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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.
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.
Afkarian,
T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells.
2002, Pubmed
Afkarian,
T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells.
2002,
Pubmed
Avni,
T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes.
2002,
Pubmed
Balasubramani,
Modular utilization of distal cis-regulatory elements controls Ifng gene expression in T cells activated by distinct stimuli.
2010,
Pubmed
Bandukwala,
Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells.
2011,
Pubmed
Blackwood,
Going the distance: a current view of enhancer action.
1998,
Pubmed
Bulger,
Looping versus linking: toward a model for long-distance gene activation.
1999,
Pubmed
Chakalova,
Replication and transcription: shaping the landscape of the genome.
2005,
Pubmed
Chen,
DNA binding by GATA transcription factor suggests mechanisms of DNA looping and long-range gene regulation.
2012,
Pubmed
Cho,
Identification of cooperative monomeric Brachyury sites conferring T-bet responsiveness to the proximal IFN-gamma promoter.
2003,
Pubmed
Coll,
Structure of the DNA-bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome.
2002,
Pubmed
,
Xenbase
Dekker,
Capturing chromosome conformation.
2002,
Pubmed
El Omari,
Structure of the DNA-bound T-box domain of human TBX1, a transcription factor associated with the DiGeorge syndrome.
2012,
Pubmed
Finotto,
Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet.
2002,
Pubmed
Garrett,
Colitis-associated colorectal cancer driven by T-bet deficiency in dendritic cells.
2009,
Pubmed
Glimcher,
Trawling for treasure: tales of T-bet.
2007,
Pubmed
Hatton,
A distal conserved sequence element controls Ifng gene expression by T cells and NK cells.
2006,
Pubmed
Herendeen,
A transcriptional enhancer whose function imposes a requirement that proteins track along DNA.
1992,
Pubmed
Hou,
CTCF-dependent enhancer-blocking by alternative chromatin loop formation.
2008,
Pubmed
Intlekofer,
Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin.
2008,
Pubmed
Intlekofer,
Requirement for T-bet in the aberrant differentiation of unhelped memory CD8+ T cells.
2007,
Pubmed
Janin,
Macromolecular recognition in the Protein Data Bank.
2007,
Pubmed
Jenner,
The transcription factors T-bet and GATA-3 control alternative pathways of T-cell differentiation through a shared set of target genes.
2009,
Pubmed
Joshi,
Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor.
2007,
Pubmed
Kaneko,
Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells.
2007,
Pubmed
Kornberg,
Mediator and the mechanism of transcriptional activation.
2005,
Pubmed
Lee,
Genome-wide studies of CCCTC-binding factor (CTCF) and cohesin provide insight into chromatin structure and regulation.
2012,
Pubmed
Lee,
A distal enhancer in the interferon-gamma (IFN-gamma) locus revealed by genome sequence comparison.
2004,
Pubmed
Li,
Chromatin looping and the probability of transcription.
2006,
Pubmed
Lugo-Villarino,
The adjuvant activity of CpG DNA requires T-bet expression in dendritic cells.
2005,
Pubmed
Maniatis,
An extensive network of coupling among gene expression machines.
2002,
Pubmed
Miele,
Mapping cis- and trans- chromatin interaction networks using chromosome conformation capture (3C).
2009,
Pubmed
Mullen,
Role of T-bet in commitment of TH1 cells before IL-12-dependent selection.
2001,
Pubmed
Müller,
Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor.
1997,
Pubmed
,
Xenbase
Ohlsson,
CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin.
2010,
Pubmed
Palstra,
Beta-globin regulation and long-range interactions.
2008,
Pubmed
Papaioannou,
The T-box gene family: emerging roles in development, stem cells and cancer.
2014,
Pubmed
Peng,
T-bet regulates metastasis rate in a murine model of primary prostate cancer.
2004,
Pubmed
Peng,
T-bet regulates IgG class switching and pathogenic autoantibody production.
2002,
Pubmed
Ravindran,
Expression of T-bet by CD4 T cells is essential for resistance to Salmonella infection.
2005,
Pubmed
Rowell,
Long-range regulation of cytokine gene expression.
2008,
Pubmed
Schoenborn,
Comprehensive epigenetic profiling identifies multiple distal regulatory elements directing transcription of the gene encoding interferon-gamma.
2007,
Pubmed
Schwede,
SWISS-MODEL: An automated protein homology-modeling server.
2003,
Pubmed
Shnyreva,
Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells.
2004,
Pubmed
Spilianakis,
Interchromosomal associations between alternatively expressed loci.
2005,
Pubmed
Spilianakis,
Long-range intrachromosomal interactions in the T helper type 2 cytokine locus.
2004,
Pubmed
Splinter,
CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus.
2006,
Pubmed
Stirnimann,
Structural basis of TBX5-DNA recognition: the T-box domain in its DNA-bound and -unbound form.
2010,
Pubmed
Sundrud,
Synergistic and combinatorial control of T cell activation and differentiation by transcription factors.
2010,
Pubmed
Szabo,
Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells.
2002,
Pubmed
Szabo,
A novel transcription factor, T-bet, directs Th1 lineage commitment.
2000,
Pubmed
Tong,
T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-gamma promoter via a conserved T-box half-site.
2005,
Pubmed
Townsend,
T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells.
2004,
Pubmed
Tuan,
Transcription of the hypersensitive site HS2 enhancer in erythroid cells.
1992,
Pubmed
Wang,
Action at a distance along a DNA.
1988,
Pubmed
Werneck,
T-bet plays a key role in NK-mediated control of melanoma metastatic disease.
2008,
Pubmed
Wijgerde,
Transcription complex stability and chromatin dynamics in vivo.
1995,
Pubmed
Wilson,
The T-box family.
2002,
Pubmed
Wilson,
BACing up the interferon-gamma locus.
2006,
Pubmed
Yang,
Identification of a distant T-bet enhancer responsive to IL-12/Stat4 and IFNgamma/Stat1 signals.
2007,
Pubmed
Yin,
Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis.
2009,
Pubmed
Yusufzai,
CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species.
2004,
Pubmed
Zhang,
Structure of the LexA-DNA complex and implications for SOS box measurement.
2010,
Pubmed
de Laat,
Spatial organization of gene expression: the active chromatin hub.
2003,
Pubmed