title: Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region.
abstract: Receptor activator of NF-κB ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Bishop
authors.forename: Heidi M
authors.initials: HM
authors.lastname: Coy
authors.forename: Robert D
authors.initials: RD
authors.lastname: Nerenz
authors.forename: Mark B
authors.initials: MB
authors.lastname: Meyer
authors.forename: J Wesley
authors.initials: JW
authors.lastname: Pike
mesh_terms: D000107:Acetylation; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D053245:RANK Ligand / Q000096:biosynthesis* / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*
chemical_list: D006657:Histones; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D012319:RNA Polymerase II