Transcription rate of noncoding roX1 RNA controls local spreading of the Drosophila MSL chromatin remodeling complex

Abstract

The dosage compensation complex in Drosophila is composed of at least five MSL proteins and two noncoding roX RNAs that bind hundreds of sites along the single male X chromosome. The roX RNAs are transcribed from X-linked genes and their RNA products "paint" the male X. The roX RNAs and bound MSL proteins can spread in cis from sites of roX transcription, but the mechanism controlling spreading is unknown. Here we find that cis spreading from autosomal roX1 transgenes is coupled to the level of roX transcription. Low to moderate transcription favors, and vigorous transcription abolishes local spreading. We constructed a roX1 minigene one third the size of wild type as a starting point for mutagenesis. This allowed us to test which evolutionarily conserved motifs were required for activity. One short repeat element shared between roX1 and roX2 was found to be particularly important. When all copies were deleted, the RNA was inactive and unstable, while extra copies seem to promote local spreading of the MSL complex from sites of roX1 synthesis. We propose that assembly of the MSL proteins onto the extreme 3' region of elongating roX1 transcripts determines whether the MSL complex spreads in cis.

Figure 1

Local spreading does not require sequences outside the roX1 transcription unit. A. The GMroX1 transgene carries 4.9 kb of genomic DNA including the native roX1 promoter. This transgene supports robust local MSL spreading when inserted on autosomes (Park et al. 2002). The H83roX1 transgene is a 3.4 kb cDNA transcribed by the hsp83 promoter (black box) (Stuckenholz et al. 2003). The UASroX1 transgene carries the same 3.4 kb roX1 cDNA transcribed from the UAS promoter (blue box). B. Polytene chromosomes from transgenic roX1 roX2 double mutant males were stained with anti-MSL1 antibodies (red) and DAPI (blue). The arrow indicates the location of the autosomal transgene and “X” indicates the X chromosome. Abundant roX1 transcription from H83roX1-87B restores MSL complex to the X chromosome and male viability, but does not support local MSL spreading around the autosomal transgenes (arrow). C. When the same roX1 cDNA is transcribed from UASroX1-29A, insufficient MSL complex is produce to paint the X chromosome leading to a collapsed morphology and weak MSL1 staining. However, small amount of MSL complex produce preferentially spreads > 1Mb along the autosome from the sites of roX1 transcription.

Figure 2

Vigorous roX1 transcription abolishes local MSL spreading. Polytene chromosomes from male larvae were stained with antibodies to MSL1 (red) and DAPI (blue). The roX genotype of the X chromosome is given at left, and arrows indicate the location of the roX1 transgene. A. Extensive local spreading is seen in roX1 roX2 /Y; [w⁺ UASroX1-66B]/+. Little MSL complex reaches the X chromosome. B. GAL4 induced roX1 transcription abolishes local spreading in roX1 roX2 /Y; [w⁺ UASroX1-66B] [w⁺ actin5C-GAL4]/+. All MSL complex is instead exported to the X. Note that no MSL complex is detectable at the autosomal roX1 transgene. C. Local MSL spreading is abolished by endogenous roX2⁺ transcription in y w roX1^ex6 /Y; [w⁺ UASroX1-66B]/+, but MSL complex is bound to the roX1 transgene under conditions of low transcription. D. y w roX1^ex6 /Y; [w⁺ UASroX1-66A] [w⁺ actin5C-GALl4]/+ shows that GAL4 induced roX1 transcription prevents MSL complex binding to transgene. E. Under low transcription conditions, paired polytene chromosomes have normal banding morphology around transgene in y w roX1^ex6 /Y; [w⁺ UASroX1-55C]/+. F. In y w roX1^ex6 /Y; [w⁺ UASroX1-55C] [w⁺ hs-GAL4]/+ males, GAL4 induces a new puff at the insertion site of the paternal transgenic homolog, but not on the snyapsed nontransgenic maternal homolog. G. w /Y; [w⁺ H83roX1-87B]/[w⁺ actin5C-GAL4] male showing that GAL4 does not inhibit MSL complex binding to a roX1 transgene lacking the UAS promoter. H. roX1 Northern. Total RNA from adults was hybridized with probes for roX1 and rp49 as a loading control. The genotypes of each sample are: Lane 1, wild type male; 2, wild type female; 3, roX1^ex6 male; lanes 4-9 are roX1^ex6 males carrying the UASroX1 transgene located at 55C (4 and 5), 28D (6 and 7), or 30C (8 and 9). The males in lanes 4, 6, and 8 lack GAL4 while the males in lanes 5, 7, and 9 express GAL4 from a weakly constitutive heat shock promoter. Lanes 1, 2, 3, 4, 6, and 8 were loaded with 10 μg total RNA. Lanes 5, 7, and 9 were loaded with 1 μg total RNA.

Figure 3

Autosomal MSL complex is lost soon after roX1 induction. A. A Northern of roX1 transcripts produced from UASroX1 following induction by heat inactivation of GAL80^ts. Lane 1, wild type male, Lane 2, wild type female, Lanes 3-10 y w roX1^ex6 males carrying no transgene (3), only the UASroX1 transgene (4), both the UASroX1 and actin5C-GAL4 transgenes (5), or the UASroX1, actin5C-GAL4, and GAL80^ts transgenes (6-10). These animals were shifted from 18° to 30° for the time (hrs) shown prior to isolation of RNA. B-E, polytene chromosome spreads of roX1 roX2 /Y; [w⁺ UASroX1-66B], [w⁺ actin5C-GAL4], and [w⁺ GAL80^ts]/+ males stained with antibodies to MSL1 (red). B. A male grown at 18° shows very little MSL staining on the X, but robust spreading around the autosomal UASroX1-66B transgene (arrow). C. After 3 hrs. at 30° the X begins to show moderate MSL painting while the staining around the UASroX transgene is still strong. D. By 6 hrs at 30° the X as acquired a nearly normal MSL binding pattern, but the staining at the transgene is weak or undetectable (arrow). E. After 14 hrs at 30° all nuclei have fully painted X chromosomes and no MSL bound near the UASroX1 transgene. F. A roX1 roX2 /Y; [w⁺ GMroX1-98A]/+ male shifted to 30° for 26 hours showing that extensive spreading around an autosomal roX transgene is resistant to temperature when transcribed from its native promoter.

Figure 4

Mutational analysis of conserved sequences at the end of roX1. A. A hypothetical RNA secondary structure of the 3′ end of roX1 based on sequences conserved across Drosophila species. B. The structure of the wild type 3.7 kb rox1 RNA is shown in light gray with the 5′ and 3′ segments retained in roX1Δ39 shown in black. C. The transgenes used to assay function of deletion mutants are indicated with the structural domains shown in A listed above the constructs. Each transgene was inserted at the same ϕC31 attP site (VK11 located at 40E), and all RNAs were transcribed from the Hsp83 promoter. The male rescue was calculated in three replicate crosses as the fraction of transgenic sons/transgenic daughters in a cross where the transgene provides the only source of roX RNA. The complete rescue data set is presented in Table S1. The extent of each deletion is indicated by the gaps bounded by ( ). The –RB123 mutant carries multiple nucleotide substitutions in RB1 and deletions of RB2 and RB3. The H83Δ39pseudo construct replaces the last 600 nt of melanogaster sequence with the corresponding region of D. pseudoobscura roX1 carrying six copies of RB. D. roX1 Northern with rp49 as a loading control. Lane 1, wild type male; 2, wild type female. Lanes 3-11 are in a y w roX1^ex6 background. Lane 3, nontransgenic male; lanes 4-6, three different random P inserted transgenic males carrying [roX1Δ39] at different locations; lane 7, a female carrying roX1Δ39; lanes 8-10, males carrying [roX1Δ39-3RB] at three different insertion sites; lane 11, a female carrying the same transgene. Lane 12, wild type male; lane13, roX1^ex6 male, lane 14, roX1^ex6 roX2⁵²/Y;[VK11 H83roX1Δ39]; lane 15, roX1^ex6 roX2⁵²/Y;[VK11 H83roX1Δ39pseudo]. E. Polytene chromosomes from a roX1 roX2/Y; [w⁺ H83roX1Δ39]/+ male showing that the 1.2 kb RNA supports normal MSL painting (red) along the X chromosome.

Figure 5

The roX1pseudoobscura minigene supports normal MSL painting along the male X and local spreading around the transgene insertion site. A. A roX1 roX2 /Y; [y⁺ attP VK11 w⁺ H83roX1Δ39p]/+ male displays a normal distribution of MSL1 (red) along the X when the only source of roX RNA is from the pseudoobscura hybrid minigene. B. A male carrying a similar transgene, roX1Δ39, except that the sequence is entirely derived from D. melanogaster. The view near the chromocenter (CC) shows the proximal regions of chromosome arms 2L and 2R with no MSL1 staining detectable at the location of the roX1Δ39 transgene (40E, arrow). C and D. When [H83roX1Δ39p] is assayed at the same integration site, a small cluster of MSL bands spreading from the site of roX transcription is visible. The arrow indicates the transgene site and the arrowhead an additional band. E. A second [H83roX1Δ39p] transgenic stock with an insertion at 48C (Arrow) again displaying local autosomal MSL spreading.

Figure 6

Model of cotranscriptional assembly of MSL complex. A. The MSL proteins appear to stimulate roX1 transcription in males by binding to the internal DHS element (Bai et al. 2004). Very high levels of transcription across the DHS displaces the MSL proteins, perhaps limiting the levels of roX1 RNA synthesis. Black rectangle=roX1 gene, gray ball=RNA polymerase. The boxed region is enlarged in B to show how the individual protein subunits; MSL1 (1), MSL2 (2), MSL3 (3), MOF (F), and the MLE helicase might recognize RNA elements emerging from RNA polymerase (RNP). MLE is the only helicase in Drosophila to contain two dsRNA binding motifs at its N-terminus which might recognized secondary structures found at the extreme 3′ end of roX1 transcripts. The success or failure of the protein subunits to assemble onto nascent transcripts while still tethered to the RNA polymerase is postulated to determine whether or not local spreading of the MSL complex occurs. Stem Loop 1 and 2 are drawn as base-paired regions, IRB is shown as a white rectangle, and the RB repeats are shown as three black rectangles.