title: DNA double-strand breaks relieve USF-mediated repression of Dβ2 germline transcription in developing thymocytes.
abstract: Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRβ locus (Tcrb) D segments, Dβ1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJβ2 segment cluster is initially restricted to the J segments and only redirected upstream of Dβ2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJβ2 recombination is less efficient than that of DJβ1. Because inefficient DJβ2 assembly offers the potential for V-to-DJβ2 recombination to rescue frameshifted V-to-DJβ1 joints, we wished to determine how Dβ2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dβ2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dβ2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dβ2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dβ2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dβ2 recombinational accessibility during thymocyte development.
authors.affiliation: Department of Microbiology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stone
authors.forename: Ruth E
authors.initials: RE
authors.lastname: McMillan
authors.forename: David A
authors.initials: DA
authors.lastname: Skaar
authors.forename: Justin M
authors.initials: JM
authors.lastname: Bradshaw
authors.forename: Randy L
authors.initials: RL
authors.lastname: Jirtle
authors.forename: Michael L
authors.initials: ML
authors.lastname: Sikes
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics / Q000276:immunology / Q000528:radiation effects; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics* / Q000276:immunology* / Q000528:radiation effects; D002460:Cell Line; D003035:Cobalt; D021901:DNA, Intergenic / Q000737:chemistry* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D016694:Gene Rearrangement, delta-Chain T-Cell Antigen Receptor / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016513:Mice, SCID; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000528:radiation effects; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology / Q000528:radiation effects; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000528:radiation effects; D051720:Upstream Stimulatory Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*
chemical_list: D020121:5' Untranslated Regions; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; D003035:Cobalt