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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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Long human TSC22DF isoforms can replace BunA function in Drosophila. (a) Schematic drawing of human and Drosophila TSC22DF proteins that were tested for their ability to rescue the lethality of bun mutants. The long isoforms possess two short conserved stretches named motif 1 and motif 2. Whereas BunA represents the long TSC22DF isoforms in Drosophila, BunB and BunC are two of the short isoforms. (b) Expression of long TSC22DF isoforms restores the viability of bun mutants. The quality of the rescue is indicated as a percentage of the expected Mendelian ratio. The Gal4 driver lines are ordered according to the strength of ubiquitous expression they direct during development, with arm-Gal4 being the weakest and Act5C-Gal4 the strongest driver line. In each experimental cross, n ≥ 200 progeny flies were analyzed. Leaky expression, without Gal4; 1 c and 2 c, one or two copies of the respective UAS construct. The ZH-attP-86Fb integration site seems to mediate strong expression as the UAS-attB-bunA constructs (ORF and cDNA) do not need to be driven by a Gal4 line for rescue, in contrast to the UAS-bunA construct (cDNA) generated by standard P-element-mediated germline transformation (inserted non-site-specifically on chromosome III). Note that too high expression of long TSC22DF members is harmful to flies. In a wild-type background, Act5C-Gal4-directed expression (n ≥ 200) of TSC22D2 and of bunA ORF kills the animals (0% survival). Expression from the bunA cDNA construct produces few escapers (3%), whereas expression from the bunA cDNA P-element construct and of TSC22D4 results in semi-viability (14% and 69%, respectively). Only TSC22D1.1 can be expressed by Act5C-Gal4 without compromising survival (>80%). Thus, there appears to be an optimal range of long TSC22DF concentration for viability.
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Figure 1: Long human TSC22DF isoforms can replace BunA function in Drosophila. (a) Schematic drawing of human and Drosophila TSC22DF proteins that were tested for their ability to rescue the lethality of bun mutants. The long isoforms possess two short conserved stretches named motif 1 and motif 2. Whereas BunA represents the long TSC22DF isoforms in Drosophila, BunB and BunC are two of the short isoforms. (b) Expression of long TSC22DF isoforms restores the viability of bun mutants. The quality of the rescue is indicated as a percentage of the expected Mendelian ratio. The Gal4 driver lines are ordered according to the strength of ubiquitous expression they direct during development, with arm-Gal4 being the weakest and Act5C-Gal4 the strongest driver line. In each experimental cross, n ≥ 200 progeny flies were analyzed. Leaky expression, without Gal4; 1 c and 2 c, one or two copies of the respective UAS construct. The ZH-attP-86Fb integration site seems to mediate strong expression as the UAS-attB-bunA constructs (ORF and cDNA) do not need to be driven by a Gal4 line for rescue, in contrast to the UAS-bunA construct (cDNA) generated by standard P-element-mediated germline transformation (inserted non-site-specifically on chromosome III). Note that too high expression of long TSC22DF members is harmful to flies. In a wild-type background, Act5C-Gal4-directed expression (n ≥ 200) of TSC22D2 and of bunA ORF kills the animals (0% survival). Expression from the bunA cDNA construct produces few escapers (3%), whereas expression from the bunA cDNA P-element construct and of TSC22D4 results in semi-viability (14% and 69%, respectively). Only TSC22D1.1 can be expressed by Act5C-Gal4 without compromising survival (>80%). Thus, there appears to be an optimal range of long TSC22DF concentration for viability.

Mentions: We hypothesized that the long isoform encoded by the TSC-22 locus, TSC22D1.1, is a functional homolog of BunA with growth-promoting capacity, and that it is antagonized by the short isoform TSC22D1.2. Therefore, we tested whether human TSC22D1.1 or any other TSC22DF member is able to replace BunA function in Drosophila. The UAS/Gal4 expression system [34] was combined with a site-specific integration system [35] to express the TSC22DF members. Ubiquitous expression of the long - but not of the short - human TSC22DF isoforms (Figure 1a) resulted in a rescue of the lethality of bun mutants carrying a deletion allele (200B) that is likely to be for all bun isoforms [12] (Figure 1b). Thus, TSC22D1.1 has the ability to replace BunA function in the fly whereas TSC22D1.2 does not. Furthermore, all long human TSC22DF isoforms can act in place of BunA in Drosophila, suggesting that sequences conserved in the long isoforms enable BunA to promote growth.


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)

Long human TSC22DF isoforms can replace BunA function in Drosophila. (a) Schematic drawing of human and Drosophila TSC22DF proteins that were tested for their ability to rescue the lethality of bun mutants. The long isoforms possess two short conserved stretches named motif 1 and motif 2. Whereas BunA represents the long TSC22DF isoforms in Drosophila, BunB and BunC are two of the short isoforms. (b) Expression of long TSC22DF isoforms restores the viability of bun mutants. The quality of the rescue is indicated as a percentage of the expected Mendelian ratio. The Gal4 driver lines are ordered according to the strength of ubiquitous expression they direct during development, with arm-Gal4 being the weakest and Act5C-Gal4 the strongest driver line. In each experimental cross, n ≥ 200 progeny flies were analyzed. Leaky expression, without Gal4; 1 c and 2 c, one or two copies of the respective UAS construct. The ZH-attP-86Fb integration site seems to mediate strong expression as the UAS-attB-bunA constructs (ORF and cDNA) do not need to be driven by a Gal4 line for rescue, in contrast to the UAS-bunA construct (cDNA) generated by standard P-element-mediated germline transformation (inserted non-site-specifically on chromosome III). Note that too high expression of long TSC22DF members is harmful to flies. In a wild-type background, Act5C-Gal4-directed expression (n ≥ 200) of TSC22D2 and of bunA ORF kills the animals (0% survival). Expression from the bunA cDNA construct produces few escapers (3%), whereas expression from the bunA cDNA P-element construct and of TSC22D4 results in semi-viability (14% and 69%, respectively). Only TSC22D1.1 can be expressed by Act5C-Gal4 without compromising survival (>80%). Thus, there appears to be an optimal range of long TSC22DF concentration for viability.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871527&req=5

Figure 1: Long human TSC22DF isoforms can replace BunA function in Drosophila. (a) Schematic drawing of human and Drosophila TSC22DF proteins that were tested for their ability to rescue the lethality of bun mutants. The long isoforms possess two short conserved stretches named motif 1 and motif 2. Whereas BunA represents the long TSC22DF isoforms in Drosophila, BunB and BunC are two of the short isoforms. (b) Expression of long TSC22DF isoforms restores the viability of bun mutants. The quality of the rescue is indicated as a percentage of the expected Mendelian ratio. The Gal4 driver lines are ordered according to the strength of ubiquitous expression they direct during development, with arm-Gal4 being the weakest and Act5C-Gal4 the strongest driver line. In each experimental cross, n ≥ 200 progeny flies were analyzed. Leaky expression, without Gal4; 1 c and 2 c, one or two copies of the respective UAS construct. The ZH-attP-86Fb integration site seems to mediate strong expression as the UAS-attB-bunA constructs (ORF and cDNA) do not need to be driven by a Gal4 line for rescue, in contrast to the UAS-bunA construct (cDNA) generated by standard P-element-mediated germline transformation (inserted non-site-specifically on chromosome III). Note that too high expression of long TSC22DF members is harmful to flies. In a wild-type background, Act5C-Gal4-directed expression (n ≥ 200) of TSC22D2 and of bunA ORF kills the animals (0% survival). Expression from the bunA cDNA construct produces few escapers (3%), whereas expression from the bunA cDNA P-element construct and of TSC22D4 results in semi-viability (14% and 69%, respectively). Only TSC22D1.1 can be expressed by Act5C-Gal4 without compromising survival (>80%). Thus, there appears to be an optimal range of long TSC22DF concentration for viability.
Mentions: We hypothesized that the long isoform encoded by the TSC-22 locus, TSC22D1.1, is a functional homolog of BunA with growth-promoting capacity, and that it is antagonized by the short isoform TSC22D1.2. Therefore, we tested whether human TSC22D1.1 or any other TSC22DF member is able to replace BunA function in Drosophila. The UAS/Gal4 expression system [34] was combined with a site-specific integration system [35] to express the TSC22DF members. Ubiquitous expression of the long - but not of the short - human TSC22DF isoforms (Figure 1a) resulted in a rescue of the lethality of bun mutants carrying a deletion allele (200B) that is likely to be for all bun isoforms [12] (Figure 1b). Thus, TSC22D1.1 has the ability to replace BunA function in the fly whereas TSC22D1.2 does not. Furthermore, all long human TSC22DF isoforms can act in place of BunA in Drosophila, suggesting that sequences conserved in the long isoforms enable BunA to promote growth.

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