| Description of regulatory DNA elements and nuclear factors governing the induction and maintenance of lineage specifying transcription factors (TF) is essential for understanding cellular differentiation. Foxp3 is the lineage specifying TF for regulatory T (Treg) cells, and is necessary for their differentiation, acquisition of suppressive function, and capacity to prevent spontaneous, fatal auto immune disorders in mice and humans. We have characterized the coordinated function of conserved non-coding sequence (CNS) regulatory elements and transacting factors in both the induction and maintenance of Foxp3 expression in vivo. We have shown that in the absence of the maintenance DNA methyltransferase, Dnmt1, T-cell receptor (TCR) signaling is sufficient for unrestrained induction of Foxp3. This finding suggests a dominant role for TCR signaling in Treg differentiation, and that Dnmt1 activity is required for restricting Foxp3 expression to the appropriate compartment of the CD4 T cell lineage. Through analysis of mice with deletion of proximal CNS elements within the Foxp3 gene we revealed modular regulation of Foxp3 expression by three CNS, each with dedicated and distinct functionality. CNS3, a c-Rel-binding pioneer element, increases the probability of Foxp3 induction in precursor cells; CNS1 facilitates the induction of Foxp3 in the lymphoid periphery but not the thymus; and CNS2, a "cellular memory module" whose binding to Foxp3 complexes potentially forms an auto-regulatory feed-forward loop, is required for the heritable maintenance of the active Foxp3 locus in the progeny of dividing Treg cells, thus defining lineage stability. We demonstrate that regulation of a lineage specifying TF, Foxp3, is mediated through multiple CNS responsive to distinct transacting factors, which cooperate to determine the appropriate induction and maintenance of Foxp3 expression, thereby controlling the composition and size of the Treg cell compartment. |
