Brannon M et al. (1999), XCtBP is a XTcf-3 co-repressor with roles throu...

XB-ART-12820

Development 1999 Jun 01;12614:3159-70. doi: 10.1242/dev.126.14.3159.

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XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.

Brannon M , Brown JD , Bates R , Kimelman D , Moon RT .

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XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes.

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Species referenced: Xenopus
Genes referenced: ctbp1 ctbp2 ctnnb1 gdf1 h4c4 lef1 lgals4.2 mmut nodal3.1 nodal3.2 not sia1 tcf4 tcf7l1 wnt8a

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	Fig. 1. XTcf-3 contains N- and C-terminal repressor domains. (A) Schematic diagram of XTcf-3, LEF-1, DC-XTcf-3 and the LEF- 1/XTcf-3 chimeric proteins (Chimeras 1-3). As indicated to the right, a protein contains repressor activity (+) or has no repressor activity (-) based on its ability to suppress dorsoanterior structure formation, to repress the siamois luciferase reporter gene (S01234), and to repress the endogenous siamois gene following dorsal injections. HMG, the HMG Box DNA-binding domain; b, the b-catenin-binding domain. (B) XTcf-3 and Chimera 3 ventralize Xenopus embryos, but LEF-1 does not. Uninjected Xenopus embryos or embryos injected into the dorsal equatorial region at the 4-cell stage with 0.5 ng of LEF-1, XTcf-3 or Chimera 3 RNA were cultured to the tadpole stage and scored for dorsoanterior structure formation. (a) Control uninjected tadpole. (b) LEF-1-injected tadpoles are indistinguishable from controls. (c) XTcf-3 injection results in severely ventralized embryos. (d) Chimera 3 injection results in ventralized embryos, although they retain rudimentary dorsoanterior structures. (C) Chimeras 1, 2 and 3, like XTcf-3, repress the siamois reporter gene, but LEF-1 does not. The wild-type siamois reporter (S01234) was injected into the dorsal equatorial region of 4-cell stage Xenopus embryos either alone (control) or in the presence of RNA encoding the indicated proteins. Three pools of five stage 10 embryos each were assayed, and the mean and standard error of the resulting luciferase activities, expressed in relative lights units (RLUs), are shown. The average fold reduction in luciferase activity relative to the S01234 control is shown above each data set. N.S., no significant reduction in activity.
	Fig. 2. The XTcf-3 C-terminal domain fused to LEF-1 (Chimera 3) has repressor activity, but LEF-1 does not. (A) Dorsal expression of the endogenous siamois gene is repressed by Chimera 3, but not by LEF-1. 4-cell stage embryos were injected at the equator of each dorsal or ventral blastomere with Chimera 3 or LEF-1 RNA. Dorsal or ventral marginal zones were explanted at stage 10 and the levels of endogenous siamois transcripts were measured by RT-PCR. Whole embryos (W.E.; lane 1); no reverse transcriptase (-RT; lane 2,) control; dorsal marginal zone (D) explants from uninjected (lane 3), LEF-1 RNA-injected (lane 5), and Chimera 3 RNA-injected (lane 7) embryos; ventral marginal zone (V) explants from uninjected (lane 4), LEF-1 RNA-injected (lane 6), and Chimera 3 RNA-injected (lane 8) embryos. (B) Endogenous siamois expression is repressed by XTcf-3. 4-cell stage embryos were injected at the equator of each blastomere with XTcf-3 RNA and levels of endogenous siamois transcripts were measured by RT-PCR at stage 10. W.E. (lane 1), -RT (lane 2) and XTcf-3 RNA-injected (lane 3). (C) Ventral injection of LEF-1 upregulates ventral siamois expression, while injection of Chimera 3 does not. The ventral marginal zone (VMZ) explants from A were subjected to additional cycles of PCR to detect ventral expression of siamois. -RT (lane 1), uninjected (lane 2), LEF-1 RNA-injected (lane 3), and Chimera 3 RNA-injected (lane 4).
	Fig. 3. Amino acid sequence of Xenopus CtBP compared with Xenopus and human CtBP1 and Drosophila CtBP. The Xenopus sequence derived from the original cDNA identified in the two-hybrid screen is shown. Amino acid identities are indicated by black boxes and similarities are indicated by gray boxes. Sequences were aligned with CLUSTALW and analyzed with BOXSHADE. The GenBank accession no. for XCtBP is AF152006.
	Fig. 4. Spatial and temporal expression of XCtBP during Xenopus development. (A) XCtBP is expressed at all stages of development. The temporal expression of XCtBP was analyzed by RT-PCR using oligonucleotide primer pairs directed against XCtBP 3¢ and 5¢ regions. EF-1a and histone H4 are included as loading controls. The developmental stage analyzed is indicated above each lane; (UF) unfertilized eggs; (6.5) pre-midblastula transition (pre-zygotic transcription); (8.5) midblastula transition (start of zygotic transcription); (10) early gastrula; (13-20) early-late neurula; (27) tailbud; (33-41) early-late tadpole. (B) XCtBP is expressed at the same level in the dorsal, ventral and animal pole regions. Dorsal and ventral equatorial and animal pole tissues were dissected from stage 10 embryos and levels of XCtBP transcripts determined by RT-PCR. Xnr3 and Xwnt-8 mark dorsal and ventral tissues, respectively. (C) XCtBP is expressed equally in the animal and vegetal pole regions. Animal and vegetal pole tissues were dissected from stage 10 embryos and levels of XCtBP transcripts determined by RT-PCR. Vg1 marks vegetal tissue.
	Fig. 5. Spatial expression pattern of XCtBP from the early gastrula to the tadpole stage. XCtBP expression was analyzed by whole-mount in situ hybridization using albinos, either cleared using Murray’s clear (A-H,J) or sectioned (I,K). (A) Vegetal and (B) lateral views of a stage 10 embryo showing ubiquitous XCtBP expression. (C) Dorsal and (D) lateral views, with anterior to the left, of a stage 13 embryo showing XCtBP transcripts localized to the anterior neural plate, dorsal midline and future tailbud. (E) An anterior view, and (F) dorsal and (G) lateral views, with anterior to the left, of a stage 20 embryo. XCtBP transcripts can be seen in the neural and mesodermal tissues of the dorsal midline, in the tailbud and in the eyes, brain and branchial arches in the head. (H) No signal is observed in a stage 10 embryo hybridized with a sense control probe. (I) A transverse section posterior to the head of a stage 20 embryo, dorsal is at the top. XCtBP transcripts mark the dorsal neural tube (nt) and lateral somitic tissues, but are excluded from the notochord (no) and medial regions of the somites (sm). (J) Lateral view of a stage 33 embryo, anterior is to the left. XCtBP expression is intense in the head, labeling the eyes, otic vesicles, CNS and branchial arches. Posterior to the head, XCtBP is expressed in the pronephros, somites and tailbud. (K) A transverse section through the head of a stage 33 embryo, dorsal is at the top. XCtBP transcripts label the dorsal and lateral regions of the neural tube, but are excluded from the ventral neural tube (an arrow indicates the limit of XCtBP expression in the neural tube).
	Fig. 6. Amino acid sequence comparison of the C-terminal region of Xenopus, zebrafish and mouse Tcf-3, human Tcf-4 and human LEF-1. Amino acid identities are indicated by black boxes and amino acid similarities are indicated by gray boxes. Sequences were aligned with CLUSTALW and analyzed with BOXSHADE. Overlining indicates the two potential CtBP binding sites, which are labeled above each line (1, site 1 and 2, site 2).
	Fig. 7. In vitro interaction of the XTcf-3 C-terminal domain with CtBP. 35S-labeled, full-length Chimera 3 protein (lane 1), binds to GST-dCtBP (lane 9). Chimera 3 proteins lacking CtBP binding site 1 (mutant 1; lane 2) or site 2 (mutant 2; lane 3), weakly interact with GST-dCtBP (lanes 10 and 11). Chimera 3 protein mutant in site 1 and site 2 (double mut.; lane 4) no longer efficiently binds to GSTdCtBP (lane 12). None of the Chimera 3 proteins bind to glutathione sepharose beads (beads; lanes 5-8) or to GST-bound beads (data not shown).
	Fig. 8. The regulation of siamois expression by XCtBP. (A) The XTcf-3 C-terminal domain requires the XCtBP binding sites in order to repress siamois expression. The siamois luciferase reporter gene (S01234) was injected into the equatorial region of the dorsal blastomeres of 4-cell stage embryos either alone or in the presence of RNAs encoding Chimera 3, Chimera 3 mutated in both XCtBP binding sites (Double Mut.) or Chimera 3 truncated to remove both sites (D-CtBP). The mean and standard error of the resulting luciferase activities, in RLUs, from three pools of five embryos each are shown. The average fold activation of S01234 relative to the activity of S01234 repressed by Chimera 3 is shown above each data set. (B) XCtBP/G4A activates siamois expression. S01234 was injected at the 4-cell stage into the equatorial region of the dorsal blastomeres (D) or into the equatorial region of the ventral blastomeres (V) in the absence or presence of XCtBP/G4A RNA. The mean and standard error of the resulting luciferase activities, in RLUs, from three pools of five embryos each are shown. The average fold activation of S01234 relative to the activity of S01234 injected into the ventral blastomeres in the absence of XCtBP/G4A is shown above each data set. A diagram of XCtBP/G4A, which is full-length XCtBP with the GAL4 activation domain fused to its C terminus, is shown above the graph. (C) XCtBP/G4A activates ventral expression of endogenous siamois. 4-cell stage embryos were injected at the equator of each dorsal or ventral blastomere with XCtBP/G4A or LEF-1 RNA. Dorsal or ventral marginal zones were explanted at stage 10 and the levels of endogenous siamois transcripts were measured by RT-PCR. Lane 1, whole embryos (W.E.); lane 2, no reverse transcriptase (-RT) control; dorsal marginal zone (D) explants from uninjected (lane 3), XCtBP/G4A RNA-injected (lane 5), and LEF-1 RNA-injected (lane 7) embryos; ventral marginal zone (V) explants from uninjected (lane 4), XCtBP/G4A RNA-injected (lane 6), and LEF-1 RNAinjected (lane 8) embryos. (D) The activation of siamois by XCtBP/G4A is mediated by XTcf-3. A siamois reporter gene with mutated Tcf/Lef binding sites that is unable to bind XTcf-3 (S24) was injected at the 4-cell stage into the equatorial region of the dorsal blastomeres (D) or into the equatorial region of the ventral blastomeres (V) in the absence or presence of XCtBP/G4A RNA. The mean and standard error of the resulting luciferase activities, in RLUs, from three pools of five embryos each are shown. The average fold activation of S24 relative to the activity of S24 injected into the ventral blastomeres in the absence of XCtBP/G4A is shown above each data set.
	Fig. 9. Role of XCtBP in Xenopus development. (A-D) The XCtBP/G4A-mediated disruption of Xenopus development is XCtBPspecific. (A) A normal, uninjected control stage 35 embryo. (B) Embryos injected with XCtBP are indistinguishable from controls. A stage 35 embryo injected with 1 ng of XCtBP RNA at the equatorial region of the dorsal blastomeres at the 4-cell stage is shown. (C) XCtBP/G4A injections disrupt normal development, resulting in head loss and a shortened anterior-posterior axis. A stage 35 embryo similarly injected with 0.2 ng of XCtBP/G4A RNA. (D) XCtBP rescues all XCtBP/G4A-mediated developmental defects. A stage 35 embryo co-injected with 1 ng of XCtBP RNA and 0.2 ng of XCtBP/G4A RNA at the equatorial region of the dorsal blastomeres at the 4-cell stage is shown. (E-H) Histological examination of the XCtBP/G4A-mediated disruptions of Xenopus development. Transverse sections through the head of a control, uninjected stage 35 embryo at the position of the eyes (E) or the otic vesicles (G) (arrows indicate the neural tube and notochord). (F) A transverse section through the anterior portion of the head of an embryo injected with 0.2 ng of XCtBP/G4A RNA shows a major reduction or loss of all head structures, including the neural tube (arrow). (H) A transverse section through the head, at the level of the otic vesicles, of an embryo injected with 0.2 ng of XCtBP/G4A RNA shows a disorganization of the neural tube, a notochord (arrow) reduced in size and laterally displaced. Dorsal is at the top in all sections. (I-J) XCtBP/G4A blocks the expression of the notochord marker Xnot. Analysis of Xnot expression is by whole-mount in situ hybridization. (I) A control, uninjected stage 11.5 embryo shows the normal pattern of Xnot transcripts along the dorsal midline (arrow). (J) A stage 11.5 embryo injected with 0.2 ng of XCtBP/G4A RNA shows a severe reduction and disorganization of Xnot expression (arrow).