During FY17 we accomplished the following: 1) Completed one phase of analysis of IGCR1-deleted IgH alleles. A manuscript describing this work was submitted for publication. 2) We continued analyses of cell lines generated during FY16 that altered additional CTCF-binding sites (beyond IGCR1) in the proximal VH region. We assayed changes in histone modifications caused by deletion of single or multiple CTCF sites using ChIP. We also used deep sequencing to identify changes in VH gene utilization during VDJ recombination in pro-B cells deleted of specific CTCF-binding sites in the proximal VH region. 3) We generated cell lines in which individual CTCF sites within IGCR1 were inverted. In these cells CTCF binding should be unaffected, permitting us to determine the role of CTCF orientation on regulating chromatin looping and VDJ recombination. 4) We found that DST4.2, a DH gene segment that is located 70kb 5 of DFL16.1 and rarely used in DH recombination on WT IgH alleles, recombined at robust levels on IGCR1-mutated IgH alleles. All observed recombination events occurred by deleting intervening DNA. This is most consistent with the recently proposed model of RAG1/2 tracking from the JH-associated recombination center. However, DST4.2 also contains a 5 RSS that could be used for inversional recombination, but is not utilized. To test the possibility that this RSS is functionally impaired we are generating several plasmids in which 5 or 3 RSSs associated with DH gene segments will be tested independently with a JH1 RSS for recombination efficiency.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000372-10
Application #
9551249
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Aging
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
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
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
Subrahmanyam, Ramesh; Sen, Ranjan (2012) Epigenetic features that regulate IgH locus recombination and expression. Curr Top Microbiol Immunol 356:39-63
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
Roy, Ananda L; Sen, Ranjan; Roeder, Robert G (2011) Enhancer-promoter communication and transcriptional regulation of Igh. Trends Immunol 32:532-9
Osipovich, Oleg A; Subrahmanyam, Ramesh; Pierce, Steven et al. (2009) Cutting edge: SWI/SNF mediates antisense Igh transcription and locus-wide accessibility in B cell precursors. J Immunol 183:1509-13

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