Regulation of roX2 While we did not specifically address transcriptional control of roX2, it must differ from roX1 in several important ways. Meller has previously shown that roX2 transcription lags roX1 by a few hours during early embryonic development and is always limited to males [16]. Here we found that roX2 transcription differs by not requiring pre-existing roX RNA and can be switched on several days later during larval development simply by ectopically expressing MSL2. MSL2 protein made by the constitutive H83M2 transgene only sporadically activates roX1, but robustly drives roX2. The roX2 gene also carries a DHS enhancer similar to that found in roX1 [45], but if it plays a comparable role in roX2 regulation, it presumably would not require complete MSL complex. The region near the proline rich domain towards the C-terminus of MSL2 is essential for this regulation [31]. Parallels between roX1 and Sxl The roX1 autoregulation loop described above shares parallels to the SXL autoregulatory loop controlling all aspects of sex determination and dosage compensation in Drosophila. X:A counting elements act upon an early establishment Sxl promoter to make SXL in early female embryos. These first SXL proteins stimulate productive splicing of Sxl mRNAs transcribed from a distinct maintenance promoter ensuring further SXL production [56]. MSL1, MSL3, MOF, and MLE are unable to package early roX1 RNA made from the embryonic promoter in female embryos to form a fully functional mature MSL complex. To do that, MSL2 protein made only in males is required. We suspect these earliest MSL complexes sustain roX1 transcription as embryos switch to the MSL- dependent promoters. Recently a new role for MSL2 in females has been described during the brief window when Sxl autoregulation is established [57]. Perhaps females also fleetingly utilize the early burst of roX1 before abundant SXL represses msl2 translation. Thousands of large noncoding RNAs have recently been discovered in vertebrates [2,58], many of which are associated with chromatin remodeling enzymes [1,59–61]. It is likely that some of these will face similar regulatory and functional demands as the roX RNAs and may have evolved comparable strategies to control their production. For instance, the short RepA sequence at the 59 end of mammalian Xist RNA may influence production of full length transcripts [59]. Materials and Methods Drosophila stocks Larvae and flies were raised on standard cornmeal-yeast-agar- molasses medium containing propanoic acid at 25uC. In all experiments the roX1 mutation is roX1ex6 and the roX2 allele is Df (1) roX252 [17]. The [w+ 4D4.3] transgene supplies essential adjacent genes lost in the roX2 deletion. The transgenic flies used have been previously described as follows: [w+H83M2-6I] [30], [w+ NOPU-M2] [21], and [w+ H83roX1- D39] [13]. The H83M2 transgene was recombined with [P{SUPor- P}KG02776] which contains y+ as a marker so larvae of the correct genotype could be recognized by mouth hook color (Flybase). The P{GAL4-Act5C(FRT.CD2).P}, P{UAS-GFP.S65T} and MKRS-hsFLP transgenic fly stocks were provided by Graeme Mardon. The UASp-MSL2 transgene was made by digesting the H83M2 transgene with EcoRI and subcloning the MSL2 ORF into the pUAS-P2 plamid vector (a gift from Pernille Rørth). Heatshock The female larvae for the flp-out experiment were kept at 25uC and heatshocked at 37uC for 1 hr each at day 4 and 5 AEL. Their Autoregulation of a Noncoding RNA Gene salivary glands were then dissected on day 6 AEL, fixed and immunostained as described below. The full genotypes of the larvae heatshocked in Figure 1 are as follows: B-F) y w; [w+ y+Act-FRT-CDC2-FRT-Gal4] [w+UAS-GFP] +/+ + [w+UASp-MSL2]; MRKS [Hs-Flp]/+ H-L) y w roX252 [w+4D4.3]; [w+ y+Act-FRT-CDC2-FRT-Gal4] [w+UAS-GFP] +/+ + [w+UASp-MSL2]; MRKS [Hs-flp]/+ The full genotypes of the larvae in Figure 2 are identical to Figure 1H–1L. The full genotypes of the larvae in Figure 3 are as follows: A) y w roX252 [w+4D4.3]; msl2; [w+H83-M2] [w+y+P{SUPor- P}KG02776]/+ + B) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+y+P{SUPor- P}KG02776]/+ + D) y w/Y E) y w F) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+y+ P{SUPor- P}KG02776]/+ + G) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+H83- roX1D39] [w+y+ P{SUPor-P}KG02776]/+ + + H) y w roX1ex6roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+H83- roX1D39] [w+y+ P{SUPor-P}KG02776]/+ + + The full genotypes of the larvae in Figure 4 are as follows: A-A0) y w; msl2; [w+H83-M2] [w+y+ P{SUPor-P}KG02776] B-B0) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+y+P{SU- Por-P}KG02776]/+ + C-C0) y w roX1ex6 roX252 [w+4D4.3]/Y; msl2/+; [w+H83-M2] [w+y+P{SUPor-P}KG02776]/+ + D-D0) Same as C The full genotypes of the parents in the crosses for Figure 5A– 5E are as follows: A) y w/Y; msl2; [w+H83-M2] [w+y+ P{SUPor-P}KG02776]/+ + X y w; msl2 B) y w roX1ex6/Y; msl2; [w+H83-M2] [w+y+ P{SUPor- P}KG02776]/+ + X y w roX1ex6; msl2 C) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+y+ P{SUPor- P}KG02776]/+ + X y w roX252 [w+4D4.3]; msl2 D) y w roX1ex6 roX252 [w+4D4.3]/Y; msl2/+; [w+H83-M2] [w+y+ P{SUPor-P}KG02776] +/+ + [w+GMroX1+] X y w roX1ex6 roX252 [w+4D4.3] E) y w roX252 [w+4D4.3]/Y; msl2; [w+H83-M2] [w+y+ P{SUPor- P}KG02776]/+ + X y w roX252 [w+4D4.3]; [w+NOPU-M2] Immunohistochemistry and in situ hybridization Immunostaining and in situ hybridization on third instar salivary glands was performed as described in [11] except for the following modification. The slides were treated with proteinase K (20 mg/ml in PBT) for 3 min. Each slide was hybridized with 5 ng of roX1, msl2 or H2A biotin-labeled single-stranded antisense riboprobes using the T7 high yield Transcription kit #K0441 (Fermentas). Fluorescent development was done as instructed by the Tyramide Signal Amplification (TSA) kit (NEL700A) (PerkinElmer) with SA- FITC (NEL720) (PerkinElmer) or SA-Texas Red (NEL 721) (PerkinElmer). For double ISH, we labeled the second antisense riboprobes with digoxygenin. Before adding the anti-digoxygenin- HRP conjugate, the first horseradish peroxidase conjugate was quenched in 3% H2O2 for 30 mins. The second color is then developed using Tyramide-Alexa488 (T20922) (Invitrogen). To inhibit MSL2 degradation, we incubate imaginal discs and salivary glands with 10 mM of MG132 (474791) (Calbiochem) dissolved in Schneider Culture Media for 3 hours before proceeding with immunostaining. PLoS Genetics | www.plosgenetics.org 11 March 2012 | Volume 8 | Issue 3 | e1002564