During FY13, we accomplished the following: 1) Used bacterial artificial chromosome (BAC) probes to validate 5C analyses of the IgH locus in pro-B cells. We identified three major interaction sites separated by approximately 1Mb that divided the VH locus into two prominent topological domains. These domains were selectively present in pro-B cells compared to non-B lineage bone marrow cells. To determine the molecular basis of this chromatin configuration, we carried out FISH studies in E- and Pax5-deficient pro-B cells expanded in culture. E-deficiency did not affect these domains, whereas the distal 1Mb domain was disrupted in Pax5-deficent pro-B cells. By incorporating these observations into our existing model, we arrived at a more refined 3D-structure of the pre-rearrangement IgH locus. 2) Two strains of newly-generated mice with modified IgH locus were bred for experimentation. Delta Emu strain replaces the intron enhancer E with short arrays of TetO and Gal4 binding sites. Site 4 strain incorporates tandem arrays of TetO sequences into a region 3 of the IgH locus. Both strains were first bred to -actin-cre mice to delete Neo gene used for targeting. Neo-deleted mmice were further bred to RAG2-deficient mice to generate Delta Emu/RAG2- and Site 4/RAG2- strains. Two independently derived lines were obtained for each genotype. Just prior to experimentation, our entire Delta Emu/RAG2- colony (both lines) was exterminated. This unfortunate incidence has put this research goal back by approproximately two years.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Institute on Aging
Zip Code
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
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
Phillips-Cremins, Jennifer E; Sauria, Michael E G; Sanyal, Amartya et al. (2013) Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153:1281-95
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
Subrahmanyam, Ramesh; Du, Hansen; Ivanova, Irina et al. (2012) Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions. Nat Immunol 13:1205-12
Subrahmanyam, Ramesh; Sen, Ranjan (2012) Epigenetic features that regulate IgH locus recombination and expression. Curr Top Microbiol Immunol 356:39-63
Roy, Ananda L; Sen, Ranjan; Roeder, Robert G (2011) Enhancer-promoter communication and transcriptional regulation of Igh. Trends Immunol 32:532-9