Thymocyte Development CD 8 Locus Nuclear Dynamics during

Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other. The Journal of Immunology , 2010, 184: 5686–5695. the second layer of Abs was applied in the same way. The Abs used in this study were: streptavidin-Alexa Fluor 594 conjugate (Invitrogen), sheep anti-DIG (Roche), and Alexa Fluor 647 donkey anti-sheep IgG (H+L) (Invitrogen), all in a dilution 1:200 in the blocking solution. The final wash was in 2 3 SSC and then DAPI stain was added before mounting them in Vectashield H-1000 (Vector Laboratories, Burlingame, CA) and sealed with a colorless nail polish.