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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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Compensation of tetO-GMR-lacZ was unaffected by additional early or constitutive expression.A) Equality of beta-galactosidase activity between the sexes was measured in adult heads of flies carrying one copy of tetO-GMR-lacZ (at the 2A attP site), and one copy of the indicated tetracycline driver. y w flies have no tTA driver. The responses of arm-lacZ and GMR-lacZ at 2A are provided for comparison. Flies were raised in the absence of tetracycline to promote transcription activation by tTA. Means of triplicates were plotted, with 1 standard error. B) Activity of the tTA drivers was detected as yellow fluorescence (through a green filter set) in embryos carrying one copy of tetO-YFP, and one copy of the indicated drivers, raised without tetracycline. White light (top of each pair), and fluorescent images of the embryos at different time points after egg laying were recorded. Dorsal up, anterior left. The solid line bisecting each embryo is the edge of tape used to mount the embryos.
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pone-0020455-g004: Compensation of tetO-GMR-lacZ was unaffected by additional early or constitutive expression.A) Equality of beta-galactosidase activity between the sexes was measured in adult heads of flies carrying one copy of tetO-GMR-lacZ (at the 2A attP site), and one copy of the indicated tetracycline driver. y w flies have no tTA driver. The responses of arm-lacZ and GMR-lacZ at 2A are provided for comparison. Flies were raised in the absence of tetracycline to promote transcription activation by tTA. Means of triplicates were plotted, with 1 standard error. B) Activity of the tTA drivers was detected as yellow fluorescence (through a green filter set) in embryos carrying one copy of tetO-YFP, and one copy of the indicated drivers, raised without tetracycline. White light (top of each pair), and fluorescent images of the embryos at different time points after egg laying were recorded. Dorsal up, anterior left. The solid line bisecting each embryo is the edge of tape used to mount the embryos.

Mentions: We crossed flies carrying tetO-GMR-lacZ to lines with arm-tTA, n1-tTA, or s1-tTA activators, and raised the offspring in the absence of tetracycline. We measured the male hyper-activity of beta-galactosidase in adult heads (Table 4), and compared the responses to those of arm-lacZ and GMR-lacZ (Figure 4A). Males and (single-copy) females of all crosses had very similar activities. The response to additional constitutive expression provided by arm-tTA was evident in the very high activities in offspring of this cross. Further, the increase in lacZ expression was dependent on tTA as there was no response when flies were raised with tetracycline (mean male activity of 2.15±0.06, female of 1.64±0.07 OD/min/mg). A separate cross to y w flies (parental strain), which lack tTA, showed that the tetO sequences alone had little effect on activity from GMR-lacZ (Table 4). To confirm that the tTA drivers were active in the early embryo, we crossed the tTA driver lines to a tetO-YFP responder line [57] and examined the offspring for yellow fluorescence (Figure 4B). We found that all tTA drivers were effective at inducing YFP expression well above background levels.


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)

Compensation of tetO-GMR-lacZ was unaffected by additional early or constitutive expression.A) Equality of beta-galactosidase activity between the sexes was measured in adult heads of flies carrying one copy of tetO-GMR-lacZ (at the 2A attP site), and one copy of the indicated tetracycline driver. y w flies have no tTA driver. The responses of arm-lacZ and GMR-lacZ at 2A are provided for comparison. Flies were raised in the absence of tetracycline to promote transcription activation by tTA. Means of triplicates were plotted, with 1 standard error. B) Activity of the tTA drivers was detected as yellow fluorescence (through a green filter set) in embryos carrying one copy of tetO-YFP, and one copy of the indicated drivers, raised without tetracycline. White light (top of each pair), and fluorescent images of the embryos at different time points after egg laying were recorded. Dorsal up, anterior left. The solid line bisecting each embryo is the edge of tape used to mount the embryos.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020455-g004: Compensation of tetO-GMR-lacZ was unaffected by additional early or constitutive expression.A) Equality of beta-galactosidase activity between the sexes was measured in adult heads of flies carrying one copy of tetO-GMR-lacZ (at the 2A attP site), and one copy of the indicated tetracycline driver. y w flies have no tTA driver. The responses of arm-lacZ and GMR-lacZ at 2A are provided for comparison. Flies were raised in the absence of tetracycline to promote transcription activation by tTA. Means of triplicates were plotted, with 1 standard error. B) Activity of the tTA drivers was detected as yellow fluorescence (through a green filter set) in embryos carrying one copy of tetO-YFP, and one copy of the indicated drivers, raised without tetracycline. White light (top of each pair), and fluorescent images of the embryos at different time points after egg laying were recorded. Dorsal up, anterior left. The solid line bisecting each embryo is the edge of tape used to mount the embryos.
Mentions: We crossed flies carrying tetO-GMR-lacZ to lines with arm-tTA, n1-tTA, or s1-tTA activators, and raised the offspring in the absence of tetracycline. We measured the male hyper-activity of beta-galactosidase in adult heads (Table 4), and compared the responses to those of arm-lacZ and GMR-lacZ (Figure 4A). Males and (single-copy) females of all crosses had very similar activities. The response to additional constitutive expression provided by arm-tTA was evident in the very high activities in offspring of this cross. Further, the increase in lacZ expression was dependent on tTA as there was no response when flies were raised with tetracycline (mean male activity of 2.15±0.06, female of 1.64±0.07 OD/min/mg). A separate cross to y w flies (parental strain), which lack tTA, showed that the tetO sequences alone had little effect on activity from GMR-lacZ (Table 4). To confirm that the tTA drivers were active in the early embryo, we crossed the tTA driver lines to a tetO-YFP responder line [57] and examined the offspring for yellow fluorescence (Figure 4B). We found that all tTA drivers were effective at inducing YFP expression well above background levels.

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