MSL-1 plays a central role in assembly of the MSL complex which mediates 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

Abstract

Dosage compensation in Drosophila melanogaster is achieved by a twofold increase of transcription of X-linked genes in males. This involves the binding of four proteins, MSL-1, MSL-2, MSL-3 and MLE (collectively known as the MSLs) which are believed to act as a multi-protein complex, to hundreds of sites along the length of the X chromosome. MOF, a putative histonc acetyl transferase, is thought to be also associated with MSLs and plays a role in hypertrascription of X-linked genes. Overexpression of either a C-terminal or N-terminal domain of MSL-1 leads to male-specific lethality which is probably due to association with other MSLs to form a non-functional complex. One aim of this study was to identify whether any known MSLs and/or unknown protein binds with the C-terminal domain of MSL-1. A second aim was to further define the domain of MSL-1 which interacts to MSL-2. Initial attempts to identify the protein which interacts the C-terminal domain of MSL-1 by either genetics analysis or co-immunoprecipitation were inconclusive. Thus, an alternative approach of affinity chromatography of epitope-tagged MSL-1/MSL-complex was followed. Transgenic flies which express either a FLAG-tagged N-terminal region of MSL-1 or FLAG tagged C-terminal domain following heat shock were generated. These lines were crossed with other transgenic lines to co-express the MSL-1 domain with Either MSL-2, MSL-3, MLE or MOF. FLAG affinity chromatography of protein extracts prepared from these flies showed that MSL-2 co-purifies with the N-terminal domain of MSL-1 (aa 85 - 263), whereas MOF and MSL-3 co-purify with the C-terminal domain of MSL-1 (aa 705 - 1039). MLE docs not appear to associate with either region of MSL-1. Further, the C-terminal domain of MSL-1 also bound specifically to a glutathione S-transferase-MOF fusion protein. Co-expression of MSL-2 rescued males from the lethal effect which was caused by overexpression of the N-terminal domain of MSL-1. However, co-expression of either or both MOF and MSL-3 with the C-terminal domain of MSL-1 did not improve male viability. This suggests that additional factors may bind to the FC/MOF/MSL-3 complex. Finally, MLE also bound to GST-MOF fusion protein, suggesting a direct interaction between MLE and MOF. These findings suggest that MSL-1 plays a central in assembly of the MSL multi-protein complex that is required to achieve dosage compensation