 Inhibitory Domain Gates Accessibility Of The V(D)J Recombinase To Chromatin. Mol Cell Biol :
Sen, Ranjan (2016) A Pioneer's Tail. Immunity 44:516-8
Lovely, Geoffrey A; Sen, Ranjan (2016) Evolving adaptive immunity. Genes Dev 30:873-5
Montefiori, Lindsey; Wuerffel, Robert; Roqueiro, Damian et al. (2016) Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire. Cell Rep 14:896-906
Feldman, Scott; Achour, Ikbel; Wuerffel, Robert et al. (2015) Constraints contributed by chromatin looping limit recombination targeting during Ig class switch recombination. J Immunol 194:2380-9
Gerasimova, Tatiana; Guo, Changying; Ghosh, Amalendu et al. (2015) A structural hierarchy mediated by multiple nuclear factors establishes IgH locus conformation. Genes Dev 29:1683-95
Kumari, Gita; Sen, Ranjan (2015) Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly. Adv Immunol 128:41-92
Selimyan, Roza; Gerstein, Rachel M; Ivanova, Irina et al. (2013) Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly. PLoS Biol 11:e1001475
Kumar, Satyendra; Wuerffel, Robert; Achour, Ikbel et al. (2013) Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny. Genes Dev 27:2439-44

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