Conlon FL et al. (2001), Determinants of T box protein specificity.

XB-ART-8312

Development 2001 Oct 01;12819:3749-58. doi: 10.1242/dev.128.19.3749.

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Determinants of T box protein specificity.

Conlon FL , Fairclough L , Price BM , Casey ES , Smith JC .

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Members of the T box family of transcription factors play important roles in early development. Different members of the family exert different effects and here we show that much of the specificity of the Xenopus T box proteins Xbra, VegT and Eomesodermin resides in the DNA-binding domain, or T box. Binding site selection experiments show that the three proteins bind the same core sequence, but they select paired sites that differ in their orientation and spacing. Lysine 149 of Xbra is conserved in all Brachyury homologues, while the corresponding amino acid in VegT and Eomesodermin is asparagine. Mutation of this amino acid to lysine changes the inductive abilities of VegT and Eomesodermin to resemble that of Xbra.

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Species referenced: Xenopus
Genes referenced: bix1.3 chrd eomes foxa4 gsc lgals4.2 tbxt vegt wnt11 wnt11b wnt8a

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	The T box proteins Xbra, VegT and Eomesodermin are expressed in similar patterns but induce the expression of different genes. (A) The structures of Xbra, VegT and Eomesodermin. Note that Eomesodermin has a larger N-terminal domain than Xbra or VegT. (B) Expression of Xbra at the early gastrula stage analysed by whole-mount in situ hybridisation. (C) Expression of VegT at the early gastrula stage. (D) Expression of Eomesodermin at the early gastrula stage. (E) Different inducing properties of Xbra, VegT and Eomesodermin. Note that all three T box proteins induce expression of Wnt11 and Bix4, that VegT and Eomesodermin induce higher levels of Pintallavis and Sox17α than does Xbra, and that Xbra cannot activate Goosecoid, chordin, Xwnt8 or Mix.1. The expression domains of the marker genes are as follows: Xwnt11, pan-mesodermal (Tada and Smith, 2000); Bix4, pan-mesendodermal (Casey et al., 1999; Tada et al., 1998); Pintallavis, dorsal mesoderm (Ruiz i Altaba and Jessell, 1992); Sox17α, endodermal (Hudson et al., 1997); Goosecoid: dorsal mesendoderm (Cho et al., 1991); chordin: dorsal mesoderm (Sasai et al., 1994); Xwnt8, ventral and lateral mesoderm (Christian et al., 1991; Smith and Harland, 1991); and Mix.1, pan-mesendodermal (Rosa, 1989).
	Xbra, VegT and Eomesodermin can function as transcriptional activators. Expression constructs encoding Xbra, VegT or Eomesodermin, or truncated versions of the proteins (see Materials and Methods), were transfected into 3T3 cells along with a reporter plasmid in which the sequence TTTCACACCT is placed upstream of a minimal promoter (below). All three T box proteins activated transcription, and activation was reduced in the truncated versions.
	Xbra, VegT and Eomesodermin specificity resides mainly in the T box. (A) Chimeric proteins comprising the T boxes of Xbra, VegT and Eomesodermin fused to the GAL4 DNA-binding domain and the VP16 activation domain. Proteins were expressed in early Xenopus embryos and their abilities to activate gene expression were assessed by RNAase protection. (B) Xbra and Xbra-VP16. (C) VegT and VegT-VP16. (D) Eomesodermin and Eomesodermin-VP16. Note that the inductive abilities of the chimeric constructs resemble those of the parent molecule.
	Sequences outside the Xbra T box restrict induction by VegT and Eomesodermin. (A) The parent Xbra, VegT and Eomesodermin proteins, and chimeric versions thereof. XVX consists of the VegT T box surrounded by Xbra non-T box sequences; XEX contains the Eomesodermin T box surrounded by Xbra non-T box sequences; and VXV consists of the Xbra T box surrounded by VegT non-T box sequences. (B) Activation of Goosecoid, Pintallavis and Chordin by XVX and XEX is lower than activation of the same genes by the parent proteins, suggesting that the non-T box sequences of Xbra reduce levels of induction. (C) Goosecoid, Pintallavis and Chordin are not induced by a protein comprising the Xbra T box surrounded by VegT non-T box sequences.
	Motifs selected by Xbra (A), VegT (B) and Eomesodermin (C) after five rounds of binding site selection. The consensus sequence is represented at the bottom of each histogram; if a nucleotide is present in greater than 10% of the selected sequences it is defined as being part of the consensus, and in this respect the sites selected by the three proteins differ. However, the core motif selected by all three T box proteins is clearly TCACACCT, and we have been unable to define sequences that are specific for a single member of the family. Note that Xbra shows a preference for a G positioned 5 nucleotides downstream of the core motif; see text for further details.
	Electrophoretic mobility gel shift assays demonstrate differences between different T box proteins to interact with different oligonucleotides. (A) Oligonucleotides used in electrophoretic gel mobility shift assays. Only one strand is shown and core motifs are boxed. Arrows indicate mutations in control oligonucleotides. Use of these oligonucleotides in electrophoretic gel mobility shift assays prevented binding (data not shown). (B) Band shift assay. Single-headed arrows indicate positions of high mobility complexes (red, Xbra; green: VegT, blue: Eomesodermin). Double-headed arrow indicates low mobility complexes.
	Identification of lysine 149 as an amino acid which may confer T box specificity. (A) Diagram of Xbra and part of the T box sequences of human, mouse, Xenopus and zebrafish Brachyury. Yellow circles indicate amino acids which contact DNA (Muller and Herrmann, 1997). The sequences are aligned with the equivalent regions of Xenopus VegT and human, mouse, Xenopus and zebrafish Eomesodermin. Note that the K residue in Xbra is replaced by an N in VegT and Eomesodermin. (B) Asterisk (on the left-hand T box) marks the position of K149 on the crystal structure of the Xbra T box bound to DNA (Muller and Herrmann, 1997). (C) Mutation of an asparagine residue in VegT and Eomesodermin to lysine causes those proteins to resemble Xbra in their inducing activities; VegTN→K and EomesoderminN→K lose the ability to induce Goosecoid and levels of Chordin and Pintallavis are significantly reduced.