Abnormal dosage compensation of reporter genes driven by the Drosophila glass multiple reporter (GMR) enhancer-promoter.

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.

The importance of location and orientation of male specific lethal complex binding sites of differing affinities on reporter gene dosage compensation in Drosophila.

The male specific lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila. The complex binds to most actively transcribed X-linked genes in males and upregulates expression. High resolution chromatin immunoprecipitation assays have identified over one hundred high affinity binding sites on the X chromosome. One of the first high affinity sites discovered is at cytological location 18D11. The MSL complex binds weakly to a single copy of a 510bp fragment from 18D11 but strongly to a tetramer of the fragment. Here we have investigated the effect of insertion of sites of differing affinities, either upstream or within the transcribed gene, on complex binding and transcription upregulation. Insertion of four copies of the 18D11 fragment upstream or at the 3' end of a reporter gene led to strong MSL complex binding and increased expression in males. In contrast, the MSL complex did not bind consistently to autosomal transgenes that contained a single copy of the 18D11 site upstream of the gene promoter. However, MSL complex binding was observed in all lines if the single 18D11 fragment was inserted into the 3' end of the reporter gene in either orientation. This is consistent with previous studies that showed gene transcription facilitates MSL complex binding. Surprisingly, transcription elevation in males was only observed if the 18D11 fragment was in the forward orientation and only in some lines. Our results suggest that MSL complex binding to weaker sites and transcription enhancement is influenced by gene transcription, binding site orientation and the local chromatin environment. In contrast, strong binding sites do not need to be transcribed to recruit sufficient complex to cause transcription elevation of nearby genes.

The MSL complex: X chromosome and beyond.

X chromosomal regulation is a process that presents systematic problems of chromosome recognition and coordinated gene regulation. In Drosophila males, the ribonucleoprotein Male-Specific Lethal (MSL) complex plays an important role in hyperactivation of the X-linked genes to equalize gene dosage differences between the sexes. It appears that X chromosome recognition by the MSL complex may be mediated through a combination of sequence-specificity and transcriptional activities. The resulting transcriptional up-regulation also seems to involve several mechanisms, encompassing both gene-specific and chromosome-wide approaches. Interestingly the histone H4 lysine 16 specific MOF histone acetyl transferase, a key MSL member that hyper-acetylates the male X chromosome, is also involved in gene regulation beyond dosage compensation. A comparison of Drosophila and mammalian systems reveals intriguing parallels in MOF behavior, and highlights the multidisciplinary nature of this enzyme.