Limits...
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.

Show MeSH

Related in: MedlinePlus

Transgenic lacZ constructs used in this study.A–H) The lacZ ORF is the solid black box, surrounded by 5′ and 3′ regulatory sequences. Transcription start points represented with bent arrows. Unlabelled elements are identical to the construct immediately above. A, F, G) Sequences identical to, or one mis-match from, the DRE consensus sequence WATCGATW [82], and GAGA (or TCTC), are indicated with points and + symbols, respectively. F) The fragment from the armadillo promoter included in arm*-GMR-lacZ is underlined in panel A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105068&req=5

pone-0020455-g002: Transgenic lacZ constructs used in this study.A–H) The lacZ ORF is the solid black box, surrounded by 5′ and 3′ regulatory sequences. Transcription start points represented with bent arrows. Unlabelled elements are identical to the construct immediately above. A, F, G) Sequences identical to, or one mis-match from, the DRE consensus sequence WATCGATW [82], and GAGA (or TCTC), are indicated with points and + symbols, respectively. F) The fragment from the armadillo promoter included in arm*-GMR-lacZ is underlined in panel A.

Mentions: To more quantifiably measure compensation of a GMR-driven transgene, we used the enhancer-promoter to drive the beta-galactosidase gene lacZ, and compared beta-galactosidase activity from several related constructs (Figure 2). To measure the degree of up-regulation and compensation supported by GMR-mediated expression, we compared the response of GMR-lacZ at X-linked sites to that of lacZ driven by the constitutive promoter from the X-linked armadillo (arm) gene. We have previously shown that X-linked insertions of arm-lacZ generally acquire dosage compensation [24], [42]. To remove position effects of integration, we used the phiC31 recombinase system to target transgenic constructs to defined attP landing sites [43]. We tested five X-linked landing sites [44] for efficiency of transformation, but could not generate insertions at attP-3B, and found that insertions at attP-3Aa were either male-lethal or ectopically integrated. Of the three remaining sites, attP-2A and attP-6E were within 20 kb of MSL binding sites noted in MSL immuno-precipitates [19], and the peri-centromeric attP-20C was devoid of bound MSL. Flies carrying GMR-hid at each of these locations had eyes similar to those with P-element insertions of the transgene (Figure 1A, lines C70, C72, C74).


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)

Transgenic lacZ constructs used in this study.A–H) The lacZ ORF is the solid black box, surrounded by 5′ and 3′ regulatory sequences. Transcription start points represented with bent arrows. Unlabelled elements are identical to the construct immediately above. A, F, G) Sequences identical to, or one mis-match from, the DRE consensus sequence WATCGATW [82], and GAGA (or TCTC), are indicated with points and + symbols, respectively. F) The fragment from the armadillo promoter included in arm*-GMR-lacZ is underlined in panel A.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020455-g002: Transgenic lacZ constructs used in this study.A–H) The lacZ ORF is the solid black box, surrounded by 5′ and 3′ regulatory sequences. Transcription start points represented with bent arrows. Unlabelled elements are identical to the construct immediately above. A, F, G) Sequences identical to, or one mis-match from, the DRE consensus sequence WATCGATW [82], and GAGA (or TCTC), are indicated with points and + symbols, respectively. F) The fragment from the armadillo promoter included in arm*-GMR-lacZ is underlined in panel A.
Mentions: To more quantifiably measure compensation of a GMR-driven transgene, we used the enhancer-promoter to drive the beta-galactosidase gene lacZ, and compared beta-galactosidase activity from several related constructs (Figure 2). To measure the degree of up-regulation and compensation supported by GMR-mediated expression, we compared the response of GMR-lacZ at X-linked sites to that of lacZ driven by the constitutive promoter from the X-linked armadillo (arm) gene. We have previously shown that X-linked insertions of arm-lacZ generally acquire dosage compensation [24], [42]. To remove position effects of integration, we used the phiC31 recombinase system to target transgenic constructs to defined attP landing sites [43]. We tested five X-linked landing sites [44] for efficiency of transformation, but could not generate insertions at attP-3B, and found that insertions at attP-3Aa were either male-lethal or ectopically integrated. Of the three remaining sites, attP-2A and attP-6E were within 20 kb of MSL binding sites noted in MSL immuno-precipitates [19], and the peri-centromeric attP-20C was devoid of bound MSL. Flies carrying GMR-hid at each of these locations had eyes similar to those with P-element insertions of the transgene (Figure 1A, lines C70, C72, C74).

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