Liu CF et al. (2016), Crystal structure of the DNA binding domain of ...

XB-ART-54531

Proc Natl Acad Sci U S A 2016 Oct 25;11343:E6572-E6581. doi: 10.1073/pnas.1613914113.

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Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites.

Liu CF , Brandt GS , Hoang QQ , Naumova N , Lazarevic V , Hwang ES , Dekker J , Glimcher LH , Ringe D , Petsko GA .

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The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the T H 1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

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Species referenced: Xenopus laevis
Genes referenced: il4 tbx1 tbx3 tbx5 tbxt usp9x

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	Fig. 1. The Tbox transcription factors bind contiguous DNA elements. (A) Structures of the Tbox DBD–DNA complexes of Xbra (PDB ID code 1XBR), TBX1 (PDB ID code 4A04), TBX3 (PDB ID code 1H6F), and TBX5 (PDB ID code 2X6V). Note that the overall folds of the four Tbox domains are essentially identical, as is their mode of recognition of the B-form DNA double helix. Xbra, TBX1, and TBX3 crystallized as homodimers and bind two consensus target sequences, respectively, but the dimers are not tightly associated and probably bind their two sites independently; TBX5 binds as a monomer to a single site. (B) Models for action at a distance by a dimeric transcription factor. The two subunits of the dimer are shown in red and blue and may be either different (heterodimer) or identical (homodimer). In a looping model, each subunit binds independently to a promoter recognition element and a distant element, which may be either another promoter or, as shown, an enhancer sequence. Formation of the dimer juxtaposes the distant sites. Tracking postulates that the different elements serve to recruit the protein subunits to the DNA, but that one of them then scans along the chromosome, leaving its site behind, until it encounters its cognate partner. Facilitated tracking combines elements of tracking and looping, in that the distant site is brought along with its binding protein. In a linking model, the two subunits and their sites remain separate and other proteins (shown in green) connect them. The previously known Tbox protein structures shown in A are either monomeric or bind contiguous DNA elements and consequently would not be expected to act at a distance as depicted in these models.
	Fig. 2. The DNA binding domain of T-bet forms a dimer that cross-links two independent DNA strands. (A) The two T-bet DBD monomers (light blue and brown) form a tight dimer. Each monomer binds a single T-bet recognition site on a separate strand of DNA. The protruding helices that make contact with the minor groove of the DNA are shown in red for the light blue monomer; the residues on the double helix that interact with this part of the Tbox domain are shown in navy blue. (B) The T-bet DBD–DNA complex with the molecular surface overlaid, and a similar surface model of the Tbox DBD–DNA complexes of Xbra and TBX3. The Xbra dimer interface is much smaller, and TBX3 binds essentially as two monomers. (C) Orthogonal views of the T-bet Tbox–DNA complex showing binding of two independent DNA strands to each subunit of the dimer. The view on the right has a surface representation overlaid, indicating the tightness of the complex.
	Fig. 3. The DNA binding domain of T-bet forms a dimer in solution. Size-exclusion chromatography data show that T-bet DBD exists as a mixture of monomeric and dimeric species in solution.
	Fig. 4. FRET experiments confirm T-bet can synapse DNA in vitro. (A) Titration of FAM- and TAMRA-labeled DNA with T-bet. A strong FRET signal is observed when T-bet is present together with both donor- and acceptor-labeled DNA, but not in the absence of T-bet. (B) Relative FRET efficiency vs. T-bet concentration. (C) Fluorescently labeled DNA FRET efficiency induced by T-bet, TBX5, lysozyme, BSA, and buffer using 10 nM FAM-labeled DNA and 10 nM TAMRA-labeled DNA. Only T-bet produced a FRET signal. Even the monomeric T-bet homolog TBX5, which binds to the same consensus sequence, fails to do so. The concentrations of T-bet, TBX5, lysozyme, and BSA used in this experiment were 1.16 mM, 3.75 mM, 5.35 mM, and 2.5 mM, respectively. (D) The binding affinity of DNA to T-bet was determined by titrating T-bet to 1.25 nM TAMRA-DNA and observing the non-FRET decrease in the fluorescence of the T-bet bound TAMRA-DNA (SI Appendix, Supplementary Methods and Materials). The dissociation constant determined by this method was ∼19 nM.
	Fig. 5. The 3C experiments show T-bet can synapse IFN-γ (Ifn-γ) enhancer and promoter sites. (A) VISTA plot comparing human and mouse sequences at the Ifng gene locus. Locations of CNS are highlighted in pink. These CNS sites are also conserved in rhesus, dog, horse, and rat. CNS-55, CNS-34, CNS-22, CNS-5, and the Ifng promoter are known to bind to T-bet as determined by ChIP (24). (B) In vitro 3C profiles of samples with T-bet (T-bet DBD) or without T-bet (BSA). (C) In vivo 3C profiles of samples with T-bet (WT mouse CD4+ T-cells) or without T-bet (T-bet−/− mouse CD4+ T-cells). In B and C, horizontal bars directly above the x axis depict the length and location of each 3C fragments analyzed. The promoter fragment, shown as a red bar, was used as the 3C anchor for both the in vitro and in vivo 3C experiments.
	Fig. 6. Proposed models for the physiological role of T-bet DNA looping activity based on the in vivo 3C data. (A) T-bet functions to stabilize and maintain existing DNA loops. No new long-range DNA interactions are created. (B) T-bet creates new long-range DNA interactions, but these new interactions were not detected uniquely as T-bet–dependent interactions because of the existence of prior T-bet–independent interactions between the same 3C fragments. (C) Schematic of T-bet molecules binding simultaneously to promoter sites on genes located on different chromosomes, activating one while repressing the other.

References [+] :

Afkarian, T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells. 2002, Pubmed