Autoregulation of a Noncoding RNA Gene with (right table, N = 170) HU treatment were scored for expression of endogenous msl2 and EdU incorporation. Nascent transcripts were detected at the H83M2 transgene in 100% of the nuclei (data not represented in the table). After sorting growing S2 cells into their respective phase of cell cycle via FACS, qPCR was done to quantify H2A (E) and msl2 (F) transcripts levels normalized to PKA. (G) The FACS profile of unsorted (U) and sorted S2 cells (G1, S and G2 cell cycle). The sorted cells have a slightly higher content of Vybrant Violet-A dye because the cells are collected into tubes containing Vybrant Violet-A. The Y-axes are drawn on different scales. doi:10.1371/journal.pgen.1002564.g006 ,200 bp male-specific DNase I hypersensitive site (DHS) found about 1.5 kb downstream of the adult roX1 promoters. The DHS is sufficient to recruit the MSL complex to ectopic sites when moved to the autosomes in a sequence specific manner [45,46]. While partial complexes lacking MOF or MSL3 remain bound to the DHS, incomplete complexes lacking either roX RNA or the MLE RNA helicase postulated to fold roX RNA bind the DHS poorly [45,46]. This element stimulates roX1 transcription when MSL complex is bound and represses basal transcription when MSL complex is absent. Also, deleting the DHS greatly reduces transcription of roX1 transgenes [33]. Together, these findings support a model where transcription from the roX1 gene requires pre-existing roX RNA within MSL complexes bound to the internal DHS enhancer. However, this view is likely an oversimplification because very large internal deletions such as roX1ex7B, comparable to our H83roX1-D39 transgene (Figure 3A), remain transcriptionally active despite the loss of the DHS enhancer [32,47]. Cell cycle regulation Translation of msl2 mRNA is normally subject to elaborate controls acting through the 59 and 39 UTRs [48–52]. Little attention has been given to its transcription control, although recently anti-MSL2 antibodies were found to precipitate msl2 mRNA [53]. We found that msl2 transcription is associated with replication, and this is likely important for its normal control. Without MSL2 protein, naked roX1 RNA is rapidly destroyed [11]. Here we found the converse; cells rapidly clear any MSL2 protein not bound to roX RNA. When free MSL2 subunits are artificially stabilized with proteosome inhibitors, they coat all the chromosomes indiscriminately. This implies that the synthesis of MSL2 and roX RNAs are closely coordinated so each component stabilizes the other ensuring that only correctly targeted molecules survive. Although MSL2 lacks known RNA binding motifs, previous work of others is consistent with an intimate interaction between MSL2 and roX RNA [31]. We suspect the loss of replication-coupled transcription may contribute to the failure of dosage compensation in some cells relying exclusively on H83M2 for MSL2 protein and roX1 for roX RNA. This defect is corrected when both roX1 and MSL2 are coordinately made from the same hsp83 promoter (Figure 3G). Cells in mosaic animals lose dosage compensation sometime between the end of embryogenesis and third instar larvae. Many tissues undergo significant changes in cell cycle near the end of embryogenesis, particularly the introduction of G1 [54], and we suspect this shift contributes to loss of dosage compensation in our mosaic animals. If MSL complex should ever drop below the level needed to sustain the autoregulatory roX1 loop, it could never recover regardless of later MSL2 production. The details remain unclear because H83M2 is vigorously transcribed at the developmental times we examined, including S phase. We do not know whether the regulatory msl2 UTR sequences removed from H83M2 disrupt additional posttranscrip- tional controls that might promote efficient translation during replication. A second issue is that the X is painted with MSL complex throughout the cell cycle. This might drive continuous rather than cyclic roX1 synthesis. While we were unable to directly determine if roX1 transcription is cell cycle regulated, we note that the replication machinery components ORC2 and MCM are bound specifically to the roX1 DHS enhancer only in male cells (Figure S8A). The significance of this is not known, but it is tempting to speculate that components of the pre-initiation complex bound to DHS compete with MSL complex thus inhibiting roX1 transcrip- tion in G1. Firing of the replication origins removes ORC2 and MCM possibly allowing MSL complex access to the DHS and so switch on transcription shortly after the onset of S phase. The replication machinery is commonly found near the promoters of many genes [55], including msl2 (Figure S8B), so further experiments will be needed to determine if such binding actually plays any regulatory role here. Figure 7. Autoregulation model. The earliest roX1 transcripts (red) made at blastoderm originate from an uncharacterized MSL-independent promoter. This RNA may assemble with MSL protein subunits to produce the first functional MSL complexes needed to bind the internal DHS enhancer that drives sustained transcription (blue) from the male-specific promoters. When present, roX2 RNA can also drive roX1 transcription. Components of the replication pre-initiation complex also bind the DHS sequence in male cells (Figure S8A). The msl2 transcripts are made predominantly during replication and new MSL2 protein is needed to assemble and stabilize newly made roX1 RNA. doi:10.1371/journal.pgen.1002564.g007 PLoS Genetics | www.plosgenetics.org 10 March 2012 | Volume 8 | Issue 3 | e1002564