The importance of the promoter in Drosophila dosage compensation : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand
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Date
2009
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Massey University
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Abstract
Dosage compensation is the equalisation of gene expression from unequal
doses of genes. Drosophila males up-regulate transcription from their single X
chromosome to equal that from the two female X chromosomes. Five malespecific
lethal (msl) genes are required in males, and encode the main agents of
the up-regulation. At least these proteins, together with either or both of two noncoding
RNAs, form the MSL chromatin-modifying complex. Female-specific
translational repression of a key component, msl2, limits the complex to males.
The MSL complex binds to the X chromosome at hundreds of distinct loci, acetylates
nucleosomes, and de-condenses the chromatin. Together with possibly many
co-factors, the transcriptional up-regulation caused by MSL complex appears to
counteract repressive factors to achieve an average effect of transcriptional doubling.
Here, I have studied the initiation of MSL regulation on the X chromosome
with a variety of approaches. In order to study early events, dosage compensation
was induced in females with ectopic expression of msl2 from the tetracycline
system. However, low background expression without activation prohibited further
studies. To identify novel factors that affect dosage compensation, a reporter
gene system based on variable eye size was evaluated. The system provided a
dose-dependent phenotype, but could not report additional up-regulation by the
MSL complex, and was thus unsuitable for the proposed mutational screen.
The quantifiable lacZ gene was measured in a strict comparison of expression
from an eye-specfic (GMR) or a constitutive (armadillo) promoter. At defined
locations on the X chromsome, armadillo-lacZ acquired local compensation, but
GMR-lacZ did not. Further modifications upstream of GMR-lacZ increased the
response, and confirmed the importance of the promoter in attraction of dosage
compensation. To corroborate this with the established importance of genic sequences
in MSL attraction, a combinatorial model of attraction is proposed. The
relative importance of early or constitutive expression was also tested, by providing
GMR-lacZ with extra expression through the tetracycline system. A burst of
embryonic expression, and constitutive expression, were both insufficient to increase
dosage compensation of the transgene. Finally, the compensation of GMRmediated
transgenes was confounded by ‘transvection’ effects of chromosome
pairing. This effect may have wider implications on the study of compensation at
individual genes.
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Keywords
Drosophila, Gene expression, Dosage compensation