Research Article: Domain Requirements and Sequence Specificity of DNA Binding for the Forkhead Transcription Factor FOXP3

Date Published: December 1, 2009

Publisher: Public Library of Science

Author(s): Kian Peng Koh, Mark S. Sundrud, Anjana Rao, Derya Unutmaz.

Abstract: The forkhead, winged-helix transcription factor FOXP3 is preferentially expressed in T regulatory (Treg) cells and is critical for their immunosuppressive function. Mutations that abolish FOXP3 function lead to systemic autoimmunity in mice and humans. However, the manner by which FOXP3 recognizes cognate DNA elements is unclear. Here we identify an in vitro optimized DNA sequence to assess FOXP3 DNA binding by electrophoretic mobility shift assay (EMSA). The optimized sequence contains two tandem copies of a core DNA element resembling, but not identical to, the canonical forkhead (FKH) binding element. The tandem nature of this optimized FOXP3-binding oligonucleotide suggests a requirement for multimerization, and EMSA experiments confirm that both the DNA-binding FKH domain and an intact leucine-zipper domain, which mediates homo-multimerization of FOXP3, are required for DNA binding. These results establish a practical framework for understanding the molecular basis by which FOXP3 regulates gene transcription and programs Treg suppressive function.

Partial Text: Forkhead box (FOX) transcription factors are a large and functionally diverse family of transcription factors, with over 100 members in mammals (reviewed in [1]). Named after the forkhead gene product in Drosophila melanogaster, the founding members of the mammalian FOX family belong to the hepatic nuclear factor-3 (HNF3/FOXA) family, which regulate the development of metabolic tissues such as the pancreas and liver [2], [3]. Many FOX transcription factors are tissue-specific regulators of development (reviewed in [4]): hair formation and keratinocyte differentiation are regulated by Foxn1 [5], cell growth and insulin responsiveness by Foxo1 [6], craniopharyngeal development by FOXE1 [4], speech and language patterning by FOXP2 [7], and auditory function by Foxi1 [8]. Additionally, several FOX proteins play key roles in the development, homeostasis and function of immune cells (reviewed in [9]). Foxo1, Foxo3 and Foxp1 all regulate B cell ontogeny, possibly through direct transcriptional regulation of the Rag1/2 locus [10]–[13]. In T cells, genetic ablation of either Foxj1 or Foxo3 precipitates a lymphoproliferative phenotype associated with variable autoimmune pathology [14], [15], suggesting that these FOX proteins negatively regulate T cell activation.

All FOX transcription factors share a common winged-helix DNA-binding domain of approximately 100 amino acids known as the forkhead (FKH) domain [1], [4], [9]. HNF3/FOXA proteins bind as monomers to DNA elements with the consensus sequence 5′–ATAACT–3′[32], [33]; however, primary sequence analyses of their FKH domains, and hence their putative sequence specificity for DNA, show a significant degree of divergence from the FKH domains of other FOX proteins [1]. Indeed Foxp1A, a close relative of FOXP3, was found to prefer modified FKH/FOX DNA elements (5′ TATTTg/aTg/aTT-3′) or its complement, 5′–AAc/tAc/tAAATA-3′) in a PCR-based site-selection assay from which the “A” oligonucleotide containing the preferred Foxp1 binding site was derived [34].

FOXP3 prevents spontaneous autoimmunity by conferring on Treg cells the transcriptional profile responsible for their immune-suppressive activity. In this study we used a systematic series of EMSA assays to define the parameters that regulate high-affinity interactions between FOXP3 and cognate DNA elements. We show that the core FOXP3 consensus element contains the sequence 5′–(G/a)TAAACA–3′; this sequence is also preferred by the closely related transcription factor Foxp1, but diverges from the classical FKH/FOX consensus site (5′–ATAACT–3′) defined for the HNF3/FOXA proteins [32]–[34]. The consensus Foxp1/FOXP3 binding element we have defined here is substantiated by previous studies that identified Foxp3 binding sites throughout the genome via ChIP-chip [29], [30]. In these reports, the predicted Foxp3 binding sequence was 5′–(A/G)(T/C)AAACA–3′. Our analysis has further defined the sequence specificity of FOXP3 as strongly preferring thymine at position 2 and favoring guanine at position 1, although adenine is also tolerated.



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