EdU labeling To block replication, salivary glands from 3rd instar larvae were first incubated in 1 mM HU for 1 h. If not, they were simply incubated in S2 cell culture media (see below). After which, the glands were transferred and incubated in EdU for 15 minutes followed immediately by fixation. The detection was then performed as instructed by the Click-iT EdU imaging kit #C10337 (Invitrogen). Both HU and EdU were dissolved in S2 cell culture media before use. Co-labeling replicating DNA and nascent transcripts via EdU and BrUTP respectively Salivary glands were initially treated the same way as described above. However, after HU treatment, the glands were incubated in a BrUTP/DOTAP/EdU mixture instead for 15 mins. The BrUTP/DOTAP mixture was previously described to allow efficient nucleotide triphosphate uptake by the cell to label nascent transcripts [62]. EdU detection was performed as described above. Rat Anti-BrdU monoclonal antibodies #NB500-169 (Novus Biological) and Goat anti-rat Alexa 594 #A11007 (Invitrogen) were then used as primary and secondary antibodies respectively, to detect BrUTP. Cell culture and FACS Schneider 2 (S2) cells were cultured in 15-cm plates at a density of 16106 cells/ml in Schneider’s Media #11720034 (Invitrogen) supplemented with 10% FCS and penicillin/streptomycin. After reaching a density of 56106 cells/ml, the cells were re-suspended in new culture media containing 5 mM of Vybrant Dye Cycle Violet #V35003 (Invitrogen) to stain the DNA. The cells were then sorted into their respective phase of cell cycle via FACSAria II (BD Biosciences) at the Cytometry and Cell Sorting Facility located in Baylor College of Medicine. Real-time PCR Total RNA was extracted from cells using TrizolH reagent 15596-018 (Invitrogen) as per the manufacturer’s protocol. cDNA was synthesized from 0.5–1 mg of total RNA using random hexamers and MMLV Reverse transcriptase #M1701 (Promega) as per manufacturer’s protocol. The cDNA was purified using MinElute PCR kit #28004 (Qiagen). Real-time PCR was performed using an Applied Biosystems 7300 Sequence Detection system. The 25 ml PCR included 1 ml cDNA, 16 SYBRH Green PCR Master Mix #4309155 (Applied Biosystems) and 1 ml of gene specifc primers. The reactions were incubated in a 96-well optical plate at 95uC for 10 min, followed by 40 cycles of 95uC for 15 s and 60u for 10 min. The Ct data was determinate using default threshold settings. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. PKA is used as an endogenous standard for normalization of histone H2A and msl2. The primers used for qPCR are: H2A forward TGGACGTG- GAAAAGGTGGCA; H2A reverse ACGGCAGCTAGG- TAAACTGGAG; MSL2 forward GGCGAGTACCAGGGCTT- CAATATC; MSL2 Reverse TGCTGCAGCTGGACACG- AATAG; PKA forward – AGCCGCACTCGCGCTTCTAC and PKA reverse - AGCCGGAGAATCTGCTGATTG. Supporting Information Figure S1 The [H83-roX1D39] transgene turns on endogenous roX1 in males. (A) roX1 transcripts (Orange) and antisense probes (green). (B) Quantification of roX1 hybridization over polytene X chromosomes: entire X (black), only distal X (gray, example E), a Autoregulation of a Noncoding RNA Gene single band at the roX1 locus (hatched, example F red arrowhead), or no staining (white). N = nuclei counted. The [H83-D39roX1] transgene does not have an effect on females. (C) The internal probe does not hybridize to D39roX1 RNA, (D) but the roX1D39 probe does. White line delineates the X chromosome. (E) Males display a diverse pattern of roX painting, ranging from local spreading from the roX1 locus to (F) just a single band (indicated by the red arrow). (G) D39roX1 RNA (not visualized) helps [H83-M2] strongly activate endogenous roX1 (detected with internal probe) in almost all cells (two nuclei shown) (H) Similar roX1 staining was found in all squashed imaginal disc cells. (PDF) Figure S2 MSL immunostaining reveals that MSL2 binds indiscriminately to all the chromosomes in the absence of any roX RNA. (A) In the presence of roX RNA, MSL2 binds and paints the X chromosome only. (B) In the absence of roX RNA, the MSL proteins form incomplete complexes and binds to all the chromosomes, albeit poorly. (C) After treatment with MG132, a proteasome inhibitor, strong MSL2 binding can now be observed to occur throughout the nucleus. roX12 roX22 males are sick and do not survive till adulthood. However, sick and rare 3rd instar larvae can be obtained for salivary squashes although the chromosomes have extremely poor morphology and easily shattered during squashes. (D) The same experiment was repeated in whole mount salivary glands and MSL2 can be seen concentrated on the X chromosome. (E) At low resolution, MSL2 staining becomes undetectable in the absence of roX RNA (compared to B). (F) MSL2 can be seen binding to the entire nucleus when degradation is being inhibited. (PDF) Figure S3 MSL immunostaining of polytene chromosomes in [H83-M2] expressing larvae reveals mosaic establishment in DC. MSL immunostaining and DAPI is represented by red and blue respectively. (A) The MSL complex is bound along the male single X chromosome at hundreds of bands. (B) Due to the lack of MSL2, female do not have MSL binding to the X chromosome. (C–D) Painting of the X can be restored by the [H83-M2] transgene in msl2 mutant animals if both roX1 and roX2 are present. (E) The MSL complex fails to paint the entire X chromosome in males if roX2 is deleted. This cis-spreading phenomenon around the roX1 locus (indicated by red arrow) is similar to the autosomal spreading of roX1 transgene observed under low transcription rate (9). (F) In females, the X chromosome is either painted (arrowhead) or not painted (arrow). See Figure 1I for quantification. (G–H) Normal painting of the X is re- established when an endogenous copy of msl2 is restored. In females, this is achieved by co-expressing the [NOPU-M2] transgene. (I) The fraction of polytene nuclei displaying complete, partial, or no X MSL1 painting is shown. N = nuclei scored. Genotypes: (1–2) roX1+roX2+;msl2;[H83-M2]/+, (3–4) roX1+- roX22;msl2;[H83-M2]/+, (5) roX1+roX22/Y;msl2/+;[H83-M2]/+, (6) roX1+roX22;msl2/+;[NOPU-M2] [H83-M2]/+. (PDF) Figure S4 MSL2 immunostaining of imaginal disc cells in [H83- M2] expressing larvae reveals mosaic establishment of DC. (A–B) Painting in all nuclei is observed in animals relying upon the [H83- M2] transgene if both roX1 and roX2 are present. (C–D) Mosaic painting of the X chromosome is observed in both males and females when roX2 is deleted. White arrows indicated unpainted nucleus. (E–F) Normal painting of the X is re-established when an endogenous copy of msl2 is restored. In females, this is achieved by co-expressing the [NOPU-M2] transgene. (G) Cells with (black) and without (white) obvious subnuclear domain MSL2 staining taken PLoS Genetics | www.plosgenetics.org 12 March 2012 | Volume 8 | Issue 3 | e1002564