A role for siRNA in X-chromosome dosage compensation in Drosophila melanogaster

Sex-chromosome dosage compensation requires selective identification of X chromatin. How this occurs is not fully understood. We show that small interfering RNA (siRNA) mutations enhance the lethality of Drosophila males deficient in X recognition and partially rescue females that inappropriately dosage-compensate. Our findings are consistent with a role for siRNA in selective recognition of X chromatin.     

Drosophila dosage compensation

Dosage compensation of X-linked genes is a phenomenon of concerted, chromosome-wide regulation of gene expression underpinned by sustained and tightly regulated histone modifications and chromatin remodeling, coupled with constrains of nuclear architecture. This elaborate process allows the accomplishment of regulated expression of genes on the single male X chromosome to levels comparable to those expressed from the two X chromosomes in females. The ribonucleoprotein Male Specific Lethal (MSL) complex is enriched on the male X chromosome and is intricately involved in this process in Drosophila melanogaster. In this review we discuss the recent advances that highlight the complexity lying behind regulation of gene expression by just two-fold.     

RNA synthesis in Drosophila melanogaster polytene chromosomes indications of simultaneous dosage compensation and dosage effect in X chromosomes

It is shown that the apparent incompleteness of dosage compensation when RNA synthesis is measured autoradiographically is not due to the existence of contiguous dosage compensated and non-dosage compensated genes. Rather this seems to be the result of peculiarities in the coordination of RNA synthesis between the X chromosomes and autosomes. The slope of the line defined by \([\bar X]_i \) and \([\overline {2R} ]_i \) (number of grains over the X and autosomal segments averaged over the different nuclei assayed in each gland) is indistinguishable in males and females (apparent complete dosage compensation). An average of the slopes obtained for different individual glands (from [X] and [2R], the grain counts over each nucleus belonging to a particular gland), on the other hand, has a value in males which is approximately half of the value attained by females (a value of one half, in males, indicates dosage effect since males have one X and females have two).     

Study of dosage compensation in Drosophila

Using a sensitive RT-QPCR assay, we analyzed the regulatory effects of sex and different dosage compensation mutations in Drosophila. To validate the assay, we showed that regulation for several genes indeed varied with the number of functional copies of that gene. We then confirmed that dosage compensation occurred for most genes we examined in male and female flies. Finally, we examined the effects on regulation of several genes in the MSL pathway, presumed to be involved in sex-dependent determination of regulation. Rather than seeing global alterations of either X chromosomal or autosomal genes, regulation of genes on either the X chromosome or the autosomes could be elevated, depressed, or unaltered between sexes in unpredictable ways for the various MSL mutations. Relative dosage for a given gene between the sexes could vary at different developmental times. Autosomal genes often showed deranged regulatory levels, indicating they were in pathways perturbed by X chromosomal changes. As exemplified by the BR-C locus and its dependent Sgs genes, multiple genes in a given pathway could exhibit coordinate regulatory modulation. The variegated pattern shown for expression of both X chromosomal and autosomal loci underscores the complexity of gene expression so that the phenotype of MSL mutations does not reflect only simple perturbations of genes on the X chromosome.     

Chromosomal basis of dosage compensation in Drosophila

3H-thymidine and 3H-uridine labeling patterns of the X-chromosome arms of Drosophila pseudoobscura have been examined autoradiographically. Results show that in all phases of replication, namely, initial, middle and terminal, both arms of the X-chromosome in the male are advanced by one or two steps of 3H-thymidine labeling in comparison with the autosomes, and both arms in the female show more or less similar labeling profile as the autosomes. Both the arms in the male show pale stainability and enlarged width ratio, as reported in other species. The 3H-uridine labeling patterns also reveal that both arms in the male incorporate twice as much precursor as the individual X in the female. Results, therefore, suggest that both arms of the X in D. pseudoobscura are faster replicating and hyperactive in the male, although it is considered that XL is homologous to the X and XR to part of the third chromosome of D. melanogaster.     

Chromosomal basis of dosage compensation in Drosophila

A critical analysis of the puffing activity and transcribing activity patterns of different sites of the X-chromosome of the male and female larval salivary glands of Drosophila hydei has been presented. The results show that within the limitations of the resolving power of the technique and variability inherent in the general chromosomal conditions the puffing activities of the different sites of the X-chromosome are very much alike in the two sexes. Of the 15 puffing sites in the X-chromosome, most of the sites either show good concordance in the two sexes or resemble in their highest class value. Only 4 sites (4CD, 8A, 16C and 20B) show considerable discordance in the activity pattern between male and female. Incorporation of ³H-uridine in the X-chromosome also reveals that there is indeed a reasonable degree of superimposition of the number of silver grains in the X-chromosomal puffs of the two sexes. Whatever disparity that exists between the grain numbers in the two sexes can be explained on the basis of sister-class compensation. These results have been interpreted as evidence in support of the piece-meal mechanism of dosage compensation in Drosophila, operating through hyperactivation in the male.This work has been supported by a grant (No. 10/14/66 G) from the Atomic Energy Establishment, Govt. of India to A.S.M. and a Senior Research Fellowship from the Bhabha Atomic Research Centre, Govt. of India to S.N.C.     

Sex determination and dosage compensation in Drosophila melanogaster: production of male clones in XX females

Sex determination and dosage compensation in Drosophila are implemented by the ratio of X-chromosomes to sets of autosomes (X:A ratio). Our aim was to change this X:A ratio during development, and to assess the response of the affected cells in sexually dimorphic structures. For this purpose, clones of XO constitution were produced in female embryos and larvae of two genotypes in which almost the entire euchromatic arm of one X-chromosome was translocated to the third chromosome. Genotype I was heterozygous for the X-linked recessive mutations Sxl^fLS, genotype II was homozygous for Sxl⁺. The Sxl⁺ gene (sex-lethal) is involved in mediating sex determination and dosage compensation. In genotype I (Sxl^fLS), male clones could be generated up to 48 h in genitalia and analia, up to 72 h in the sex comb region and up to 96 h in 5th and 6th tergites. In genotype II (Sxl⁺), male clones only appeared in the tergites, and only up to 24 h. The difference in these results is ascribed to the presence of Sxl^fLS in genotype I: when homozygous, this mutation causes XX clones to differentiate male structures; most of the male clones produced in genotype I must therefore be XX. In contrast, male clones produced in genotype II must be XO. Since these were only found when generated in embryos we conclude that the X:A ratio expresses itself autonomously in clones by setting the state of activity of the Sxl gene around blastoderm stage. Once this is achieved, the X:A signal is no longer needed, and the state of activity of the Sxl⁺ gene determines sex and dosage compensation.