of whole animals. As previously reported, ectopic MSL2 produced by H83M2 is toxic to females (Figure 5A) [30] and toxicity requires roX RNA (Figure 5B) [17]. We found that roX2 alone was almost Figure 5. roX2 mutant females escape the toxic effects of H83M2. Adult females eclosing each day were counted from a population of eggs laid on day one. White bars show non-transgenic females without ectopic MSL2 (except E), and black bars show females carrying the H83M2 transgene. Each experiment varied the roX genotype. (A) roX1+ roX2+, (B) no roX, (C) roX2+ only, (D) roX1+ only, (E) roX1+ only. Cumulative viability of H83M2 females is shown as a percentage of their non-transgenic sisters. doi:10.1371/journal.pgen.1002564.g005 Autoregulation of a Noncoding RNA Gene equally toxic to females as when both RNAs were present (compare Figure 5A–5C). The surviving adults produced very few eggs and were sterile. However, roX1 alone was much less toxic to females (Figure 5D) as would be expected if many cells lacked dosage compensation and thus escaped the toxic effects of MSL2 production. These surviving females were fertile. We conclude that the mosaic dosage compensation pattern is not limited to polytene tissues, but instead reflects a widespread failure of MSL2 protein made by the constitutive H83M2 transgene to activate the endogenous roX1 gene. H83M2 does not turn on the expression of roX1 reliably Males that lack roX2, but are in all other ways wild type, have normal dosage compensation in all cells utilizing the remaining wild type roX1 gene [17]. Finding spotty roX1 activation only in animals relying on H83M2 prompted us to examine why the transgene did not behave like the endogenous msl2 locus in only this unusual genetic background. MSL2 made from the endoge- nous locus must activate roX1 RNA production more effectively than transgenic MSL2. This was shown by introducing single wild type allele of endogenous msl2 to roX2; H83M2 males and observing dosage compensation in all cells (Figure S3G and S3I lane 5, Figure S4E and S4G lane 5). Next, we turned to the NOPU- M2 transgene that also escapes SXL repression in females, but differs from H83M2 by using the endogenous msl2 promoter [21]. NOPU-M2 is not toxic to females because it makes less MSL2 compared to H83M2. However, addition of the NOPU-M2 transgene to roX2 mutant females makes them sensitive to the toxic effects of H83M2 (Figure 5E). Females carrying both msl2 transgenes also drive activation of roX1 expression in all cells (Figure S3H and S3I lane 6, Figure S4F and G lane 6). This is unlikely to be a simple additive effect of two pools of MSL2 protein since the H83M2 transgene itself was fully capable of driving roX1 expression in 100% of the cells when roX2 was present (Figure S3C–S3D, and S3I lane 1–2 and Figure S4A–S4B and S4G lane 1–2). We propose that H83M2 differs from the endogenous msl2 gene in some way needed to reliably drive the roX1 autoregulatory loop. The mosaic pattern might arise from the H83M2 transgene suffering from positional effect so MSL2 protein was not made in some cells. This is not the case since MSL2 expression can be clearly observed in all cells when both roX RNAs are present (Figure 4A). A second possibility is that H83M2 initiates expression too late in embryogenesis to capture the early MSL-independent roX1 transcripts we postulate are needed to begin the autoregu- latory loop. We performed both MSL2 immunostaining and roX1 FISH on roX2; msl2; H83M2 embryos from 2 h to 20 h AEL. These embryos showed H83M2 expression and subnuclear punctate roX1 accumulation in essentially all cells at early gastrulation and remained on through at least until 20 hrs AEL when cuticle formation prevents reliable staining of internal tissues (Figure S5 and Figure S6). These results suggest that H83M2 does come on during early embryogenesis and is effective in driving roX1 transcription during the rapid cell divisions of embryonic development. However, many cells subsequently lose roX1 transcription, and thus dosage compensation, later during larval stages. Cell cycle regulation of msl2 expression The MSL complex associates with many hundreds of actively transcribed genes along the male X. We considered the possibility that like core histone proteins, the MSL complex might need to abruptly increase its abundance following DNA replication. We set out to test if the transcription of msl2 might be coupled to S phase. PLoS Genetics | www.plosgenetics.org 7 March 2012 | Volume 8 | Issue 3 | e1002564