FoxD3 and Grg4 Physically Interact to Repress Transcription and Induce Mesoderm in Xenopus

Sergey Yaklichkin,Aaron B Steiner,Qun Lu,Daniel S Kessler

doi:10.1074/jbc.m607412200

Sergey Yaklichkin, Aaron B Steiner + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m607412200

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Abstract

FoxD3 is a forkhead-related transcriptional regulator that is essential for multiple developmental processes in the vertebrate embryo, including neural crest development and maintenance of mammalian stem cell lineages. Recent results demonstrate a requirement for FoxD3 in Xenopus mesodermal development. In the gastrula, FoxD3 functions as a transcriptional repressor in the Spemann organizer to maintain the expression of Nodal-related members of the transforming growth factor-beta superfamily that induce dorsal mesoderm formation. Here we report that the function of FoxD3 in mesoderm induction is dependent on the recruitment of transcriptional corepressors of the TLE/Groucho family. Structure-function analyses indicate that the transcriptional repression and mesoderm induction activities of FoxD3 are dependent on a C-terminal domain, as well as specific DNA-binding activity conferred by the forkhead domain. The C-terminal domain contains a heptapeptide similar to the eh1/GEH Groucho interaction motif. Deletion and point mutagenesis demonstrated that the FoxD3 eh1/GEH motif is required for both repression of transcription and induction of mesoderm, as well as the direct physical interaction of FoxD3 and Grg4 (Groucho-related gene-4). Consistent with a functional interaction of FoxD3 and Grg4, the transcriptional repression activity of FoxD3 is enhanced by Grg4, and reduced by Grg5, a dominant inhibitory Groucho protein. The results indicate that FoxD3 recruitment of Groucho corepressors is essential for the transcriptional repression of target genes and induction of mesoderm in Xenopus.

Highlights

2548 JOURNAL OF BIOLOGICAL CHEMISTRY expressed in the preimplantation mouse embryo, as well as mammalian embryonic and trophoblast stem cells [9, 21,22,23]
FoxD3 Mesoderm Induction Is Dependent on the Specific DNA-binding Activity of the Forkhead Domain—Fox family proteins are characterized by a conserved 100-residue forkhead domain that is required for DNA-binding activity [1, 52]
A strong transcriptional repression domain was identified in the C terminus of Xenopus FoxD3 that is required for biological activity

Summary

EXPERIMENTAL PROCEDURES

Embryos and Microinjection—Xenopus embryos were collected, fertilized, injected, and cultured, and animal pole explants prepared and cultured as previously described [46]. A previously described pCS2-FoxD3 subclone containing the open reading frame (nucleotides 172–1287) of Xenopus FoxD3 was used to generate the constructs used in this study [30]. Gal4-UAS-Luciferase Reporter Transcriptional Assays—Xenopus embryos were injected in the animal pole at the one-cell stage with in vitro transcribed RNA encoding the Gal DNAbinding domain or Gal4-FoxD3 fusion proteins. Protein Interaction Assays and Western Blotting Analysis— One-cell stage embryos were injected with in vitro transcribed RNA encoding glutathione S-transferase (GST), or GST-FoxD3 fusions proteins alone, or in combination with myc-Grg mRNA [43]. Cleared supernatants and bead eluates were subjected to Western blot analysis using a 1:1000 dilution of anti-GST polyclonal antibody (Amersham Biosciences) or anti-myc monoclonal antibody (Sigma), and detected with a 1:3000 dilution of peroxidase-coupled secondary antibody by chemiluminescence (Amersham Biosciences). Duplicate blots were analyzed with a monoclonal antibody against the ubiquitous hnRNPK at a 1:1000 dilution [51]

RESULTS

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DISCUSSION