Domain requirements and sequence specificity of DNA binding for the forkhead transcription factor FOXP3.
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.
Koh
Kian Peng
KP
Department of Pathology, Harvard Medical School and Immune Disease Institute, Boston, Massachusetts, United States of America.
Sundrud
Mark S
MS
Rao
Anjana
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D020080:Tandem Repeat Sequences / Q000235:genetics
C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C432917:Foxp1 protein, mouse; D012097:Repressor Proteins; D004247:DNA