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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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The promoter affected compensation of lacZ-mediated beta-galactosidase activity.beta-galactosidase activity was measured in transgenic males and females carrying the indicated lacZ constructs at defined attP landing sites (2A, 6E, 20C) on the X chromosome. Top panels: Hemizygous male activity was compared to heterozygous female activity to measure level of male hyperactivation (A 2-fold increase  =  log2 of 1). Bottom panels: Hemizygous male activity was compared to homozygous female activity to measure efficiency of dosage compensation (complete compensation  =  log2 of 0). Means of triplicates were plotted, with 1 standard error.
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pone-0020455-g003: The promoter affected compensation of lacZ-mediated beta-galactosidase activity.beta-galactosidase activity was measured in transgenic males and females carrying the indicated lacZ constructs at defined attP landing sites (2A, 6E, 20C) on the X chromosome. Top panels: Hemizygous male activity was compared to heterozygous female activity to measure level of male hyperactivation (A 2-fold increase  =  log2 of 1). Bottom panels: Hemizygous male activity was compared to homozygous female activity to measure efficiency of dosage compensation (complete compensation  =  log2 of 0). Means of triplicates were plotted, with 1 standard error.

Mentions: We targeted both arm-lacZ and GMR-lacZ to each of the three X-linked attP sites, and measured beta-galactosidase activity (Table 1). We compared activities in (hemizygous) males to those in females heterozygous for the insert, as a measure of male hyper-activity (Figure 3, top panels). Separately, we compared males to homozygous females, to measure dosage compensation (Figure 3, bottom panels). We took the log2 of the ratios, to enable a more accurate comparison of ratios above and below zero. A two-fold increase would give a log2 of 1.0, whereas equal expression would give 0. As expected, arm-lacZ males had nearly twice the activity of single-copy females (top panels), nearly sufficient to compensate for deficiency against two-copy females (bottom panels), although the response at 6E was slightly lower. However, male activity in lines with GMR-lacZ was only slightly above single-copy female activity, providing effectively no dosage compensation against two-copy females. The apparent discrepancy between the two ratios (slight hyper-activity but no dosage compensation) is explored below. As the lacZ sequences were identical between arm-lacZ and GMR-lacZ, these results imply that the GMR-hsp70 regulatory sequences were responsible for poor dosage compensation.


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)

The promoter affected compensation of lacZ-mediated beta-galactosidase activity.beta-galactosidase activity was measured in transgenic males and females carrying the indicated lacZ constructs at defined attP landing sites (2A, 6E, 20C) on the X chromosome. Top panels: Hemizygous male activity was compared to heterozygous female activity to measure level of male hyperactivation (A 2-fold increase  =  log2 of 1). Bottom panels: Hemizygous male activity was compared to homozygous female activity to measure efficiency of dosage compensation (complete compensation  =  log2 of 0). Means of triplicates were plotted, with 1 standard error.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020455-g003: The promoter affected compensation of lacZ-mediated beta-galactosidase activity.beta-galactosidase activity was measured in transgenic males and females carrying the indicated lacZ constructs at defined attP landing sites (2A, 6E, 20C) on the X chromosome. Top panels: Hemizygous male activity was compared to heterozygous female activity to measure level of male hyperactivation (A 2-fold increase  =  log2 of 1). Bottom panels: Hemizygous male activity was compared to homozygous female activity to measure efficiency of dosage compensation (complete compensation  =  log2 of 0). Means of triplicates were plotted, with 1 standard error.
Mentions: We targeted both arm-lacZ and GMR-lacZ to each of the three X-linked attP sites, and measured beta-galactosidase activity (Table 1). We compared activities in (hemizygous) males to those in females heterozygous for the insert, as a measure of male hyper-activity (Figure 3, top panels). Separately, we compared males to homozygous females, to measure dosage compensation (Figure 3, bottom panels). We took the log2 of the ratios, to enable a more accurate comparison of ratios above and below zero. A two-fold increase would give a log2 of 1.0, whereas equal expression would give 0. As expected, arm-lacZ males had nearly twice the activity of single-copy females (top panels), nearly sufficient to compensate for deficiency against two-copy females (bottom panels), although the response at 6E was slightly lower. However, male activity in lines with GMR-lacZ was only slightly above single-copy female activity, providing effectively no dosage compensation against two-copy females. The apparent discrepancy between the two ratios (slight hyper-activity but no dosage compensation) is explored below. As the lacZ sequences were identical between arm-lacZ and GMR-lacZ, these results imply that the GMR-hsp70 regulatory sequences were responsible for poor dosage compensation.

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