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First published online 11 October 2006
doi: 10.1242/dev.02620

Development 133, 4475-4483 (2006)
Published by The Company of Biologists 2006
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DNA supercoiling factor contributes to dosage compensation in Drosophila

Hirofumi Furuhashi*, Mikage Nakajima and Susumu Hirose{dagger}

Department of Developmental Genetics, National Institute of Genetics, and Department of Genetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan.


Figure 1
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  		Fig. 1. Effect of scf RNAi on expression of SCF and viability. (A) Comparison of the RNAi effect on expression of SCF between males and females. To generate control individuals (carrying only GAL4) or RNAi-induced individuals (carrying both GAL4 and UAS-IRscf), the Act5C-GAL4 driver was crossed to yw or yw; [UAS-IRscf] (line 2-1) at 25°C (lanes 1 to 8) or 18°C (lanes 9 to 12). Two individuals (late third-instar larvae) of each sample type indicated were randomly selected. Proteins in a 12 µl or 1.5 µl aliquot of the extract from each animal were resolved by SDS-PAGE, blotted onto a membrane, and the membrane probed with anti-SCF (upper panel) or anti-{alpha}-tubulin antibody (lower panel, loading control). (B) Effect of scf RNAi on viability of males and females. Each UAS-IRscf line indicated (balanced with CyO, y+ or TM3, y+) was crossed to Act5C-GAL4 driver. F1 animals carrying only GAL4 (control), or carrying both GAL4 and UAS-IRscf (RNAi), were sorted and counted. Numbers above the bars indicate viability relative to the control sisters carrying only GAL4. The reduction in male viability observed at 25°C was significantly suppressed at 18°C in the experiment using the 2-1 line. The number of control sisters examined for the lines 2-1, 3-1, 3-2, and 2-1 at 18°C was 180, 164, 59 and 160, respectively.

 

Figure 2
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  		Fig. 2. Genetic interactions between SCF and the MSL complex. (A) Expression levels of MSL proteins and rox RNAs are not affected by knockdown of SCF. Control individuals (carrying only GAL4) and RNAi-induced individuals (carrying both GAL4 and UAS-IRscf) were sampled from third-instar male larvae. The expression levels of MSL-1, MSL-2, MSL-3, MLE, MOF and tubulin were examined by immunoblotting (left panel), and rox1 and rox2 RNA expression levels were analyzed by RT-PCR (right panel) with rp49 as a control. First-strand cDNA synthesized from each RNA sample was diluted (1:3, 1:9 and 1:27) and used as PCR template. (B) Effect of the scf deficiency on the survival of females expressing msl-2 under the control of a constitutive promoter. To generate the progeny females listed in the boxes, [H83MSL2]/+ males were crossed to {Delta}scf/TM6B females (left) or [Hsp83-SCF];{Delta}scf/TM6B females (right). Day 1 represents the day when the first population eclosed. Females bearing the [H83MSL2] transgene displayed developmental delay and decreased viability compared with their sisters lacking the transgene. The scf deficiency significantly rescued these females (left graph). Moreover, the rescue was suppressed by introducing Hsp83-driven scf cDNA (right graph).

 

Figure 3
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  		Fig. 3. Localization of SCF along the dosage-compensated male X chromosome. (A-E) Extra SCF signals are associated with the male X chromosome when compared with autosomes or the female X chromosomes. Polytene chromosomes prepared from males (A-C) or females (D,E) were stained with antibodies or DAPI as indicated. Arrowheads indicate examples of the intensely stained SCF bands, which are identical in males and females. Note that the `milky way'-like signals between the intense bands are specific to the male X chromosome. (F-I) Most of the faint signals (but not the intense bands) of SCF colocalize with the MSL complex. Boxed regions in B and C are enlarged in F and G, and merged and split images are shown in H and I, respectively. (J-M) MSL-1 localization (J,K) and acetylation of histone H4K16 (L,M) along the male X chromosome are not affected by knockdown of SCF (J,L). To generate control individuals (carrying only GAL4) or RNAi-induced individuals (carrying both GAL4 and UAS-IRscf), the Act5C-GAL4 driver was crossed to yw or yw; [UAS-IRscf] (line 2-1) at 25°C. Polytene chromosomes from RNAi (J,L) or control (K,M) third-instar larvae were stained with DAPI (green in J,K) and anti-MSL-1 antibody (magenta in J,K), or with anti-acetylated H4K16 antibody (L,M). (N,O) The presence of SCF on the male X chromosome is reduced in a mof1 mutant background. Polytene chromosomes were labeled with DAPI (green in N) and anti-SCF antibody (O; magenta in N). The tip of the X chromosome is marked by an arrow, and the X chromosome is boxed.

 

Figure 4
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  		Fig. 4. Expression levels of X-linked genes are reduced in males deficient in SCF function. To generate control individuals (carrying only GAL4) or RNAi-induced individuals (carrying both GAL4 and UAS-IRscf), the Act5C-GAL4 driver was crossed to yw or yw; [UAS-IRscf] (line 2-1) at 25°C. The effect of SCF reduction on the expression levels of the X-linked genes (Zw, Sgs4, Pgd, BR-C, Gs2 and mRpL16) and the autosomal genes (Dspt4, Rp49, blue and trio) was examined by quantitative RT-PCR. The level of transcripts from each gene was normalized to an internal standard (ß1-tubulin) and then individuals deficient in SCF function via RNAi were compared with control individuals. Relative level of mRNA means the ratio of values relative to the value of the control female; error bars represent s.d. of at least three different samples.

 

Figure 5
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  		Fig. 5. Overexpression of SCF results in abnormal morphology of the male X chromosome but not of autosomes or the female X chromosomes. (A) Polytene chromosomes prepared from an yw control male were labeled with DAPI. (B) Chromosomes prepared from a female of the transgenic SCF-overexpression line were labeled with DAPI. (C-F) Chromosomes prepared from males of the transgenic SCF-overexpression line were labeled with DAPI (C,E; green in F) and anti-MSL-1 (D) or anti-SCF (magenta in F) antibody. (G,H) The male X chromosome appears normal in the line overexpressing both SCF and ISWI (G), and upon overexpresssion of SCF in the mof1 mutant background (H). (I) Acetylation of H4K16 was not detectable on polytene chromosomes in mof1. The chromosomes shown in I were labeled with anti-acetylated H4K16 antibody. Arrows mark the X chromosome.

 

Figure 6
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  		Fig. 6. Immunoblotting analyses. Proteins from the indicated lines were resolved by SDS-PAGE, blotted onto a membrane, and probed with antibodies against SCF, ISWI or {alpha}-tubulin (loading control). Numbers in parenthesis indicate the ratio of the band intensity of yw; p[Hsp83-SCF] to that of yw, normalized to the ratio of the {alpha}-tubulin control.

 

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© The Company of Biologists Ltd 2006