A study of CIS-acting elements required for dosage compensation in Drosophila Melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
Dosage compensation (the equalisation of X-linked gene products) occurs in Drosophila melanogaster by a two fold transcriptional up-regulation of X-linked gene expression in males. This involves the binding of five proteins, MSL-1, MSL-2, MSL-3, MLE, MOF, and potentially an RNA (roXl or roX2), to hundreds of sites along the male X chromosome. The cis-acting X-linked DNA sequences required for dosage compensation (called dosage compensation regulatory elements or DCREs) remain elusive, despite numerous attempts of identify them. An insulated reporter gene assay system has been developed to minimise problems previously encountered with identification of these elements. The reporter system consists of the constitutive armadillo promoter fused to the lacZ reporter gene (called arm-lacZ). This reporter construct is flanked by SCS/SCS' insulator elements to block potential repressive effects of an autosomal chromatin environment. The role of the roX genes during dosage compensation was investigated. Initially both the roXl and roX2 RNAs were expressed from within the arm-lacZ insulated system. Expression of either RNA lead to a significant increase in lacZ expression in males, although consistently less than two-fold. These results suggested that either the MSL complex was binding to the roX genes or the expression of the roX RNAs in cis lead to male-specific hypertranscription of lacZ. To test these possibilities roX1 and roX2 cDNAs were inserted into the arm-lacZ reporter. Insertion of either cDNA lead to a significant increase in lacZ expression in males, suggesting that the transcribed regions of the roX genes contain binding site(s) for the MSL complex. Interestingly the level of lacZ hypertranscription in males was significantly higher in homozygous roX1 cDNA lines than homozygous roX1 gene lines. This may indicate that too high a local concentration of roX1 RNA has a dampening effect on the level of hypertranscription meditated by the MSL complex. In a set of experiments designed to identify the MSL binding site(s) in roX1, two regions of the cDNA sequence were amplified and inserted into the arm-lacZ system. One of these fragments, containing a proposed DNAseI hypersensitivity site and possible GAGA binding sites, increased lacZ expression in males, but to levels lower than the entire cDNA. This suggests there may be more than one MSL biding site in roX1. A second method of dosage compensation is thought to occur in Drosophila, independently of the MSL proteins. The arm-lacZ insulated reporter system was used to investigate the hypothesis that some genes may be dosage compensated due to repression by Sex-lethal (Sxl) in females. Several genes have been found to contain three or more Sxl binding sites in their 3' UTRs. with some also carrying Sxl binding sites in the 5' UTR. Fragments from the Sxl, Cut and Small Forked genes, containing numerous Sxl binding sites from the 3' UTR, were inserted into the 3' UTR region of arm-lacZ. Males carrying autosomal insertions of the construct had on average 1.07 - 1.50 times the level of β-galactosidase in females. This suggests that some genes could be partially compensated through Sxl repression in females. In addition to inserting 3' UTR fragments into arm-lacZ, a synthetic oligonucleotide containing a long Sxl binding site was inserted into the 5' region of an arm-lacZ construct already carrying the Runt 3' UTR fragment. Males carrying autosomal insertions of the construct had levels of β-galactosidase activity similar to those lines carrying autosomal insertions of the 3' UTR fragments alone. This suggests that other factors such as RNA binding proteins or RNA secondary structure may be required in order to obtain efficient translation repression by Sxl. Finally three X-linked DNA fragments, from the 1C region, were inserted individually between the SCS' element and the armadillo promoter. If the X-linked fragment contained a DCRE then males carrying autosomal insertions of the construct would produce twice the β-galactosidase activity of females. However, males and females expressed the same levels of lacZ.