Sequence-specific targeting of MSL complex regulates transcription of the roX RNA genes

Abstract

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of at least five proteins and two noncoding RNAs, roX1 and roX2. The roX RNAs function in targeting MSL complex to the X chromosome, and roX transgenes can nucleate spreading of the MSL complex into flanking chromatin when inserted on an autosome. An MSL-binding site (DHS, DNaseI hypersensitive site) has been identified in each roX gene. Here, we investigate the functions of the DHS using transgenic deletion analyses and reporter assays. We find that MSL interaction with the DHS counteracts constitutive repression at roX1, resulting in male-specific expression of roX1 RNA. Surprisingly, the DHS is not required for initiation of cis spreading of MSL complex, instead local transcription of roX RNAs correlates with extensive spreading.

Figure 1

DHS positively regulates roX RNA levels in males. (A) Structures of the transgenes. GMroX1 and GMroX2 contain full-length roX genomic sequences along with partial segments from flanking genes as indicated by an arrow. In both ΔDHS transgenes, ∼300 bp encompassing the DHS are deleted. In GMroX2-DHS-mut, three blocks of consensus sequences are mutagenized, as indicated by ‘^***'. (B–D) Northern blots from adult males to compare RNA level among GMroX, GMroX-ΔDHS and GMroX2-DHS-mut. All transgenic lines carry either a mutant allele (roX1^ex6) at their endogenous roX1 locus (B), or are deleted for endogenous roX2 (C, D). Hybridization for rp49 is the loading control in all Northern blots. (B) roX1 Northern. Lane 1: wild-type males; lane 2: GMroX1-67B; lanes 3–12: different GMroX1-ΔDHS lines with the site of insertion indicated. Quantification of roX1 RNA by a phosphoimager is shown as relative to the wild-type level (designated as 1.0), after normalization to rp49 levels. The roX1-ΔDHS transcript is ∼300 nt shorter than wild-type roX1 RNA. (C) roX2 Northern. Lane 1: wild-type males; lanes 2 and 3: GMroX2-86F and 97F, respectively; lanes 4–10: different roX2-ΔDHS transgenic lines with the site of insertion and RNA quantification indicated. (D) roX2 Northern. Lanes 1 and 2: GMroX2-86F and 97F, respectively; lanes 3–9: GMroX2-DHS-mut transgenic lines.

Figure 2

DHS is not essential for spreading of the MSL complex from roX loci. Polytene chromosome immunostaining by anti-MSL1 antibody (red). DNA is stained with DAPI (blue). GMroX1 (A) and GMroX2 (E) transgenes provide nucleation sites for extensive spreading (arrowhead) of the MSL complex in ∼100% of nuclei in roX1roX2 mutants. (B) GMroX1-ΔDHS-68A, no binding and spreading at the transgene (arrow). (C) The same transgenic line as in (B) but a different nucleus showing extensive spreading (arrowhead). (D) GMroX1-ΔDHS-25B with no binding and spreading detected (arrow). (F) GMroX2-ΔDHS-50A with no binding and spreading at the transgene (arrow). (G) GMroX2-ΔDHS-23B shows extensive spreading in some nuclei (arrowhead). (H) GMroX2-DHS-mut-70B with limited spreading (arrowhead).

Figure 3

DHS directly regulates roX1 promoter activity. (A) Reporter constructs. ProX1 contains 750 bp sequences (gray) from the roX1-5′ region inserted upstream of a promoterless eGFP reporter (green). ProX1-DHS is the same as ProX1 except that the DHS (pink) is added downstream of the SV40 polyA site at the 3′ end of eGFP. The blue bar under the reporter construct represents the probe used in Northern experiments and the red bar indicates the amplified region in real-time PCR. The 750 bp roX1-5′ region contains three roX1 5′ ends mapped by RNase protection assay (see Supplementary information), indicated by arrows, two of which correspond to the start sites of roX1 cDNAs c3 and c20, respectively. (B) Northern blot of reporter RNA from transgenic males (m) or females (f). Lane 1: Hsp70-eGFP; lanes 2 and 3: promoterless eGFP; lanes 4 and 5: ProX1-1; lanes 6 and 7: ProX1-5; lanes 8 and 9: ProX1-DHS-1; lanes 10 and 11: ProX1-DHS-4. Note: The reporters express a roX1-eGFP fusion RNA that is slightly larger (see Supplementary information) than eGFP mRNA in lane 1. (C) Northern blot of reporter RNA from males (m) or females (f), in the presence (+) or absence (−) of an Hsp83-MSL2 transgene. Lanes 1–3: ProX1-4; lanes 4–6: ProX1-DHS-1; lanes 7–9: ProX1-DHS-5; hybridization for rp49 acts as the loading control. (D) Quantitative real-time RT–PCR of ProX1 and ProX1-DHS males (M), females (F) and females with ectopic expression of MSL2 (F+MSL2). For each sample, eGFP transcription has been normalized to the RNA level of the internal control gene pka. Relative eGFP transcription is presented as a ratio to the RNA level of the calibrator, which is the sample with the lowest expression level of eGFP. Here, ProX1-DHS-1 has the lowest expression and is chosen as the calibrator (designated as 1). For each sample, data represent the mean±s.d. from three independent experiments.

Figure 4

DHS-mediated regulation requires MSL proteins but not roX RNA. (A) Quantitative real-time RT–PCR of ProX1-DHS males (M) or females (F) in wild-type or different msl or roX1roX2 mutant backgrounds (as indicated at the bottom). Results are represented as in Figure 3D, with the transcription level of wild-type female designated as 1. (B) Left panel: in situ hybridization of roX1 RNA (red) in msl3 [Hsp83-MSL2] females. No roX1 RNA was detected on X. We also failed to detect transcription at the endogenous roX1 locus (3F), as indicated by the arrow. Right panel: in the presence of an Hsp83-roX1 transgene on an autosome, roX1 RNA was detected at many CESs on the X chromosome. (C) Polytene chromosomes from ProX1-DHS-1 stained with anti-MSL1 antibody (red). The top panel shows the male chromosomes in a wild-type background; the bottom panel shows the chromosomes from females with ectopic MSL2 protein but mutant for roX1 and roX2. The arrows indicate the transgene loci. X: X chromosome. DNA was stained with DAPI (blue) in (B, C).

Figure 5

The roX1 promoter is repressed in msl mutant males. Real-time RT–PCR of ProX1 or ProX1-DHS reporter transcription from mle (A) and msl3 (B) mutant male larvae. Results are represented as in Figures 3 and 4, with the transcription level of ProX1-DHS-1 and ProX1-DHS-6 designated as 1 in (A) and (B), respectively.

Figure 6

Model for regulation of roX1 transcription. See text for details.