title: TGF-β-mediated Foxp3 gene expression is cooperatively regulated by Stat5, Creb, and AP-1 through CNS2.
abstract: Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3(-) T cells. These sequences are, however, demethylated in Foxp3(+) Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect and shown that transcription factors AP-1, Stat5, and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-β signaling through Smad3-mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during induced Treg induction, whereas AP-1 and Creb maintain Enhancer 2 activity.
authors.affiliation: Research Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Ogawa
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Tone
authors.forename: Masato
authors.initials: M
authors.lastname: Tsuda
authors.forename: Christian
authors.initials: C
authors.lastname: Peter
authors.forename: Herman
authors.initials: H
authors.lastname: Waldmann
authors.forename: Masahide
authors.initials: M
authors.lastname: Tone
mesh_terms: D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D051858:Forkhead Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006657:Histones / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D006657:Histones; D050799:STAT5 Transcription Factor; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta