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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000372-06
Application #
8736545
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$513,893
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
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