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Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate


Immune homeostasis is dependent on tight control over the size of a population of regulatory T (Treg) cells capable of suppressing over-exuberant immune responses. The Treg cell subset is comprised of cells that commit to the Treg lineage by upregulating the transcription factor Foxp3 either in the thymus (tTreg) or in the periphery (iTreg)1,2. Considering a central role for Foxp3 in Treg cell differentiation and function3,4, we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the Treg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1–3) in Treg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of Treg cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-β–NFAT response element, is superfluous for tTreg cell differentiation, but has a prominent role in iTreg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing Treg cells. Foxp3 binds to CNS2 in a Cbf-β–Runx1 and CpG DNA demethylation-dependent manner, suggesting that Foxp3 recruitment to this ‘cellular memory module’ facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, Treg lineage stability. Together, our studies demonstrate that the composition, size and maintenance of the Treg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic or -intrinsic cues.

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Figure 1: Conserved non-coding sequences and chromatin modifications at the Foxp3 locus.
Figure 2: CNS3 controls de novo Foxp3 expression.
Figure 3: CNS1 controls peripheral, but not thymic, induction of Foxp3 expression.
Figure 4: CNS2 controls the heritable maintenance of Foxp3 expression.


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We thank T.-T. Chu and L. Karpik for expert technical assistance and mouse colony management, S. Roh for embryonic stem cell culture and screening, J. Rasmussen and A. Kas for bioinformatics support, A. Beg for providing c-Rel knockout mice, P. Treuting for histopathology analysis, J. Gerard for assistance in luciferase reporter assays, and C. Wilson, S. Tarakhovsky and L.-F. Lu for critical comments on the manuscript. This work was supported by grants from the National Institutes of Health (to A.Y.R.). Y.Z. and A.C. were supported by the CRI-Irvington Institute postdoctoral fellowship. S.Z.J. was supported by the CRI pre-doctoral training grant. A.Y.R. is an investigator with the Howard Hughes Medical Institute.

Author Contributions Y.Z. and S.J. performed and analysed the experiments, with assistance from A.C. in oligonucleotide pull-down and from X.P.P. in ChIP experiments. K.F. assisted with blastocysts injections. S.J., Y.Z. and A.Y.R. designed experiments and wrote the paper.

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Correspondence to Alexander Y. Rudensky.

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Zheng, Y., Josefowicz, S., Chaudhry, A. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).

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