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. 2017 Sep 1;49(9):792-799.
doi: 10.1093/abbs/gmx079.

DNA-binding properties of FOXP3 transcription factor

Jun Li  1   2 , Longying Jiang  1 , Xujun Liang  1 , Lingzhi Qu  1 , Daichao Wu  1 , Xiaojuan Chen  1   2 , Ming Guo  1 , Zhuchu Chen  1   3 , Lin Chen  1   4 , Yongheng Chen  1   2   3
Affiliations

DNA-binding properties of FOXP3 transcription factor

Jun Li et al. Acta Biochim Biophys Sin (Shanghai). .

Abstract

FOXP3, a lineage-specific forkhead (FKH) transcription factor, plays essential roles in the development and function of regulatory T cells. However, the DNA-binding properties of FOXP3 are not well understood. In this study, FOXP3 fragments containing different domains were purified, and their DNA-binding properties were investigated using electrophoretic mobility shift assay and isothermal titration calorimetry (ITC). Both the FKH and leucine-zipper domains were required for optimal DNA binding for FOXP3. FOXP3 protein not only binds with DNA sequences containing one FKH consensus sequence, but also binds with DNA sequences with two direct repeats of consensus sequences separated by three-nucleotides (DRE3). Our results shed lights on the mechanisms by which FOXP3 recognizes cognate DNA elements, and would facilitate further structural and functional studies of FOXP3.

Keywords: DNA binding; FOXP3; electrophoretic mobility shift assay; isothermal titration calorimetry; protein purification.

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Figures

Figure 1.
Figure 1.
FOXP3 domain structures and binding sequences (A) Domain composition of full-length FOXP3. (B) Structural representation of FOXP3-LZ + ZF fragment. The ZF loop was not defined in the structure, while the ZF helix and adjacent LZ domain formed an extended single long helix. (C) Structural representation of FOXP3-FKH domain in complex with DNA. Two FOXP3-FKH monomers form a domain-swapped dimer. (D) FOXP3 consensus-binding site. (E) Oligonucleotide sequence used in the EMSA experiments (the core recognition element ‘AAACA’ is shown in red). DBE2, Daf-16 family-binding element 2; ARRE2, a DNA sequence from human IL2 promoter; DRE3, a direct repeat of FKH consensus element separated by three-nucleotides.
Figure 2.
Figure 2.
Expression and purification of different FOXP3 fragments (A) Purification of FOXP3-FKH + LZ + ZF fragment. Lane 1, supernatant of cell lysate; Lane 2, elution fractions from amylose-resin affinity column; Lane 3, after digestion with PreScission protease; Lane 4, flow-through fraction from mono S column; and Lanes 5–7, elution fraction from mono S column. (B) Purification of FOXP3-FKH + LZ fragment. Lane 1, supernatant of cell lysate; Lane 2, pellet of cell lysate; Lanes 3-4, elution fractions from amylose-resin affinity column; Lane 5, after digestion with PreScission protease; Lanes 6 and 7, flow-through fraction from mono S column; and Lanes 8–10, elution fractions from mono S column. (C) Purity analysis of different FOXP3 fragments. Lane 1, F FOXP3-FKH + LZ + ZF; Lane 2, FOXP3-FKH + LZ; Lane 3, FOXP3-LZ + ZF; and Lane 4, FOXP3-FKH. (D) Purification of FOXP3-FKH + LZ using Mono S 5/50 column. (E) Gel filtration elution profiles of different FOXP3 fragments using Superdex 75 10/300 column. The elution volumes of molecular size markers were reported on top of the figure. The elution volume of FOXP3 fragments suggested that all FOXP3 fragments are predominantly dimers in solution.
Figure 3.
Figure 3.
DNA binding of FOXP3 with DBE2 DNA (A) Comparison of DBE2 DNA binding with FKH domains of FOXO1, FOXA2, and FOXP3. (B) Comparison of DBE2 DNA binding with different FOXP3 fragments.
Figure 4.
Figure 4.
Quantitative analysis of DBE2 DNA with different FOXP3 domain using ITC (A–C) Representative ITC isotherms of the binding of DBE2 DNA with different FOXP3 fragments. The raw curves showed the change in thermal power with regard to time in the period of titration (top). The bottom curves showed the heat of reaction normalized with the molar ratio. Standard free energies of binding and entropic contributions were also obtained from the bottom curves. (D) Thermodynamic parameters of the interaction between FOXP3 fragments and DBE2 DNA. All data were measured at 298 K in 25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM MgCl2, and 1 mM TCEP.
Figure 5.
Figure 5.
DNA binding of FOXP3 with DRE3 DNA (A) FOXP3-FKH + LZ fragment bound to different DNA sequences: DBE2, ARRE2, and DRE3. (B) DNA binding of DRE3 DNA with different FOXP3 fragments.
Figure 6.
Figure 6.
DNA binding of DRE3 DNA variants with FOXP3-FKH + LZ fragment Nucleotide substitution was indicated using arrows.
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