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A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion.

ABSTRACT: In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene's DNA regulatory elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A peptide. This method requires explicit knowledge only of the endogenous gene's open reading frame and not its regulatory elements.

SUBMITTER: Diao F

PROVIDER: S-EPMC3296248 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion.

Diao Fengqiu F White Benjamin H BH

Genetics 20111229 3

In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene's DNA regulatory elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A pe ...[more]

PMID: 22209908

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Project description:In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes.Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-DsRed but not UAS-arm-lacZ genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.

Dataset Information

A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion.

Publications

A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion.

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