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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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GMR-hid constructs were expressed similarly in both sexes.A, C–F) Drosophila eyes from transgenic lines (e.g. C32) carrying one copy of the indicated constructs. A) Lines C70, C72 and C74 are phi-C31 mediated insertions at the attP sites 2A, 6E and 20C respectively. B) An example eye from a female homozygous for GMR-hid construct. G) Eye from wild-type (Canton S). In all panels right eyes are shown, dorsal up, anterior right. Bar  = 0.2 mm.
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pone-0020455-g001: GMR-hid constructs were expressed similarly in both sexes.A, C–F) Drosophila eyes from transgenic lines (e.g. C32) carrying one copy of the indicated constructs. A) Lines C70, C72 and C74 are phi-C31 mediated insertions at the attP sites 2A, 6E and 20C respectively. B) An example eye from a female homozygous for GMR-hid construct. G) Eye from wild-type (Canton S). In all panels right eyes are shown, dorsal up, anterior right. Bar  = 0.2 mm.

Mentions: We followed two strategies to adapt the GMR-hid system to report on Drosophila dosage compensation, with the aim of then conducting a mutagenesis screen for modifiers of dosage compensation. To obtain a sex-specific difference in eye size, we created P-element transgenic lines carrying GMR-hid, and screened for those genetically linked to the X chromosome (Figure 1A). Transgenic insertions on the X chromosome usually acquire a degree of compensation [22], [23], [24], meaning that males with one transgene copy express more than single-copy females, and perhaps as much as two-copy females. In all cases, flies homozygous for the GMR-hid transgene had ablated eyes (Figure 1A) representing high levels of transgene expression, but little information as to what level of expression. All lines homozygous for any GMR-hid transgene were weak, presumably due to leaky expression of hid from the basal hsp70 TATA, or expression in other neuronal tissues. Flies heterozygous for all autosomal GMR-hid insertions had similar eye sizes in each sex (Figure 1B). Surprisingly, lines with X-linked insertions of GMR-hid also showed very little difference in eye size between the sexes. In most cases, (hemizygous) male eyes were only slightly smaller than heterozygous female eyes; both sexes having a small range of sizes that just overlapped. The line with greatest difference and least overlap between the sexes (line C60) was selected for further analysis.


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)

GMR-hid constructs were expressed similarly in both sexes.A, C–F) Drosophila eyes from transgenic lines (e.g. C32) carrying one copy of the indicated constructs. A) Lines C70, C72 and C74 are phi-C31 mediated insertions at the attP sites 2A, 6E and 20C respectively. B) An example eye from a female homozygous for GMR-hid construct. G) Eye from wild-type (Canton S). In all panels right eyes are shown, dorsal up, anterior right. Bar  = 0.2 mm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020455-g001: GMR-hid constructs were expressed similarly in both sexes.A, C–F) Drosophila eyes from transgenic lines (e.g. C32) carrying one copy of the indicated constructs. A) Lines C70, C72 and C74 are phi-C31 mediated insertions at the attP sites 2A, 6E and 20C respectively. B) An example eye from a female homozygous for GMR-hid construct. G) Eye from wild-type (Canton S). In all panels right eyes are shown, dorsal up, anterior right. Bar  = 0.2 mm.
Mentions: We followed two strategies to adapt the GMR-hid system to report on Drosophila dosage compensation, with the aim of then conducting a mutagenesis screen for modifiers of dosage compensation. To obtain a sex-specific difference in eye size, we created P-element transgenic lines carrying GMR-hid, and screened for those genetically linked to the X chromosome (Figure 1A). Transgenic insertions on the X chromosome usually acquire a degree of compensation [22], [23], [24], meaning that males with one transgene copy express more than single-copy females, and perhaps as much as two-copy females. In all cases, flies homozygous for the GMR-hid transgene had ablated eyes (Figure 1A) representing high levels of transgene expression, but little information as to what level of expression. All lines homozygous for any GMR-hid transgene were weak, presumably due to leaky expression of hid from the basal hsp70 TATA, or expression in other neuronal tissues. Flies heterozygous for all autosomal GMR-hid insertions had similar eye sizes in each sex (Figure 1B). Surprisingly, lines with X-linked insertions of GMR-hid also showed very little difference in eye size between the sexes. In most cases, (hemizygous) male eyes were only slightly smaller than heterozygous female eyes; both sexes having a small range of sizes that just overlapped. The line with greatest difference and least overlap between the sexes (line C60) was selected for further analysis.

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