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First published online January 23, 2009
doi: 10.1242/10.1242/dev.027656

Development 136, 689-698 (2009)
Published by The Company of Biologists 2009
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Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation

Solenn Patalano1, Marija Mihailovich1, Yaiza Belacortu2, Nuria Paricio2 and Fátima Gebauer1,*

1 Centre de Regulació Genòmica (CRG-UPF), Gene Regulation Programme, Dr Aiguader 88, 08003 Barcelona, Spain.
2 Departamento de Genética, Facultad Ciencias Biológicas, Universitad de Valencia, Dr Moliner 50, 46100 Burjasot, Spain.


Figure 1
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  		Fig. 1. UNR overexpression preferentially affects male viability. (A) Schematic of the UNR-GFP constructs FLUNR and UNR1+2 that were overexpressed in flies. The relevant domain (CSD1) for msl-2 mRNA and SXL interactions is indicated, as well as the amino acids of UNR that are contained in each construct. CSD, cold-shock domain; Q, glutamine-rich region. (B) FLUNR and UNR1+2 were systemically overexpressed in flies using promoters of increasing strength. FLUNR and UNR1+2 homozygous flies were crossed to Actin5C (Act5C)-Gal4 or engrailed (en)-Gal4 driver lines balanced with CyO. F1 flies carrying CyO (control) or carrying the corresponding Gal4 gene (Gal4) were scored. Numbers above the bars indicate the male-to-female ratio. Flies were grown at 25°C. In some cases, an increased temperature (29°C) was used to boost expression of the transgene.

 

Figure 2
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  		Fig. 2. Overexpression of UNR causes DCC targeting and chromatin defects. Polytene chromosome stainings of FLUNR male flies in which overexpression of UNR was specifically activated in the salivary glands (UAS-FLUNR/Sgs3-Gal4). The non-activated FLUNR line (UAS-FLUNR/UAS-FLUNR) was carried as control. The temperature of activation is indicated. Arrows indicate chromatin defects. Selected areas (a-d) are shown at higher magnification in the bottom row.

 

Figure 3
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  		Fig. 3. Insertion of a PiggyBac element in the Unr locus. (A) Schematic of the PiggyBac (PBac) insertion in the Drosophila Unr gene. Introns and exons are represented as thin and thick lines, respectively. The locations of the sequences encoding the various domains of UNR are indicated within the exons. The PBac element is shown as a gray arrow. The insertion was mapped by PCR using the primers indicated by the small black arrows. The amplification products are shown in the inset (lane PBac). The domain structure of the truncated UNR fragment resulting from the insertion (PBacUNR) is shown. (B) Analysis of UNR products by western blot. Immunoblots were performed on cytoplasmic extracts from wild-type (+/+) or heterozygous (PBac/+) adult flies, or from homozygous (PBac/PBac) third-instar larvae. Tubulin is shown as a loading control.

 

Figure 4
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  		Fig. 4. UNR inhibits DCC formation in females. (A) MSL-2 expression in Unr hypomorphic mutant females. Eye imaginal discs (a-f) and polytene chromosomes (g-k) of third-instar Drosophila larvae were stained with anti-MSL-2 antibodies. MSL-2 was derepressed and assembled on the X chromosomes of mutant females (b,e,h,k). Staining of wild-type females (a,d,g,j) and males (c,f,i) is shown for comparison. The boxed regions in g and h are shown at higher magnification in j and k, respectively. a-c and d-f represent independent staining sets. a-e correspond to maximal projections and f represents a single section. (d-f) Higher magnification images in which the background has been increased to highlight the nuclei (also revealed by DAPI staining in f). Arrowheads indicate MSL-2 on the X chromosome of cells from female mutant eye imaginal discs. MSL-2 staining is stronger in wild-type male cells, where the X chromosome domain is detected as an extended signal on one side of the nucleus (f). Asterisks denote MSL-2 assembled on X chromosome high-affinity sites. The bar chart indicates the geometric averages of MSL-2 light intensity from eye imaginal disc images from arbitrary values calculated with Leica LCS software. Quantifications were performed on 6, 9 and 8 images (same exposure time) for +/+ females, PBac/PBac females and +/+ males, respectively. (B) The DCC assembles on X chromosome high-affinity sites of female Unr hypomorphic mutants. Except for a few sites (arrowheads), most of the MSL-2 signal colocalizes with MLE (asterisks).

 

Figure 5
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  		Fig. 5. Truncation of UNR decreases DCC binding to the male X chromosome. (A) Staining of male Drosophila eye imaginal discs with anti-MSL-2 antibodies. (B) Staining of male polytene chromosomes with Hoechst (a-c) and with anti-MSL-2 antibodies (d-f). The arrowhead in c indicates a bloated region of the X chromosome. The bar chart shows quantification of MSL-2 intensity corrected for the total amount of DNA. Quantifications were performed using ImageJ software on 25, 14 and 30 images (same exposure time) for +/+, PBac/+ and PBac/PBac males, respectively. (C) Decreased recruitment of MSL-2 to the male X chromosome is accompanied by reduced binding of other DCC components. Co-staining of MLE (green) with histone H3 (red) is also shown. A black-and-white image of histone H3 staining alone is shown to illustrate the intensity of the signal. Arrows indicate the X chromosome, the arrowhead shows a region of the X chromosome with knotted chromatin, and asterisks denote MLE binding sites on the autosomes.

 

Figure 6
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  		Fig. 6. UNR depletion in SL2 cells abrogates DCC binding to the X chromosome without affecting DCC protein levels or distribution. (A) Staining of untreated and UNR RNAi-treated Drosophila SL2 cells with antibodies against MSL-2 (green) and Mtor (red). DNA is shown in blue (Hoechst). The left panel shows a wide-field view of the cell preparation; the middle and right-hand panels show staining of a single cell. (B) UNR depletion does not alter the levels or nucleocytoplasmic distribution of DCC components or of nuclear pore proteins involved in DCC targeting. Western blots were performed on total (T), nuclear (N) and cytoplasmic (C) extracts of the indicated cells. Tubulin is shown as a loading control. Untreated Kc female Drosophila cells were carried in parallel as a negative control for MSL-2 and MSL-1 detection.

 

Figure 7
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  		Fig. 7. UNR binds to roX RNAs. (Left) Immunoprecipitation (IP) of UNR from adult male and female Drosophila extracts followed by RT-PCR amplification of roX RNAs from the pellet. Two other abundant mRNAs in males, Dad and LIMK1 (limk), were amplified as controls. Beads with no antibody, as well as parallel reactions with no reverse transcriptase (RT), were also included as negative controls. Left-hand lane, 1 kb DNA ladder (Invitrogen). (Right) Amplification of transcripts from total RNA preparations to show their relative abundance. Left-hand lane, 50 bp DNA ladder (Invitrogen).

 

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© The Company of Biologists Ltd 2009