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Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

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A genetic eyFLP/FRT-based screen in Drosophila identifies Madm as a positive growth regulator. (a-d) Dorsal view of mosaic heads generated by means of the eyFLP/FRT system. (a) The isogenized FRT82 chromosome used in the genetic screen produces a control mosaic head. (b, c) Heads largely homozygous mutant for an EMS-induced Madm mutation display a pinhead phenotype that can be reverted by one copy of a genomic Madm rescue construct (d). (e) Graphic representation of the Drosophila Madm protein (top) and gene (bottom). In the protein, the BunA-binding region and the NES and NLS sequences are indicated (netNES 1.1 [63], ELM [64], PredictNLS [65]). The seven alleles isolated in the genetic screen and the sites of their EMS-induced mutations are in red. Amino acid changes in the protein are indicated. In alleles 3Y2 and 7L2, the first nucleotide downstream of the first Madm exon is mutated, thus disrupting the splice donor site. In allele 2D2, a deletion causes a frameshift after amino acid 385, resulting in a premature translational stop after an additional 34 amino acids. Alleles 3Y2, 4S3, and 7L2 lead to a pinhead phenotype of intermediate strength (b) whereas 2D2, 2U3, and 3G5 produce a stronger pinhead phenotype (c). The hypomorphic allele 3T4 generates a weak pinhead phenotype (data not shown). Genotypes of the flies shown are: (a) y, w, eyFlp/y, w; FRT82B/FRT82B, w+, cl3R3; (b, c) y, w, eyFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3; (d) y, w, eyFlp/y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3G5/FRT82B, w+, cl3R3.
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Figure 3: A genetic eyFLP/FRT-based screen in Drosophila identifies Madm as a positive growth regulator. (a-d) Dorsal view of mosaic heads generated by means of the eyFLP/FRT system. (a) The isogenized FRT82 chromosome used in the genetic screen produces a control mosaic head. (b, c) Heads largely homozygous mutant for an EMS-induced Madm mutation display a pinhead phenotype that can be reverted by one copy of a genomic Madm rescue construct (d). (e) Graphic representation of the Drosophila Madm protein (top) and gene (bottom). In the protein, the BunA-binding region and the NES and NLS sequences are indicated (netNES 1.1 [63], ELM [64], PredictNLS [65]). The seven alleles isolated in the genetic screen and the sites of their EMS-induced mutations are in red. Amino acid changes in the protein are indicated. In alleles 3Y2 and 7L2, the first nucleotide downstream of the first Madm exon is mutated, thus disrupting the splice donor site. In allele 2D2, a deletion causes a frameshift after amino acid 385, resulting in a premature translational stop after an additional 34 amino acids. Alleles 3Y2, 4S3, and 7L2 lead to a pinhead phenotype of intermediate strength (b) whereas 2D2, 2U3, and 3G5 produce a stronger pinhead phenotype (c). The hypomorphic allele 3T4 generates a weak pinhead phenotype (data not shown). Genotypes of the flies shown are: (a) y, w, eyFlp/y, w; FRT82B/FRT82B, w+, cl3R3; (b, c) y, w, eyFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3; (d) y, w, eyFlp/y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3G5/FRT82B, w+, cl3R3.

Mentions: The BunA-binding domain in Drosophila Madm was reciprocally mapped by means of co-IP and Y2H experiments to the carboxy-terminal amino acids 458-566 (Figure 2f and Additional file 3). Furthermore, we found that amino acids 530-566, including a nuclear export signal (NES) and a predicted nuclear-receptor-binding motif (LXXLL) in mammals, were not dispensable for the binding to BunA [see Additional file 4]. In addition, a point mutation leading to the arginine to histidine substitution R525H disrupted BunA-binding (the point mutation derived from the Madm allele 4S3; Figure 3e). Thus, Madm is a Bun-interacting protein that specifically binds the long Bun isoforms.


Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

A genetic eyFLP/FRT-based screen in Drosophila identifies Madm as a positive growth regulator. (a-d) Dorsal view of mosaic heads generated by means of the eyFLP/FRT system. (a) The isogenized FRT82 chromosome used in the genetic screen produces a control mosaic head. (b, c) Heads largely homozygous mutant for an EMS-induced Madm mutation display a pinhead phenotype that can be reverted by one copy of a genomic Madm rescue construct (d). (e) Graphic representation of the Drosophila Madm protein (top) and gene (bottom). In the protein, the BunA-binding region and the NES and NLS sequences are indicated (netNES 1.1 [63], ELM [64], PredictNLS [65]). The seven alleles isolated in the genetic screen and the sites of their EMS-induced mutations are in red. Amino acid changes in the protein are indicated. In alleles 3Y2 and 7L2, the first nucleotide downstream of the first Madm exon is mutated, thus disrupting the splice donor site. In allele 2D2, a deletion causes a frameshift after amino acid 385, resulting in a premature translational stop after an additional 34 amino acids. Alleles 3Y2, 4S3, and 7L2 lead to a pinhead phenotype of intermediate strength (b) whereas 2D2, 2U3, and 3G5 produce a stronger pinhead phenotype (c). The hypomorphic allele 3T4 generates a weak pinhead phenotype (data not shown). Genotypes of the flies shown are: (a) y, w, eyFlp/y, w; FRT82B/FRT82B, w+, cl3R3; (b, c) y, w, eyFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3; (d) y, w, eyFlp/y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3G5/FRT82B, w+, cl3R3.
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Related In: Results  -  Collection

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Figure 3: A genetic eyFLP/FRT-based screen in Drosophila identifies Madm as a positive growth regulator. (a-d) Dorsal view of mosaic heads generated by means of the eyFLP/FRT system. (a) The isogenized FRT82 chromosome used in the genetic screen produces a control mosaic head. (b, c) Heads largely homozygous mutant for an EMS-induced Madm mutation display a pinhead phenotype that can be reverted by one copy of a genomic Madm rescue construct (d). (e) Graphic representation of the Drosophila Madm protein (top) and gene (bottom). In the protein, the BunA-binding region and the NES and NLS sequences are indicated (netNES 1.1 [63], ELM [64], PredictNLS [65]). The seven alleles isolated in the genetic screen and the sites of their EMS-induced mutations are in red. Amino acid changes in the protein are indicated. In alleles 3Y2 and 7L2, the first nucleotide downstream of the first Madm exon is mutated, thus disrupting the splice donor site. In allele 2D2, a deletion causes a frameshift after amino acid 385, resulting in a premature translational stop after an additional 34 amino acids. Alleles 3Y2, 4S3, and 7L2 lead to a pinhead phenotype of intermediate strength (b) whereas 2D2, 2U3, and 3G5 produce a stronger pinhead phenotype (c). The hypomorphic allele 3T4 generates a weak pinhead phenotype (data not shown). Genotypes of the flies shown are: (a) y, w, eyFlp/y, w; FRT82B/FRT82B, w+, cl3R3; (b, c) y, w, eyFlp/y, w; FRT82B, Madm7L2 or 3G5/FRT82B, w+, cl3R3; (d) y, w, eyFlp/y, w; gen.Madm(LCQ139)/+; FRT82B, Madm3G5/FRT82B, w+, cl3R3.
Mentions: The BunA-binding domain in Drosophila Madm was reciprocally mapped by means of co-IP and Y2H experiments to the carboxy-terminal amino acids 458-566 (Figure 2f and Additional file 3). Furthermore, we found that amino acids 530-566, including a nuclear export signal (NES) and a predicted nuclear-receptor-binding motif (LXXLL) in mammals, were not dispensable for the binding to BunA [see Additional file 4]. In addition, a point mutation leading to the arginine to histidine substitution R525H disrupted BunA-binding (the point mutation derived from the Madm allele 4S3; Figure 3e). Thus, Madm is a Bun-interacting protein that specifically binds the long Bun isoforms.

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

Show MeSH
Related in: MedlinePlus