How T-bet Binds to DNA

Gabriel Brandt*, Quyen Hoang*, Ce-Feng Liu*, Dagmar Ringe*, Eun Sook Hwang^, Laurie H. Glimcher^, and Gregory A. Petsko^ * Departments of Biochemistry and Chemistry and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street MS 029, Waltham, MA 02454-9110 USA ^ Department of Immunology and Infectious Diseases, Harvard School of Public Health and department of Medicine, Harvard Medical School, Boston, MA 02115 USA The transcription factor T-bet is one of the master regulators of both the innate and adaptive immune response (1). It plays a central role in T-cell lineage commitment, where it controls the Th-1 response, and also in gene regulation in plasma B-cells and dendritic cells (2). T-bet is a member of the Tbox family of transcription factors, a family that includes the TBX3 and Brachyury proteins, but T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question for master transcription regulators like T-bet is how a single protein molecule is able to bind to both a promoter recognition element on one gene, and a recognition element on a second gene or an enhancer element, either of which may be located thousands of base pairs away (3,4). We have determined the three-dimensional structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a 24 base pair palindromic consensus DNA recognition site, by the method of X-ray crystallography. The structure of the T-bet DBD-DNA complex differs dramatically from that of the other Tbox proteins whose structures have been determined. Two different fluorescence-based assays confirm that T-bet binds DNA in solution in the same manner as that observed in the crystal structure. Mutational analysis of conserved tyrosine residues in T-bet is also consistent with the observed structure. We present a looping model for transcriptional regulation by T-bet that explains how one transcription factor recognizes distinct genetic elements (5), either a promoter site and a distant enhancer site, or promoter sites on two different genes, as has been suggested for so-called “transcriptional factories” (6). References 1) Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000 Mar 17;100(6):655-69. 2) Glimcher LH. Trawling for treasure: tales of T-bet. Nat Immunol. 2007 May;8(5):448-50. 3) Cho JY, Grigura V, Murphy TL, Murphy K. Identification of cooperative monomeric Brachyury sites conferring T-bet responsiveness to the proximal IFN-gamma promoter. Int Immunol. 2003 Oct;15(10):1149-60. 4) Tong Y, Aune T, Boothby M. T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-gamma promoter via a conserved T-box half-site. Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):2034-9. 5) E. Rowell, M. Merkenschlager and C.B. Wilson. Long-range regulation of cytokine gene expression. Curr Opin Immunol 20 (2008), pp. 272–280. 6) Amsen D, Spilianakis CG, Flavell RA. How are T(H)1 and T(H)2 effector cells made? Curr Opin Immunol. 2009 Apr;21(2):153-60.