Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation

Abstract

Regulatory T cells (T reg ) expressAcquired immune tolerance depends upon a subset of T cells that suppress the differentiation and function of pathogenic effector T cells. These regulatory T cells (T reg ) specifically express Foxp3, a member of the forkhead family of DNA-binding proteins that appears to be necessary and sufficient for T reg lineage choice (1-3). Humans with mutations in the foxp3 gene suffer from an X-linked complex of immune dysregulation, polyendocrinopathy, and enteropathy, a syndrome that results in part from the lack of T reg and leads to eventual death of these patients in childhood (4). Similarly, T reg are crucial for the inhibition of immunopathology in experimental models of organ transplantation and early onset autoimmune disorders and have been implicated in the control of childhood (type 1) autoimmune diabetes mellitus (5, 6).Recent studies have shown that Foxp3 overexpressed in transformed cell lines can repress transcription from artificial forkhead, nuclear factor of activated T cell (NFAT), 3 or NFB consensus binding elements in transiently transfected plasmids (7-9). These data suggest that Foxp3, like Foxp1 and Foxp2 (10), can function as a transcriptional repressor. However, expression of Foxp3 also leads to the induction of multiple genes, but the molecular basis for this activity is not known. It is also clear that Foxp3 expression alone is not sufficient for regulatory activity, as Foxp3ϩ T reg must receive antigenic signaling to exert suppressor function (11), but the basis for this activation requirement is not clear.We have used chromatin immunoprecipitation (ChIP) to analyze binding and remodeling of the chromatin at several Foxp3-responsive genes. Our studies provide important insight into how Foxp3 may regulate anergy in T reg and suggest a potential explanation for why T reg require T cell receptor (TCR) activation for their suppressive functions. EXPERIMENTAL PROCEDURESCells-A Jurkat human T cell line expressing the ecotropic retroviral receptor (mCAT-1) (12) was used for these studies. CD4ϩCD25Ϫ and CD4ϩCD25ϩ were purified from C57BL/6 mice using a T reg isolation kit (Miltenyi). T reg were also induced in vitro by culturing CD4ϩCD25Ϫ for 3-4 days on platebound anti-CD3 in the presence of soluble anti-CD28 (0.5 g/ml each) and TGF␤ (5 ng/ml).Retroviral Transduction-Murine Foxp3 cDNA was amplified from C57BL/6 thymus and cloned into the murine stem cell virus (MSCV)-based MIGR1 and MINR1 retroviral vectors (13), and versions of each vector were constructed containing an in-frame, N-terminal FLAG epitope. For generation of retrovirus, constructs were cotransfected with the pCLeco (Invitrogen) helper plasmid into the 293T-based Phoenix ecotropic packaging cell line (provided by G. Nolan, Stanford University). CD4ϩCD25Ϫ T cells were activated with phorbol 12-myristate 13-acetate (3 ng/ml), ionomycin (1 M), and IL-2 (10 units/ml) for 24 h, washed, and transduced by spinfection

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

“…Recently, FOXP3 was shown to directly repress IL-2 as well as IFN-g gene expression after TCR-mediated stimulation by its binding to the promoter/enhancer regions of the respective genes, as part of a FOXP3-histone deacetylase-histone acetyltransferase complex that induces chromatin remodeling through histone deacetylation [31,32]. Any reduction in FOXP3 expression is therefore likely to interfere with correct assembly of such complexes and ultimately leads to loss of their repressive activity [31].…”

Unravelling the molecular basis for regulatory T‐cell plasticity and loss of function in disease

“…The modifiable mediators of this regulation include methylation,37, 38, 39, 40, 41, 42, 43, 44 acetylation45, 46, 47 and microRNA‐mediated control of transcript translation 28, 48, 49, 50, 51, 52, 53, 54, 55. The global regulation of FOXP3 is influenced by chromatin organises, including SATB1 acts both during development and in mature cells 48, 58.…”

Human FOXP3 and cancer

“…This dual activity of FOXP3 defines characteristic features of Treg cells, which have high expression of CTLA-4 and CD25, but low expression of IL-7R and lack IL-2 production. FOXP3 also binds to the IFNg locus, suppressing IFNg expression (Chen et al, 2006). The molecular mechanisms by which FOXP3 controls gene expression in Treg cells involve interactions of FOXP3 with other transcription factors.…”

“…Binding of FOXP3 to the IL-2 and IFNg genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling, impeding gene transcription. Conversely, binding of FOXP3 to the Il2ra and Ctla4 promoters results in increased histone acetylation, enhancing gene transcription (Chen et al, 2006).…”