Regional control of chromatin organization by noncoding roX RNAs and the NURF remodeling complex in Drosophila melanogaster

Abstract

Dosage compensation in Drosophila is mediated by a histone-modifying complex that upregulates transcription of genes on the single male X chromosome. The male-specific lethal (MSL) complex contains at least five proteins and two noncoding roX (RNA on X) RNAs. The mechanism by which the MSL complex targets the X chromosome is not understood. Here we use a sensitized system to examine the function of roX genes on the X chromosome. In mutants that lack the NURF nucleosome remodeling complex, the male polytene X chromosome is severely distorted, appearing decondensed. This aberrant morphology is dependent on the MSL complex. Strikingly, roX mutations suppress the Nurf mutant phenotype regionally on the male X chromosome. Furthermore, a roX transgene induces disruption of local flanking autosomal chromatin in Nurf mutants. Taken together, these results demonstrate the potent capability of roX genes to organize large chromatin domains in cis, on the X chromosome. In addition to interacting functions at the level of chromosome morphology, we also find that NURF complex and MSL proteins have opposing effects on roX RNA transcription. Together, these results demonstrate the importance of a local balance between modifying activities that promote and antagonize chromatin compaction within defined chromatin domains in higher organisms.

Figure 1.—

An allelic series of Nurf301 mutants display regional puffing on the male X chromosome. (A) Top: domain structure of NURF301 protein. Bottom: wild-type male X chromosome stained by anti-MSL2 antibody (green). DNA is counterstained by DAPI (purple). The approximate cytological positions of roX1 and roX2 are indicated by arrows. (B) Severely truncated alleles of Nurf301 (top) dramatically disrupt the morphology of the male X chromosome (bottom). (C) Overall X morphology is retained in the two hypomorphic Nurf301 mutant males, although regional puffing is observed (arrowheads). (D) Higher magnification images of hypomorphic Nurf301alleles highlighting regional puffing.

Figure 2.—

roX mutations result in a regional reversal of the Nurf mutant phenotype on the male X chromosome. Male polytene chromosomes were stained with anti-MSL1 antibody (red) and counterstained with DAPI (blue). (A) In Nurf301 mutant males, deletion of roX2 strongly reduces chromosome puffing of a large portion of the proximal X, including around the roX2 locus. (B) Similarly, puffing of the distal X including the roX1 locus is lost when roX1 is deleted. The genotypes are (A) yw Df (1) roX2⁵² [w⁺4Δ4.3]; nurf301³ and (B) yw roX1^ex6; nurf301³.

Figure 3.—

Chromosomal decondensation induced by a GMroX2 transgene in nurf301 mutant males. In the absence of X-linked roX genes, local spreading is seen at the GMroX2 autosomal insertion sites at cytological positions 50B and 34A (A and C, respectively, arrows). Loss of NURF function causes local chromosomal puffing at these same transgene insertion sites (B and D, arrows), and X-chromosome puffing is less severe. Male polytene chromosomes were stained with anti-MSL1 antibody (red) and counterstained with DAPI (blue). The genotype of the double mutant X chromosome is yw roX1^ex6 Df (1) roX2⁵² [w⁺4Δ4.3].

Figure 4.—

NURF represses transcription of roX genes in females. (A) Schematic of roX gene reporter constructs with and without the DHS. Real-time RT–PCR was used to measure the transcription from either the roX reporters (B and C) or the endogenous roX genes (D and E) in Nurf301 mutants. RNA levels of three other X-linked genes were not affected in Nurf mutants (F). For each sample, data represents the mean ± SD from three independent experiments.

Figure 5.—

ChIP using antibodies against the ISWI subunit of NURF indicates that NURF binds to the DHS of roX1. (A) Organization of the roX1 locus showing primer pairs used in ChIP and location of the MSL-binding DHS. (B) Comparison of ISWI ChIP signals in wild type and Nurf301² mutant tissue reveals that NURF binds to the DHS (primer pair 2), but is not localized to the promoter (primer pair 1) of roX1. (C) ISWI ChIP was performed in sorted samples of male and female salivary glands and demonstrates that NURF is recruited to the roX1 DHS in both males and females. (D) Organization of the roX2 locus showing primer pairs used in ChIP and location of the MSL-binding DHS. (E) NURF is not detected at the roX2 DHS or on the body of the gene. B, C, and E show representative ChIP PCR data. Graphs show the fold enrichment of ChIP signals relative to the input and are derived from the ratio of ChIP to input band signal intensities averaged over three separate determinations. Input and IP samples were titrated and PCR cycle number was optimized to ensure that PCR products quantified using a PhosphorImager were in the linear range.