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Abnormal dosage compensation of reporter genes driven by the Drosophila glass multiple reporter (GMR) enhancer-promoter.

Laverty C, Li F, Belikoff EJ, Scott MJ - PLoS ONE (2011)

Bottom Line: Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation.Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females.We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3' end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex.

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tetO-GMR-lacZ responded to tTA in brain and other tissues but not imaginal discs.Third instar larvae of the indicated strains, raised with (+) or without (−) tetracycline (tet) in the diet, were dissected and stained with X-gal for lacZ expression. Additional induction of tetO-GMR-lacZ occurs with arm-tTA in the absence of tetracycline. y w and arm-lacZ provided as negative and positive controls for lacZ expression, respectively. All staining and photographic conditions were equal across all sets. Bar  = 0.2 mm.
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pone-0020455-g005: tetO-GMR-lacZ responded to tTA in brain and other tissues but not imaginal discs.Third instar larvae of the indicated strains, raised with (+) or without (−) tetracycline (tet) in the diet, were dissected and stained with X-gal for lacZ expression. Additional induction of tetO-GMR-lacZ occurs with arm-tTA in the absence of tetracycline. y w and arm-lacZ provided as negative and positive controls for lacZ expression, respectively. All staining and photographic conditions were equal across all sets. Bar  = 0.2 mm.

Mentions: To confirm that regulation via the tetracycline system was capable of shifting the expression pattern of tetO-GMR-lacZ, we crossed the reporter line to arm-tTA flies and observed the altered expression pattern in climbing third instar larvae (Figure 5). The GMR enhancer/promoter is active in all cells in, and posterior to, the morphogenetic furrow in the developing eye-antennal disc in third instar larvae [6]. In a control cross to y w flies, beta-galactosidase staining could be detected in the developing eye, posterior to the morphogenetic furrow, and to a limited extent in the brain, consistent with Glass expression [30]. When crossed to arm-tTA, beta-galactosidase could also be detected in the brain and most other larval tissues (Figure 5). This response was specific to arm-tTA as it was inhibited with the addition of tetracycline to the culture medium. Given that the tetO-GMR-lacZ reporter could respond to additional regulation through the tetracycline system, and that the activators were all active in the early embryo, we concluded that the above test of effect on dosage compensation was valid. Therefore, additional early or constitutive expression of GMR-lacZ was insufficient to affect dosage compensation. The time and specificity of expression may have been relatively less important than the nature of the promoter/enhancer.


Abnormal dosage compensation of reporter genes driven by the Drosophila glass multiple reporter (GMR) enhancer-promoter.

Laverty C, Li F, Belikoff EJ, Scott MJ - PLoS ONE (2011)

tetO-GMR-lacZ responded to tTA in brain and other tissues but not imaginal discs.Third instar larvae of the indicated strains, raised with (+) or without (−) tetracycline (tet) in the diet, were dissected and stained with X-gal for lacZ expression. Additional induction of tetO-GMR-lacZ occurs with arm-tTA in the absence of tetracycline. y w and arm-lacZ provided as negative and positive controls for lacZ expression, respectively. All staining and photographic conditions were equal across all sets. Bar  = 0.2 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105068&req=5

pone-0020455-g005: tetO-GMR-lacZ responded to tTA in brain and other tissues but not imaginal discs.Third instar larvae of the indicated strains, raised with (+) or without (−) tetracycline (tet) in the diet, were dissected and stained with X-gal for lacZ expression. Additional induction of tetO-GMR-lacZ occurs with arm-tTA in the absence of tetracycline. y w and arm-lacZ provided as negative and positive controls for lacZ expression, respectively. All staining and photographic conditions were equal across all sets. Bar  = 0.2 mm.
Mentions: To confirm that regulation via the tetracycline system was capable of shifting the expression pattern of tetO-GMR-lacZ, we crossed the reporter line to arm-tTA flies and observed the altered expression pattern in climbing third instar larvae (Figure 5). The GMR enhancer/promoter is active in all cells in, and posterior to, the morphogenetic furrow in the developing eye-antennal disc in third instar larvae [6]. In a control cross to y w flies, beta-galactosidase staining could be detected in the developing eye, posterior to the morphogenetic furrow, and to a limited extent in the brain, consistent with Glass expression [30]. When crossed to arm-tTA, beta-galactosidase could also be detected in the brain and most other larval tissues (Figure 5). This response was specific to arm-tTA as it was inhibited with the addition of tetracycline to the culture medium. Given that the tetO-GMR-lacZ reporter could respond to additional regulation through the tetracycline system, and that the activators were all active in the early embryo, we concluded that the above test of effect on dosage compensation was valid. Therefore, additional early or constitutive expression of GMR-lacZ was insufficient to affect dosage compensation. The time and specificity of expression may have been relatively less important than the nature of the promoter/enhancer.

Bottom Line: Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation.Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females.We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3' end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex.

Show MeSH
Related in: MedlinePlus