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FOXP3 promoter demethylation reveals the committed Treg population in humans.

Janson PC, Winerdal ME, Marits P, Thörn M, Ohlsson R, Winqvist O - PLoS ONE (2008)

Bottom Line: We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy.In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.

Methodology/principal findings: Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.

Conclusions/significance: The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

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Identification of cross-species conserved FOXP3 promoter elements.(A) FOXP3 transcript and m-Vista alignment showing conservation between human and mouse genomic sequences. Dark blue boxes display exons, outlined boxes are UTR's. Conserved regions are in red and conserved regions corresponding to exons are in light blue. (B) Schematic view of the conserved region upstream of the transcription start site indicating the location of promoter elements and CpG dinucleotides. (C) Clustal W alignment of human, mouse and rat genomic sequences showing a detailed view of the conservation at the FOXP3 core promoter. The broken arrow shows the position of the human transcription start site. The location of the TATA box is indicated by a black box. Red boxes indicate the CpG dinucleotides analyzed in this study. Transcription factor binding sites are marked with grey boxes.
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pone-0001612-g002: Identification of cross-species conserved FOXP3 promoter elements.(A) FOXP3 transcript and m-Vista alignment showing conservation between human and mouse genomic sequences. Dark blue boxes display exons, outlined boxes are UTR's. Conserved regions are in red and conserved regions corresponding to exons are in light blue. (B) Schematic view of the conserved region upstream of the transcription start site indicating the location of promoter elements and CpG dinucleotides. (C) Clustal W alignment of human, mouse and rat genomic sequences showing a detailed view of the conservation at the FOXP3 core promoter. The broken arrow shows the position of the human transcription start site. The location of the TATA box is indicated by a black box. Red boxes indicate the CpG dinucleotides analyzed in this study. Transcription factor binding sites are marked with grey boxes.

Mentions: The functional human FOXP3 promoter has been previously defined [11]. The region preceding the 5′ untranslated region (UTR) is highly conserved and contains important promoter elements such as TATA, GC and CAAT boxes as well as binding sites for NFAT and AP-1, well-established mediators of T cell activation (Figure 2B). The high degree of conservation of such cis-acting motifs in this region (Figure 2A) indicated that it might be under epigenetic influence. Therefore we investigated the FOXP3 promoter region with regards to CpG dinucleotides using ClustalW alignment comparing human, mouse and rat sequences (Figure 2C). The whole promoter region showed a mouse to man conservation of 87% (online supplemental material Figure S1), whereas the CpG containing region was 92% conserved, stressing its evolutionary importance (online supplemental material Figure S2). Moreover, ClustalW alignment determined that out of eight tightly positioned CpG dinucleotides just upstream of the transcription start site, positions −43, −65 and −77 were totally conserved between species. As these CpGs mapped just downstream of the binding sites for the transcription factors NFAT and AP-1 (Figure 2B–C), a special interest in these positions was rendered.


FOXP3 promoter demethylation reveals the committed Treg population in humans.

Janson PC, Winerdal ME, Marits P, Thörn M, Ohlsson R, Winqvist O - PLoS ONE (2008)

Identification of cross-species conserved FOXP3 promoter elements.(A) FOXP3 transcript and m-Vista alignment showing conservation between human and mouse genomic sequences. Dark blue boxes display exons, outlined boxes are UTR's. Conserved regions are in red and conserved regions corresponding to exons are in light blue. (B) Schematic view of the conserved region upstream of the transcription start site indicating the location of promoter elements and CpG dinucleotides. (C) Clustal W alignment of human, mouse and rat genomic sequences showing a detailed view of the conservation at the FOXP3 core promoter. The broken arrow shows the position of the human transcription start site. The location of the TATA box is indicated by a black box. Red boxes indicate the CpG dinucleotides analyzed in this study. Transcription factor binding sites are marked with grey boxes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238816&req=5

pone-0001612-g002: Identification of cross-species conserved FOXP3 promoter elements.(A) FOXP3 transcript and m-Vista alignment showing conservation between human and mouse genomic sequences. Dark blue boxes display exons, outlined boxes are UTR's. Conserved regions are in red and conserved regions corresponding to exons are in light blue. (B) Schematic view of the conserved region upstream of the transcription start site indicating the location of promoter elements and CpG dinucleotides. (C) Clustal W alignment of human, mouse and rat genomic sequences showing a detailed view of the conservation at the FOXP3 core promoter. The broken arrow shows the position of the human transcription start site. The location of the TATA box is indicated by a black box. Red boxes indicate the CpG dinucleotides analyzed in this study. Transcription factor binding sites are marked with grey boxes.
Mentions: The functional human FOXP3 promoter has been previously defined [11]. The region preceding the 5′ untranslated region (UTR) is highly conserved and contains important promoter elements such as TATA, GC and CAAT boxes as well as binding sites for NFAT and AP-1, well-established mediators of T cell activation (Figure 2B). The high degree of conservation of such cis-acting motifs in this region (Figure 2A) indicated that it might be under epigenetic influence. Therefore we investigated the FOXP3 promoter region with regards to CpG dinucleotides using ClustalW alignment comparing human, mouse and rat sequences (Figure 2C). The whole promoter region showed a mouse to man conservation of 87% (online supplemental material Figure S1), whereas the CpG containing region was 92% conserved, stressing its evolutionary importance (online supplemental material Figure S2). Moreover, ClustalW alignment determined that out of eight tightly positioned CpG dinucleotides just upstream of the transcription start site, positions −43, −65 and −77 were totally conserved between species. As these CpGs mapped just downstream of the binding sites for the transcription factors NFAT and AP-1 (Figure 2B–C), a special interest in these positions was rendered.

Bottom Line: We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy.In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.

Methodology/principal findings: Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.

Conclusions/significance: The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

Show MeSH
Related in: MedlinePlus