X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3

Abstract

In Drosophila, dosage compensation of X-linked genes is achieved by transcriptional upregulation of the male X chromosome. Genetic and biochemical studies have demonstrated that male-specific lethal (MSL) proteins together with roX RNAs regulate this process. Here, we show that MSL-3 is essential for cell viability and that three domains in the protein have distinct roles in dosage compensation. The chromo-barrel domain (CBD) is not necessary for MSL targeting to the male X chromosome but is important for male viability and equalization of X-linked gene transcription. The polar region cooperates with the CBD in MSL-3 function, whereas the MRG domain is responsible for targeting the protein to the X chromosome. Our results demonstrate that MSL-3 localization to the male X chromosome and transcriptional upregulation of X-linked genes are two separable functions of the MSL-3 protein.

Figure 1

Generation of MSL-3 derivatives and rescue test. (A) Schematic representation of MSL-3 domains and transgenic flies generated for this study. wt, enhanced green fluorescent protein (EGFP)-tagged and FLAG-tagged wild-type MSL-3 were generated as controls; chromo-barrel domain (CBD) mutants, deletion in the CBD and point mutation in the CBD of MSL-3; polar region mutants, deletion in the polar region and point mutation; CBD+polar region mutant, deletion of the CBD and the polar region; MRG mutant, deletion of the MRG motif. (B) Rescue test of msl-3⁰⁸³ lethality by MSL-3 carboxy-terminal-tagged proteins (lanes 1–3), CBD MSL-3 mutant (lanes 4,5), polar region MSL-3 mutants (lanes 6,7), CBD and polar region MSL-3 mutant (lane 8) and MRG mutant (lane 9). The numbers of insertion analysed, the number of males and females counted and the average of the rescue frequency are indicated for each mutant. (C) Homozygous males expressing ΔCBD–EGFP in a homozygous msl-3⁰⁸³ mutant background show different male-specific phenotypic abnormalities, including rough eyes (compare panels b,c,d with panel a); missed bristles (compare panels f,g,h with panel e) and misshapen wings (compare panels j,k,l with panel i). Panels a,e,i show normal phenotype of homozygous females expressing ΔCBD–EGFP.

Figure 2

The MRG domain is required for MSL-3 localization to the X chromosome. (A) Polytene chromosomes from MSL-3–EGFP (top) or ΔMRG–EGFP (bottom) male larvae were stained with antibodies against EGFP and MSL-1. (B) Salivary glands from MSL-3–EGFP (top) or ΔMRG–EGFP (bottom) male larvae were stained with antibodies against EGFP and MSL-1.

Figure 3

Deletion of MSL-3 chromo-barrel domain does not affect targeting of MSL proteins to the male X chromosome. (A) Polytene chromosomes from ΔCBD–EGFP male larvae were stained with antibodies against EGFP, MSL-1, MOF, MSL-2 and MLE. Mislocalization of MSL proteins to autosomes is indicated by arrows. (B) Polytene chromosomes from F56A-EGFP male larvae were stained with antibodies against EGFP and MSL-1. (C) Intact salivary glands from MSL-3–EGFP (top) or ΔCBD–EGFP (bottom) male larvae were hybridized with roX1- and roX2-specific probes or hybridized with roX1- and roX2-specific probes and simultaneously stained with MSL-1 antibody. (D) Polytene chromosomes from MRG–EGFP male larvae were stained with antibodies against EGFP and MSL-1.

Figure 4

ΔCBD–EGFP males are not able to fully compensate X-linked genes. Quantitative PCR analysis of pgd, dspt6, sl, vap33-1, crm, CG32721, para, dspt4 and pol2 in wild-type (wt) females (black), wt males (grey), MSL-3–EGFP females (dark green) and males (light green) and ΔCBD–EGFP females (dark blue) and males (light blue). Error bars represent the standard deviation of three different biological samples. Expression levels were normalized against the autosomal gene rp49 and set to 100% for each gene in females.

Figure 5

MSL-3 CBD is involved in DNA and chromatin interaction. (A) Schematic representation of recombinant MSL-3 derivatives generated for the DNA and nucleosome electrophoretic mobility shift assay. (B) Increasing amounts (9.21 and 13.82 pmol) of MSL-3 derivatives were incubated with radioactively labelled 146 bp DNA fragment (lanes 1–8) or with nucleosomes assembled on the same radioactively labelled DNA fragment (lanes 10–17). Full-length MSL-3 (lanes 1,2; 10,11), ΔCBD (lanes 3,4; 12,13), ΔPOLAR (lanes 5,6; 14,15) or CBD alone (lanes 7,8; 16,17) are shown. Lane 9, free DNA; lane 18, nucleosomes. Arrows indicate the free probes. The protein–DNA and protein–nucleosome complexes are also indicated.