MLE functions as a transcriptional regulator of the roX2 gene

(Invitrogen), and 50 fmol of annealed Prox probe. After 30 min incubation at 37 O C, reaction mixtures were resolved by electrophoresis for 90 min at 19 mA on a 4.5% polyacrylamide gel (30:1) containing 5% glycerol and 0.5x TBE. Complexes formed with Prox were visualized by autoradiography.


INTRODUCTION
In higher eukaryotes, females contain an extra X chromosome. The transcription activity of the X chromosome must be equalized between the sexes during development, and any failure in this process leads to embryonic lethality (1,2). Diverse mechanisms have evolved to reconcile the differences in X chromosome dosage. Eutherian mammals suppress the transcription activity of one of their two X chromosomes in females (3,4).
In humans and Drosophila, dosage compensation is achieved by opposite processes, i. e., by transcriptional repression and activation, respectively. Despite this difference, it is intriguing to find that both processes involve non-coding RNAs that associate with the X chromosome. In humans, XIST (X inactivation specific transcript), synthesized from the inactive X chromosome, physically coats the entire inactive X chromosome, conferring an inactive status (10)(11)(12). In contrast, in Drosophila, roX1 1 and roX2, two RNAs originating from the male X chromosome, associate with hundreds of sites on the X chromosome, making it transcriptionally hyperactive (13,14).
To date, genetic analysis has identified 8 transacting factors that are necessary for the onset or maintenance of dosage compensation in Drosophila. Based on observations that loss of their functional allele leads to a male-specific lethal phenotype, these factors 4 are named the MSL (male-specific lethal) proteins. They include MSL1 (15), MSL2 (16,17), MSL3 (18), MLE (maleless) (19), MOF (male-absent on the first) (20), and a more recently identified factor JIL-1 (21). It is generally believed that dosage compensation occurs in several discernible steps, starting with the targeted association of roX RNAs and MSL complex with the X chromosome. Upon the expression of MSL2 protein, MSL proteins assemble a complex on approximately 35 sites of the X chromosome, called "chromatin entry sites" (22)(23)(24). Once the chromatin entry sites are fully occupied with the MSL complex in the presence of MLE, the flanking chromatin region becomes competent in binding the MSL complex. This "spreading" or "nucleation" process appears to require the histone acetyltransferase (HAT) activity of MOF (25).
We previously reported that mle GET , a mutant MLE defective in ATPase activity, led to a poor association of the MSL complex with the male X chromosome and lethality of male embryos (26). Recently, it has been shown that the expression of mle GET in place of the wild type MLE resulted in the formation of the MSL complex devoid of roX2 RNA (25). Furthermore, Mof 1 , which lacks HAT activity, was unable to support the spreading of MSL complexes on the X chromosome and also drastically reduced the association of MLE with the X chromosome (25). However, lack of HAT activity did not influence the association of MSL complex with roX RNAs in nucleoplasm and with the chromatin entry sites. Although the underlying mechanism remains unknown, it is likely that MLE plays a critical role in determining the intracellular level of roX2 RNA or its interaction 7 using the standard calcium phosphate method. After 24 hr incubation, cells were supplied with fresh media containing 0.5 mM copper sulfate. On the following day, cells were harvested in ice-cold phosphate-buffered saline (1x PBS), and resuspended in 200 µl of a lysis buffer consisting of 50 mM potassium phosphate buffer, pH 7.4, 1% Triton X-100, 5 mM β-glycerophosphophate, and 2 mM DTT. Lysates were cleared by centrifugation for 15 min at 15,000 rpm at 4 o C. Luciferase and β-galactosidase activities were measured as described previously (27).  To confirm the above sequence information obtained from the fly genome database, we performed PCR using two primers, Prox2-5 and Prox2-3, and genomic DNA isolated from S2 cells as a template. PCR primers were designed to contain restriction enzyme cleavage sites, NheI and HindIII (Fig. 1B). The PCR product was subsequently treated with NheI and HindIII, re-isolated (Fig. 1C, lane 2), and inserted into pGL3-basic that was pre-cleaved with BamH1 and HindIII (Fig. 1C, lane 1). Three independent clones, containing 0.5 kb insert, were sequenced, which verified the 12 identified sequence presented in Fig. 1A, including the 54 bp repeat present within the upstream region of the roX2 gene. We named this newly identified 54 bp repeat "Prox".

Detection of a Prox binding activity in S2 cell extracts
The 54 bp repeat (Prox) present in the intergenic region upstream of the roX2 gene prompted us to determine whether it serves as a cis-acting element for transacting factors. We tested this possibility, employing electrophoresis mobility shift assay (EMSA) using whole cell extracts (WCE) prepared from S2 cells and 32 P-labeled Prox as probe. Indeed, WCE of S2 cells exhibited an activity to form stable complexes with 32 Plabeled Prox in the presence of 200-fold molar excess of non-specific dsDNA competitor ( Fig. 2A, lane 2). To further explore the specificity of Prox-protein complexes, we supplemented the EMSA reactions with 25-and 100-fold molar excess of dsDNA competitors, as shown in Fig. 2B, in addition to 250 ng of non-specific DNA. Drastic reduction in the complex formation was observed with Prox ( Fig. 2A, lanes 3 & 4). XorP, a 54 bp synthetic dsDNA containing reverse sequence of Prox, was less efficient in competing with 32  Employing M2-affinity chromatography, MSL1 was successfully isolated from whole cell extracts (Fig. 3B). An additional protein of about 55 kDa in size is believed to be a major degradation product of MSL1 since it cross-reacts with anti-MSL1 antibodies (data not shown). To compare their activity to interact with Prox, either MSL1 (120 ng) or MLE (65 ng) was incubated in reaction mixture containing 50 fmol (approximately 1.8 ng) of 32 P-labeled Prox and 250 ng of non-specific dsDNA in the presence or absence of ATP, and subsequently analyzed on native gel. As shown in Fig. 3C, MLE but not MSL1 exhibited an activity to form complexes with Prox in an ATP-independent manner. MSL1 did not react with Prox even in the absence of non-specific DNA competitor (data not shown). Our results indicate that at least MLE is capable of interacting with Prox in a sequence-specific fashion.

Interaction of Prox and roX2-DHS with MLE in vitro
It is generally accepted that MSL proteins assemble a complex on the male X chromosome in a sequence-specific manner, including approximately 35 sites of X chromosome, called "chromatin entry sites" (22)(23)(24). Treatment of total RNA with DNase I was essential for the reproducibility of semiquantitative RT-PCR containing a mixture of primers (data not shown). The two PCR products for roX2, obtained with pre-mRNA containing an intron and a mature mRNA (24), are presented as roX2-1 and roX2-2, respectively. PCR reactions were performed for 25 cycles using total RNAs, isolated from S2 cells expressing the indicated protein, as template in the presence of α-32 P-dCTP.
Aliquots (2 µl) were analyzed on 2% agarose gel. Subsequent to staining with ethidium bromide, PCR products were visualized by autoradiography (Fig. 5C) It is intriguing to note that the relative extent of increase in endogenous roX2 gene expression by MLE, MSL1, or MSL2 is proportional to that of luciferase expression driven by Prox (compare Fig. 5A with Fig. 5D). These results suggest that the activation of Prox-driven transcription in either a reporter construct or in a chromosomal context might be mechanistically similar and that MLE plays an active role in that process as a transacting factor specific for Prox.

DISCUSSION
Two X-linked genes, roX1 and roX2, encode non-coding RNAs (13,14). It is generally believed that roX1 and roX2 RNAs exert their function through the interaction with MLE and MSL proteins. Although earlier studies, employing embryos lacking roX1, roX2 or both genes, show that they are functionally redundant (14,33,34), at least two lines of evidence support their independent functions. First, in the absence of MSL3 protein, roX2 RNA, but not roX1, spreads to other sites on the X chromosome, a cytological indicator of normal dosage compensation (34). Second, in embryos, roX1 RNA is transcribed in both sexes in the absence of MSL2, whereas transcription of roX2 RNA is preceded by MSL2 expression in males and activated by exogenous expression of MSL2 in females (35). These observations suggest that the roX1 and roX2 genes are differentially regulated at the transcription level during development and that MSL proteins play active roles in transcription of the roX2 gene, but not the roX1 gene.
At present, the molecular mechanisms underlying roX gene transcription remain unknown. It has been shown that mutant embryos, lacking MLE, normally synthesize roX1 RNA, but roX1 RNA appears to be concentrated at its site of synthesis (35). Based on this observation, it has been proposed that MLE is required for the stability of roX1 RNA and its movement from the transcription site but not for its synthesis. MLE has also been implicated in stable maintenance of the steady-state level of roX2 RNA. Expression of mle GET in place of the wild type MLE results in drastic reduction in the roX2 RNA level and the formation of the MSL complex devoid of roX2 RNA (25). In addition to this post-transcriptional function, the present study suggests a direct involvement of MLE in transcription regulation of the roX2 gene. First, MLE interacts with the upstream promoter region (i. e, Prox2) of the roX2 gene through association with a 54 bp repeat, Prox. Second, over-expression of MLE activates transcription driven by Prox2 either in a reporter construct or in chromosomal context.
Our study shows that ATP is not essential for the interaction of MLE with Prox.
In addition, MLE ATPase activity is dispensable for transcriptional activation supported by Prox2 (data not shown). These results are consistent with the findings that MLE retains X chromosome binding ability in spite of various mutations introduced in the ATPase motifs (26,36) and that the ATPase activity is dispensable for transcriptional activation of the X-linked genes (37). Since mutations in the ATPase motifs of MLE affect the viability of male flies, the ATPase activity seems to be required for normal development of male flies (36). Then, by what mechanism does the ATPase activity of MLE influence dosage compensation? Based on poor binding of roX1 RNA to the X chromosome in flies expressing mle GET , Kuroda and coworkers have proposed that MLE ATPase activity plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes (36). In support of this hypothesis, a recent study has shown that in the absence of an ATP-dependent function of MLE, MSL complex can be assembled but are devoid of roX RNA (25). Thus, it is likely that in addition to transcriptional regulation by an ATP-independent function of MLE, roX2 RNA is post-transcriptionally regulated through association with MLS proteins, which require an ATP-dependent function of MLE. MOF, male-absent on the first; PBS, phosphate-buffered saline; Prox2, the upstream promoter of the roX2 gene; roX, RNA on the X.