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* Department of Immunology,
Department of Pulmonary Medicine, and
Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands
Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-
production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.
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1 This work was supported by the Dutch Cancer Society KWF, the European Science Foundation EUROCORES Programme EuroDYNA (ERAS-CT-2003-980409), Fundação para a Ciência e a Tecnologia, and the Association for International Cancer Research.
2 C.R.d.A. and H.H. contributed to this study equally.
3 Address correspondence and reprint requests to Dr. Niels Galjart, Department of Cell Biology and Genetics, Erasmus MC Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands and Dr. Rudi H. Hendriks, Department of Pulmonary Medicine, Erasmus MC, Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail addresses: n.galjart{at}erasmusmc.nl and r.hendriks{at}erasmusmc.nl
4 Abbreviations used in this paper: Treg, regulatory T; SATB1, special AT-rich binding protein 1; CTCF, CCCTC-binding factor; CBS, CTCF binding site; ChIP, chromatin immunoprecipitation; BM-DC, bone marrow-derived dendritic cell; TNP-KLH, 2,4,6-trinitrophenyl-keyhole limpet hemocyanin; DN, double negative; DP, double positive; ISP, immature single positive; SP, single positive; WT, wild type.
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