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miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells

miR-206 integrates multiple components of differentiation pathways to control the transition from... Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state. Methods: Potential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results: Modulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions: These findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation). Keywords: Rhabdomyosarcoma, RUNX1, ZNF238, miR-206, MSC, myogenesis Background differentiation [6]. However, in RMS the ability of MyoD Rhabdomyosarcoma (RMS) is a soft tissue sarcoma char- to induce differentiation is impaired [7]. acterized by expression of myogenic regulatory factors, Our recent study indicated that rhabdomyosarcomas especially MyoD, and other skeletal muscle genes [1-3]. represent an arrested progress through a normal transi- MyoD is capable of converting multiple cell types into tional state that is regulated by the formation of hetero- terminally differentiated skeletal muscle [4,5] and nor- dimers between MyoD and the full-length E-proteins mally acts as a nodal point in differentiation to integrate [8]. MyoD binds DNA as a heterodimer with an E-pro- multiple signals to balance regulative growth and cell tein (E2A, E2-2, or HEB) [9]. Normal myoblasts and RMS express a transcriptionally less active splice form of E2A as well as the bHLH (basic helix-loop-helix) proteins ID and Musculin (MSC), both of which * Correspondence: stapscot@fhcrc.org compete with the full-length E-proteins for heterodi- Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 merization with MyoD. The demonstration that a Fairview Ave N, C3-168, Seattle, WA 98109, USA Department of Neurology, University of Washington, Seattle, WA 98105, USA forced heterodimer of MyoD and a full-length E-protein Full list of author information is available at the end of the article © 2012 MacQuarrie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 2 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 suppressed multiple inhibitory mechanisms and all conditions. Transient transfections of pre-microRNA induced differentiation in the RD and other rhabdo- constructs were performed using 25 μMofpre-miR con- myosarcomas suggested that a central integrating structs (Ambion, Grand Island, USA) and siPORT NeoFX mechanism might regulate the switch from regulative (Ambion) according to the manufacturer’sdirections. growth to differentiation [8]. If a central integrating mechanism does exist, then mul- qPCR and RT-PCR tiple pathways should regulate its activity and multiple fac- All qPCR was performed using SybrGreen from Bio-Rad tors should be able to induce differentiation in RMS. In this on an Applied Biosystems 7900HT. Relative expression manuscript we demonstrate that modulation of multiple levels were calculated using cDNA dilution standard different myogenic factors can induce differentiation in curves or delta-delta Ct calculations. All values are rhabdomyosarcoma cells and that their activity is inte- reported as the mean ± SEM of at least three independent grated, at least in part, through the activation of miR-206, biological experiments and significance tested with t-tests. which acts as a genetic switch to transition the cell from a All RT-PCRs were performed simultaneously with minus proliferative growth phase to differentiation. These findings reverse transcriptase controls to check the absence of sig- suggest that multiple components of differentiation path- nal. Primers used for amplification are listed in (Additional ways that converge on miR-206 might be targeted for dif- file 1: Table S1). ferentiation therapies in at least some rhabdomyosarcomas. Methods microRNA microarrays Plasmid construction RNA was isolated using acid-phenol purification from Coding sequences of RUNX1 and ZNF238 were ampli- two biologically independent sets of RD cells transduced fied by PCR from human myotube cDNA, and cloned with either MD ~ E or empty vector pCLBabe retro- into pRRLSIN.cPPT.PGK/GFP.WPRE, pCLBabe, and viruses and differentiated for 24 h after puromycin selec- pCS2. The miR-206 lentivirus was purchased from Open tion. miRNAs were labeled using Exiqon’s miRCURY Biosystems. Lentiviral supernatant was produced by the labeling kit, and then competitively hybridized to in- FHCRC core viral facility, and viruses from pCLBabe house spotted miRNA arrays (FHCRC core facility). Cut- plasmids packaged using BBS-mediated calcium precipi- offs for significant changes were a FDR <0.05 and a tation into Phoenix cells. MD ~ E2/5 was cloned into the fold-change >2. Data are available under GEO accession pCLBabe backbone. For the miR-206 promoter lucifer- number GSE35921. ase reporter, a piece of approximately 2.5 kb of DNA up- stream of human miR-206 was amplified using primers microRNA northerns located in (Additional file 1: Table S1). microRNA northern blots were performed as described previously [10]. Probe sequences used are listed in (Add- Cell culture, transient transfections, and luciferase assays itional file 1: Table S1). RD cells were obtained from ATCC (American Type Cul- ture Collection) in approximately 1990, and all analyses have been performed on cells that originated from low pas- Expression microarrays sage number frozen aliquots. RhJT cells were obtained from RNA was isolated using the RNeasy mini kit (Qiagen) PJ Houghton in 1990 and, as with RD cells, all experiments from RD cells infected with either RUNX1-, ZNF238-, have used cells from original low passage number frozen miR-206-, or GFP-expressing lentiviruses and allowed to cells. RD and RhJT cells were maintained in DMEM with differentiate for 72 h. Each condition was performed 10% bovine calf serum and 1% Pen-Strep (Gibco, Grand Is- with three independent biological replicates. RNA was land, USA). Low-serum differentiation media consisted of hybridized to Illumina Human HT-12 v4 BeadChips. DMEM with 1% horse serum, 1% Pen-Strep and 10 μg/mL Analysis was performed in R/Bioconductor using the insulin and transferrin. Transient transfections for lucifer- lumi and limma packages with annotations found in the ase assays were performed using Superfect according to lumiHumanAll.db package. P values were adjusted to ac- manufacturer’s directions with a total of 3 ug of plasmid count for multiple testing using Benjamini and Hochberg’s DNA in each well (Qiagen, Valencia, USA). Luciferase method, and cutoffs for significant changes were a FDR assays used the Dual-Luciferase Assay kit (Promega, Madi- <0.05 and a fold-change >2. GO category enrichment son, USA) according to manufacturer’s directions. All tests were performed using the conditional algorithm of results were corrected to co-transfected Renilla-pCS2 and the GOstats package and a gene ‘universe’ of any gene are reported as the mean ± SEM of at least three independ- with a GO annotation that was called as ‘present’ in at ent experiments, with significance calculated using a t-test, least one of the 12 array datasets. Data are available and each experiment having three biological replicates of under GEO accession number GSE35921. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 3 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 ChIPs (Chromatin immunoprecipitations) confirmed that both RUNX1 and ZNF238 increased with ChIPs were performed as has been described previously differentiation induced in the RD RMS line by the forced [11]. Antibodies used were as follows: RUNX1 (Abcam, MD ~ E dimer and that myotubes formed from MyoD- ab23980), MyoD [12], MSC (Santa Cruz, sc-9556X), induced myogenesis in normal fibroblasts had higher ex- Acetylated Histone H4 (Upstate 06-866). Primers used pression levels of both factors compared to RD cells for amplification are listed in (Additional file 1: Table S1). (Figure 1A and 1B). Therefore, we hypothesized that RUNX1 and/or ZNF238 might cooperate with MyoD to EdU and BrdU labeling, western blots, and cell stains drive muscle gene expression and tested each factor for After 24 h in low-serum differentiation media, cells were its ability to induce differentiation in RD cells. shifted to differentiation media supplemented with EdU Lentiviral-mediated expression of each factor in RD at a final concentration of 50 μM (Invitrogen) and incu- cells (Additional file 2: Figure S1) induced muscle differ- bated for a further 24 h. Cells were then fixed and entiation, as measured by morphology and expression of stained according to the manufacturer’s protocols using myosin heavy chain (MHC) (Figure 1C), muscle-specific the Click-iT kit, and total nuclei and EdU positive nuclei creatine kinase (CKM) (Figure 1D), and withdrawal from counted by hand. the cell cycle based on EdU incorporation (Figure 1E). RD cells were labeled with BrdU at a final concentra- As in normal myogenesis, expression of ZNF238 tion of 30 μg/mL in differentiation media for 24 h. Cells decreased both ID2 and ID3 (Figure 1F). This differenti- were treated with hydrochloric acid before incubation ation is not restricted to the embryonal RMS RD cell with anti-BrdU antibody (Invitrogen A21300), and fluor- line; the RhJT alveolar line expressing RUNX1 showed escent secondary antibody (Jackson Immunoresearch). similar results (Additional file 3: Figure S2). Nuclei were detected with DAPI, and cells counted by hand. miR-206 expression correlates with factor-induced Western blots were performed on whole cell differentiation in RMS cells lysates collected in Laemelli buffer containing 10% We previously demonstrated that the expression of the beta-mercaptoethanol. All blots were blocked in 3% milk MD ~ E dimer in RD cells down-regulated multiple myo- (w/v) in 0.5% Tween-20-containing PBS before incubation genic inhibitors [8], consistent with induction of a with primary antibody (MHC: MF-20, MyoD: 5.8A, microRNA. MicroRNA microarrays from MD ~ E trans- RUNX1: Abcam, ab23980), a HRP-conjugated secondary duced RD cells identified several microRNAs that chan- antibody (Jackson, West Grove, USA), and chemilumines- ged expression and miR-206, a microRNA that has been cent detection (Amersham, Pittsburgh, USA). shown to induce myogenic differentiation in normal Cells were fixed with 2% paraformaldehyde for cells and RMS [19,20], was the most consistently 6 min at room temperature before permeabilization increased (Additional file 4: Table S2) and was confirmed with Triton X-100. Myosin heavy chain was detected by miRNA Northern blotting (Figure 2A, upper panel), with the MF-20 antibody, and nuclei detected with as was miR-133b, a miRNA from the same primary tran- DAPI. script (Figure 2A, second panel). RT-PCR for the pre- sumptive primary transcript also showed a substantial Electrophoretic mobility shift assays increase (Figure 2B). Northern blotting confirmed that Electrophoretic mobility shift assays were performed as the forced dimer decreased miR-199a* expression, as sug- described previously [13]. Probe sequences are listed in gested by the array results (Additional file 5: Figure S3A), (Additional file 1: Table S1). and that miR-29b, previously implicated in RMS differ- entiation, was expressed at low levels and did not Results change in response to MD ~ E expression (Additional RUNX1 and ZNF238 differentiate rhabdomyosarcoma cells file 5: Figure S3B) [21]. Furthermore, RD cells differen- RUNX1 is a runt-related transcription factor with a well- tiated through RUNX1 and ZNF238 expression showed characterized hematopoietic role [14] that is expressed an increase in mature miR-206 in both cases (Figure 2C), in muscle cells [15], functions in denervated muscle along with an increase in primary transcript (data not [16], and is regulated by the MyoD network [8,17], but shown). miR-206 levels in the myogenic C2C12 cell line has an uncharacterized role in developing muscle. showed that miR-206 expression in proliferative and dif- ZNF238 (aka RP58) is induced by MyoD and directly ferentiated RMS resembled the changes as C2C12 cells down-regulates the inhibitory Id factors [18]. MyoD shift from beginning myogenesis (90% confluency) to ChIP-seq (chromatin immunoprecipitation coupled to myotubes (DM) (Figure 2D). high-throughput sequencing) found an association be- In agreement with previous reports demonstrating that tween the RUNX1 and ZNF238 binding motifs and miR-206 alone is sufficient to differentiate RMS cells MyoD bound sites in muscle cells [11] and qPCR [20,22,23], transient transfection of pre-miR-206 constructs MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 4 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 1 Expression of RUNX1 or ZNF238 leads to terminal differentiation of RMS cells. (A) qPCR for RUNX1 was performed in RD cells either infected with a control virus, or the forced MyoD ~ E dimer (MD ~ E) as well as control (0 h) human fibroblasts and fibroblasts differentiated into myotubes (96 h). (B) RT-PCR for the two isoforms of ZNF238 in RD cells and fibroblasts as in 1A. (C) Myosin heavy chain (MHC) immunostains in RD cells either not infected (no infection), infected with a control GFP-expressing lentivirus (GFP control) or RUNX1 or ZNF238 expressing lentivirus. All cells were infected at approximately equivalent MOIs, and cells were allowed to differentiate for 72 h in low-serum media before staining. GFP was detected directly, without the use of an antibody. (D) qPCR for muscle-specific creatine kinase (CKM) in RD cells infected with either ZNF238 or RUNX1 viruses compared to cells with control retroviruses. (E) After 24 h of differentiation, RD cells were pulsed for a further 24 h with EdU-containing differentiation media, before fixation and quantification of the percentage of EdU-positive cells. (F) qPCR for ID2 and ID3 in control and ZNF238-expressing RD cells. All qPCR data are normalized to TIMM17b expression, and the level in control cells is set to 1. All -4 bar graphs represent the mean ± SEM of at least three independent experiments. *: P< 0.05; **: P< 0.01; ***: P< 0.001; ****: P< 1×10 into RD cells resulted in myotube formation (Figure 2E), data not shown). As would be expected from prior reports an increase in CKM expression (Figure 2F), and evidence of its effect on myogenic cells [24], introduction of miR- that cells undergoing morphological change withdrew 133b did not lead to RMS differentiation as judged by ei- from the cell cycle (Figure 2G, H), with similar results in ther morphology or gene expression (Additional file 6: the alveolar RhJT cells (Additional file 6: Figure S4A and Figure S4B, C). MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 5 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 MyoD directly regulates miR-206 [25] and a putative MyoD to enhance the expression of miR-206. ZNF238 RUNX1 binding site exists near the MyoD binding site. did not activate the miR-206 reporter (data not shown), RUNX1 alone showed minor activation of a miR-206 suggesting that the reporter does not have the elements luciferase reporter, while RUNX1 combined with MyoD or context for ZNF238 regulation. showed synergistic activation (Figure 2I, black bars), RUNX1 also induced the expression of ZNF238 in RD which was dependent on the integrity of the RUNX1 cells and ChIP identified RUNX1 binding close to the binding site (Figure 2I, grey bars). ChIP experiments in TSS of ZNF238, suggesting it functions directly to acti- RD cells confirmed that RUNX1 binds the miR-206 pro- vate ZNF238 (Figure 3A and 3B). Consistent with moter (Figure 2J). Therefore, RUNX1 cooperates with this model, ID2 and ID3 expression were decreased Figure 2 (See legend on next page.) MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 6 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 (See figure on previous page.) Figure 2 MyoD ~ E, RUNX1, and ZNF238 increase miR-206. (A) microRNA northern blots to detect the mature form of the indicated microRNAs in RD cells infected with either empty (control) retrovirus, or retrovirus expressing MyoD ~ E (MD ~ E). (B) RT-PCR using primers located in the pre- and pri-miR-206 sequence to detect the primary miR-206 transcript. TIMM17b is the internal control. (C) microRNA northerns as in 2A, in RD cells expressing a transcription factor as indicated. (D) microRNA northerns in C2C12 cells at various stages of differentiation ranging from undifferentiated myoblasts (50% GM), through beginning differentiation (90% GM) to myotubes (DM). (E) Immunostains for MHC in RD cells transiently transfected with either a pre-miR-206 RNA construct, or a negative control construct. Nuclei were stained with DAPI. (F) qPCR for CKM in RD cells treated as in E. (G) RD cells treated as in E were pulsed with BrdU for 24 h and then stained and counted to determine the extent of co-localization of MHC + myotubes and BrdU + nuclei. (H) RD cells transiently transfected as in E were pulsed for 24 h with BrdU-containing differentiation media before fixation and quantification of the percentage of BrdU positive cells. The percent reduction of BrdU + nuclei almost exactly equals the percent of cells found to be MHC + (not shown). (I) Luciferase activity in RD cells using a miR-206 promoter driven reporter and transiently transfected factors as indicated. ‘206 RUNX mutant’ indicates that the reporter has had a putative RUNX1 binding site mutated to prevent RUNX1 binding. Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co- transfected renilla plasmid. (J) RUNX1 ChIP assays, both with normal PCR and qPCR results, at the miR-206 promoter and a control locus before (Control) and after (RUNX1) infection of the cells with empty or RUNX1-expressing retrovirus. All PCRs in the imaged gel (upper) were performed for the same number of cycles. qPCR results (lower) represent the mean ± SEM of two independent ChIP experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. All other graphs in this figure represent the mean ± SEM of at least three independent experiments. * : P< 0.05; ** : P< 0.01; *** : P< 0.001. in RD cells expressing RUNX1 (Figure 3C). In con- Together, this shows that MyoD functions in nested trast, ZNF238 did not upregulate RUNX1 expression, feed-forward circuits with RUNX1 and ZNF238 to but we confirmed the prior report [18] that MyoD activate miR-206 expression and induce differenti- activates ZNF238 (Additional file 7: Figure S5). ationinthe RD cells. Figure 3 ZNF238 is a downstream target of MyoD and RUNX1. (A) qPCR for RUNX1 and ZNF238 in RD cells transduced with virus expressing the converse factor. (B) RUNX1 ChIP results at the intron of ZNF238 and a control locus before (Control) and after (RUNX1) infection of the cells with empty or RUNX1-expressing retrovirus. (C) qPCR for ID genes in control and RUNX1-expressing RD cells. PCRs in the imaged gel in 3B were performed for the same number of cycles. The graph in 3B represents qPCR data showing the mean ± SEM of two independent experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. qPCRs in 3A and 3C are represented as the mean ± SEM of at least three independent experiments. *: P< 0.05; **: P< 0.01. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 7 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 RUNX1, ZNF238, and miR-206 activate a common We have previously shown that the inhibitory bHLH differentiation program factor Musculin (MSC) inhibits MyoD activity in RD Gene expression arrays were performed on RD cells cells and other RMS [8]. Furthermore, since miR-206 transduced individually with RUNX1-, ZNF238-, and has been shown to inhibit MSC expression [19], we miR-206 expressing viruses. GO analysis ranked by hypothesized that the opposing activities of MyoD and P values identified multiple muscle-related categories MSC might constitute an on-off switch at the miR-206 induced by each factor, with four of the ten most signifi- promoter. ChIP showed that MSC was bound to the same cant categories shared between all factors (Additional region as MyoD in undifferentiated RD cells (Figure 5A), file 8: Table S3). In agreement with our deduced epistatic and MSC occupancy diminished in RD cells that under- relationship, the number of genes significantly regulated went RUNX1- (Figure 5B) or MD ~ E-mediated differenti- (fold change> 2, FDR <0.05) by each factor became se- ation (Additional file 11: Figure S7). High-throughput quentially reduced from RUNX1 (734) to ZNF238 (616) sequencing of the MyoD and MSC ChIP material to miR-206 (355) and showed substantial overlap (ChIP-seq) from undifferentiated RD cells with analysis (Figure 4A) and correlation (Figure 4B). Loosening the restricted to sequences mapping to the miR-206 promoter fold-change threshold showed that nearly all genes regu- revealed distinct MyoD and MSC peaks over two adjacent lated by miR-206 similarly changed expression in re- E-boxes (Figure 5C), indicating a MyoD bound E-box adja- sponse to RUNX1 and/or ZNF238 (Figure 4C, top). cent to a MSC bound E-box. Similarly, a large portion of the ZNF238 genes were also Electrophoretic mobility shift assaysdemonstrated that regulated by RUNX1 (Figure 4C, bottom). the E-box associated with the MyoD peakhad a higher RUNX1, ZNF238, and miR-206 affected the expression affinity for MyoD compared to the E-box underthe MSC of a variety of transcription factors and signaling path- peak, but that both E-boxes can be bound by either- ways involved in myogenesis (Additional file 9: Table S4), MyoD or MSC in vitro (Additional file 12: Figure S8). and a subset of the changes were confirmed by RT-PCR Whenthe E-protein is in excess, gel shift assays show that (Figure 4D). The MRF myogenin, a MyoD target [26], MyoD:Eheterodimers can compete with MSC:E heterodi- increased in response to all three of the factors. MEF2C mers to bindthe E-box under the MyoD ChIP-seq peak and MEF2D, cooperative factors for MyoD activity [17], (Figure 5D, leftpanel, lane 1) and that decreasing the were up-regulated. Down-regulation of positive regula- amount of MSC shiftsthe binding progressively toward tors of the cell cycle (MYCN, RCOR2, E2F2)and various MyoD:E protein heterodimers(lanes 2–5). Even with a rela- members of the HES/HEY family (HEY1, HES6, HEYL, tive excess of E-protein,MSC:E heterodimers outcompete HES1) was observed as well. It has previously been MyoD:E heterodimers onthe E-box under the MSC demonstrated that interference with HES1 contributes ChIP-seq peak andMyoD:E bindingto this site occurred to RMS proliferation [27], and the HES/HEY family is only when MSC levels were decreased (Figure 5D, right known to be Notch responsive [28], a signaling pathway panel, lanes 1–5). Therefore the relative affinitiesof MSC:E with myogenic inhibitory effects [29-31]. Among miR- and MyoD:E heterodimers for the two E-boxesin the 206’s most strongly down-regulated targets were two miR-206 regulatory regions likely account for theob- members of the Notch signaling pathway, DLL3 and served endogenous binding (Figure 5C) and suggest that NOTCH3. decreasinglevels of MSC would result in occupancy of both E-boxes by MyoD. Co-transfection experiments showed that MyoD:E12 The miR-206 promoter integrates multiple inputs to heterodimers robustly activated the miR-206 reporter switch from inhibition by MSC to activation by MyoD containing both E-boxes and this was prevented by We have previously shown that MyoD binds and acti- MSC (Figure 5E, black bars). MSC also repressed ac- vates the expression of the miR-206 gene in normal tivation by the MD ~ E dimer, suggesting the effect of myogenesis [25], whereas our current data indicate that MSC is due to DNA binding, not interference with the miR-206 promoter integrates multiple inputs to the formation of MyoD:E dimers (Additional file 13: modulate its activation by MyoD. Somewhat to our sur- Figure S9). Mutation of the MSC binding site made prise, ChIP in RD cells showed that MyoD was bound to the reporter insensitive to activation by MyoD and the miR-206 promoter in undifferentiated RD cells that E12 (Figure 5E, grey bars). This suggests that MSC is repressing miR-206 by physically occluding an E-box expressed only low levels of miR-206 (Additional file 10: Figure S6A) and regional enrichment for acetylated H4 that MyoD needs to occupy for full activation. The histones indicated active regional histone acetyltransfer- MyoD and MSC ChIP-seq data also supports this conclusion. Compared to the MyoD peak in RD cells, ase activity (Additional file 10: Figure S6B). Therefore, a factor, or factors, was preventing efficient transcriptional there is a broadening of the MyoD peak in myotubes activation by the bound MyoD. that appears to widen to include E-boxes located MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 8 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 4 RUNX1, ZNF238, and miR-206 function through common mechanisms. (A) 3-way Venn diagram representing the overlap between significantly regulated (fold-change >2, FDR <0.05, overlap considers only changes in same direction) gene targets in RD cells differentiated either through RUNX1, ZNF238, or miR-206 expression relative to GFP-infected controls. The table indicates the breakdown of upregulated versus down-regulated genes for each portion of the Venn diagram. (B) Scatter plots showing pairwise comparisons of gene expression. All values are plotted as the log of the fold-change relative to GFP-infected controls, as indicated along the x- and y-axes. Correlation is listed for each comparison in the matrix. (C) (upper panel) Bar graph demonstrating that the majority of the 95 genes listed as being ‘uniquely’ regulated by miR-206 in 4A are also regulated by RUNX1 and/or ZNF238, but at lower levels of expression change. FDR was kept constant (<0.05) in this analysis, and to be included as a ‘shared’ target, the change had to occur in the same direction (either up- or down-regulated) in RUNX1 and/or ZNF238 as in miR-206. (bottom panel) Analysis as in the top panel for genes in the ZNF238 unique and ZNF238:miR-206 intersection groups relative to RUNX1 changes. (D) RT-PCR for select gene targets from Additional file 9: Table S4. TIMM17b serves as the internal control. more proximally to the start of the miR-206 tran- activation domain by splicing exon 2 directly to exon 5 script (Figure 5C, bottom panel), suggesting that in that we called E2A-2/5. To determine whether the E2A- differentiated muscle, MyoD occupies additional positions. 2/5 splice form contributed to the bHLH balance at the Consistent with a model in which miR-206 activity miR-206 promoter, we tested the response of RD cells to requires multiple E-boxes to be bound by MyoD for full expression of a forced protein dimer consisting of MyoD activation, mutation of the MyoD-binding E-box also and the E12-2/5 splice form of E12 (MD ~ E2/5). MD ~ resulted in a dramatic reduction in the ability of the re- E2/5 expressing cells formed substantially fewer myo- porter to be activated by MyoD and E12 (Additional file tubes (Additional file 15: Figure S11A) while expressing 14: Figure S10). roughly equivalent amounts of dimer (Additional file 15: Figure S11B) compared to cells expressing the full-length An alternative splice form of E2A modulates MyoD MD ~ E dimer. MD ~ E expressing cells expressed sub- activation of miR-206 stantially more CKM (Figure 5F), RUNX1 (Figure 5G), We previously described a developmentally regulated and miR-206 (Figure 5H) than MD ~ E2/5 cells. However, splice form of E2A that removes a portion of the first in all cases, MD ~ E2/5 expressing cells still expressed MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 9 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 5 (See legend on next page.) MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 10 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 (See figure on previous page.) Figure 5 MSC interferes with the ability of MyoD to positively regulate the microRNA miR-206 by blockading a necessary MyoD-binding site. (A) Site-specific ChIP for MSC in the miR-206 promoter and at a control locus. (B) MSC ChIP as in (A) in RD cells infected either with an empty retrovirus (Control), or RD cells differentiated through expression of RUNX1 (RUNX1). (C) Screenshot from the human UCSC Genome Browser of the region that corresponds to the miR-206 promoter. Mapped reads from ChIP-seq for MyoD in RD and HFF cells are indicated, with the number on the left-hand y-axis indicating the number of reads mapped at the peak of occupancy. The location of E-boxes are indicated at the bottom of the panel by the black rectangles. Vertical lines are drawn through the apparent highest points of occupancy for MyoD (red) and MSC (green) in RD cells. (D) Electrophoretic mobility shift assays using in vitro transcribed and translated proteins as indicated and probes that correspond to either the E-box located at the peak of MyoD binding as indicated by the red mark in 5C, or the E-box located at the peak of MSC binding, indicated by the green mark. The position of MyoD:E and MSC:E heterodimers are indicated by the arrows. The lane marked 2x E12 indicates protein mixtures that included double the amount of E12 compared to other lanes, and the triangles indicate decreasing amounts of either MSC or MyoD in the mixtures as other proteins were maintained at constant levels and total protein amounts were balanced with translation of empty CS2. (E) Luciferase assays in RD cells with constructs as indicated below the figure using either the miR-206 promoter luciferase reporter (206) or one in which the E-box that the peak of MSC occupancy is located over has been mutated (206 MSC-binding Ebox mutant). Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co-transfected renilla plasmid. (F) qPCR for CKM from RD cells transduced with either an empty virus (control), or one expressing either the MD ~ E or MD ~ E2/5 forced dimer. Cells were differentiated for 24 h before collection of RNA for use in qPCR. (G) qPCR for RUNX1 from the RD cells assayed in E. (H) qPCR for pri-miR-206 from the RD cells assayed in E and F. For all ChIPs, relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. The control locus is located at hemoglobin beta, a silent gene in myogenic cells. Corrected relative enrichment (5B) is the ratio of enrichment at miR-206 to enrichment at the control locus. All graphs represent the mean ± SEM of at least three independent experiments. All qPCR of gene expression was corrected to TIMM17b and control cells set to 1. *: P <0.05; ** : P< 0.01; *** : P< 0.001; † : P = 0.058. more of the MyoD targets than control cells, suggesting induction of this inhibitory factor serves two purposes: that, while the specific E-protein partner is important for (1) to down-regulate genes that inhibit myogenesis; and full activation of miR-206, the inhibitory effect of MSC is (2) to interfere with MyoD binding at genes it might crucial. regulate in myoblasts. Despite differences in their direct targets, RUNX1 and ZNF238 both increase miR-206 Discussion transcription and lead to a terminally differentiated state. We have previously proposed that RMS represent an For RUNX1 we demonstrated a direct binding of the arrested phase of normal development at the transition miR-206 promoter, whereas the mechanism for ZNF238 point between regulative growth and terminal differenti- remains speculative, possibly through its suppression of ation, a transition regulated in part through the balance ID gene expression, which would increase the propor- of repressive and activating bHLH protein dimers [8]. tion of productive MyoD:E-protein heterodimers. While We showed that increasing the abundance of MyoD:E- in vitro in nature, our data on the relative ability of protein heterodimers tipped the balance to differentiation MyoD:E and MSC:E to shift the regulatory E-box and proposed that this heterodimer might target an un- sequences that control miR-206 expression (Figure 5D known central integrating function since multiple myo- and Additional file 12: Figure S8) suggest that even rela- genic repressors were down-regulated. One prediction of tively small changes in the availability of E-protein part- this earlier model was that modulating the abundance of ners could make a dramatic difference in the expression any of the factors that impinge on the integrating func- of miR-206. tion might be sufficient to induce differentiation in RMS. MSC, a bHLH that inhibits myogenesis [32], sup- Our present study supports and extends this model by presses the activation of miR-206 by binding an E-box demonstrating that miR-206 integrates the activity of required for induction by MyoD. A requirement for multiple proliferative and myogenic factors and acts as a more than one MyoD-bound E-box to drive full target switch that transitions the RMS from growth to activation has been described before [33-37], and it is differentiation. currently unclear whether MSC is simply preventing To test whether different myogenic co-factors can in- MyoD binding or recruiting repressive factors to the duce differentiation in RMS, we chose one transcrip- locus. The fact that the miR-206 locus has acetylated H4 tional activator and one repressor, RUNX1 and ZNF238, even when not robustly expressed suggests that MSC respectively. RUNX1 enhances MyoD activation at a var- may have a simple obstructionist role at this locus. Fu- ture work will be necessary to determine the relative iety of MyoD targets, including ZNF238 and possibly MYOG and the MEF genes. In contrast, ZNF238 down- roles of MSC and MyoD at miR-206 and other myogenic regulates multiple members of the inhibitory HES and targets. Data reported in this manuscript, when combined with HEY protein family and factors that drive proliferation. Our data on ZNF238 regulation, motif analysis, and gene previous data from us and others [8,11,18,19] suggests a targets in this and previous work [11] suggest that the specific model for the regulation of miR-206 that involves MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 11 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 nested positive and negative feed-forward and feed-back output of oscillating circuits and acts as a genetic switch to loops to create a molecular switch for regulating the tran- transition the cell from a proliferative growth phase to sition from growth to differentiation in myogenic cells differentiation. (Figure 6). In replicating myoblasts, a MyoD:E-protein het- Hematological malignancies are often categorized based erodimer binds an E-box site in the regulatory regions of on an arrested transition between stages of cellular differen- ID2 and ID3 [11] creating the potential for an oscillating tiation. Our work suggests that the same might apply to circuit: any increase in MyoD activity would increase ID RMS and possibly other solid tumors. bHLH factors control expression, which would dampen MyoD activity by de- cell fate and differentiation in multiple cell types and a bal- creasing the availability of the E-protein dimer partner. ance among bHLH dimer partners and other co-factors However, if MyoD:E-protein heterodimers pass a threshold might establish similar ‘tipping points’ at critical genes that of activity sufficient to initiate a feed-forward circuit acti- regulate the transition from regulative growth to differenti- vating RUNX1 and ZNF238, then ZNF238 shuts off ID ation. Our emerging model of multiple pathways (some production by occluding the MyoD binding sites and functioning as oscillating circuits) integrated by switch- thereby relieving the negative-feedback regulation of points for differentiation has significant implications for MyoD. The increased MyoD and RUNX1 activity can then drug therapies to induce differentiation. Different cell types more effectively compete with MSC on the miR-206 regu- may not exhibit identical convergence of pathways. There- latory regions and the increased miR-206 levels feed back fore, combining multiple drugs that each has a small to inhibit MSC, and likely other growth promoting factors effect on different components might induce differen- [8,19,20,24,38], thereby locking the cell into a committed tiation in the target cells while exhibiting low toxicity differentiation program. Therefore, miR-206 integrates the and few off-target effects. Figure 6 miR-206 integrates the output of oscillating circuits and acts as a genetic switch to transition from growth to differentiation. The experimental data support a network model composed of coupled oscillators with miR-206 functioning as a switch regulating the transition from one stable state to another. In myoblasts, MyoD, E-proteins, and ID proteins compose the first oscillating circuit: (1) MyoD:E heterodimers bind an E-box in the regulatory regions of the ID2 and ID3 genes and drive ID transcription; (2) the ID protein competitively forms dimers with the E-protein, limiting the production of active MyoD:E-protein heterodimers; (3) the decline in active MyoD:E-heterodimers results in decreased ID production; and (4) the decreased ID permits an increase in active MyoD:E-protein heterodimers and more ID production. The second oscillating circuit is composed of MyoD, E-proteins, MSC, and miR-206: (1) MyoD:E-protein and MSC:E-protein heterodimers compete for binding at the E-box in the miR-206 regulatory region, which oscillates between MyoD-activated and MSC-repressed states; (2) limiting amounts of E- protein prevent full activation by MyoD; and (3) low levels of miR-206 prevent full suppression by MSC. These circuits are coupled by their shared response to the concentration of active MyoD:E-protein heterodimers. The oscillating circuits bifurcate to a new determined state when the concentration and/or activity of the MyoD:E-proteins become sufficient to activate the expression of RUNX1 and ZNF238 in a feed-forward circuit that blocks the expression of the ID genes and permits the accumulation of active MyoD:E-protein complexes. The increase of MyoD:E-protein heterodimers together with RUNX produces higher miR-206 expression, and the increased miR-206 suppresses MSC and other inhibitors of differentiation. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 12 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Conclusions Our results in a rhabdomyosarcoma cell line provide evi- Additional file 8: Table S3. Top GO categories of genes regulated by dence for feed-forward and feed-back regulatory circuits RUNX1, ZNF238, and miR-206. in myogenic cells that regulate miR-206 expression as a Additional file 9: Table S4. Select potential regulators of myogenesis affected by RUNX1, ZNF238, and miR-206. genetic switch to transition a muscle cell from growth to Additional file 10: Figure S6. The miR-206 promoter in RD cells is differentiation. One circuit is composed of MyoD, bound by MyoD and has acetylated histone H4. (A) ChIP for MyoD in RD RUNX1, ZNF238, and ID2/3 and might function, at least cells in differentiation media shows MyoD enrichment upstream of miR- in part, to regulate the availability of E-proteins to form 206, compared to a control locus at a non-expressed gene (hemoglobin beta). (B) Site-specific ChIPs in RD cells for acetylated histone H4, at heterodimers with MyoD; whereas the second circuit hemoglobin beta (control), miR-206, and the myogenin promoter composed of MyoD, RUNX1, and MSC regulates the ex- (MYOG). ChIP results are represented as the mean ± SEM of at least three pression of miR-206. These have characteristics of independent experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified coupled oscillatory circuits that can dampen the activity of with no antibody. *: P<0.05; **: P<0.01. MyoD during expansionary growth, or, under differenti- Additional file 11: Figure S7. MSC occupancy in the miR-206 ation promoting conditions, can be switched off and promoter diminishes with MD~E differentiation. ChIP for MSC in the miR- thereby enhance MyoD activity and drive differentiation. 206 promoter in RD cells either transduced with empty virus (Control), or differentiated through the expression of the forced MD~E protein dimer Modulating the abundance of multiple different compo- (MD~E). Values are the means ± standard deviation of two independent nents of these coupled circuits can drive differentiation, experiments. Corrected relative enrichment equals the relative suggesting that multiple targets within these circuits enrichment at miR-206/the relative enrichment at the control locus. Relative enrichment is calculated as the ratio of the % of input amplified might be candidates for targeting differentiation-inducing with antibody to the % of input amplified with no antibody. therapeutics. Additional file 12: Figure S8. In vitro assessment of MyoD and MSC binding in the miR-206 promoter. Electrophoretic mobility shift assays were performed using in vitro translated proteins as indicated and probes Additional files that represent the DNA sequence under either the E-box occupied most prominently by MyoD in RD cells as assessed by ChIP-seq results (MyoD- Additional file 1: Table S1. Primer and oligonucleotide sequences. binding E-box) or the E-box occupied most prominently by MSC (MSC- binding E-box). Bound complexes were competed with cold competitor Additional file 2: Figure S1. RD cells infected with ZNF238 and RUNX1 probes prepared at the indicated excess. viruses increase expression of the appropriate factor. (A) RT-PCR for ZNF238 in RD cells infected with either a control virus or the ZNF238- Additional file 13: Figure S9. MSC inhibits the activation of the miR- containing virus. TIMM17b is used as a loading control. (B) Western blot 206 reporter by the forced MD~E dimer. Luciferase assay results in RD using whole cell lysates for RUNX1 in control and RUNX1 virus infected cells using the miR-206 promoter reporter with constant amounts of RD cells. The blot was then stripped and reprobed for alpha-tubulin as a MyoD and E12 introduced individually or as the forced dimer, in the loading control. Bands were confirmed to be of the correct size through presence of varying amounts of co-transfected MSC. - indicates no MSC a protein size ladder (not shown). was added, 1x indicates that the MSC transfected was equal by mass to the amount of MyoD or MD~E, and 0.1x indicates that the MSC Additional file 3: Figure S2. RUNX1 differentiates alveolar subtype RMS transfected was equal to 1/10th that amount. Results are indicated as the cells. (A) Western blots on whole cell lysates from RhJT cells infected with means ± SEM from three independent experiments. Control indicates the either a RUNX1-expressing or control virus. MHC is myosin heavy chain, a results from transfection with empty vector. marker of myogenesis, and alpha-tubulin is the loading control. Blots were serially stripped and reprobed, and bands confirmed to be of the Additional file 14: Figure S10. Strong miR-206 activation is dependent correct size. (B) RT-PCR for CKM (muscle specific creatine kinase) on cells on multiple E-boxes. Luciferase assay results in RD cells with transient treated as in A. TIMM17b is the internal control. transfection as indicated using the miR-206 promoter and a reporter in which the E-box exhibiting the peak of MyoD occupancy in RD cells Additional file 4: Table S2. miRNA changes in response to MD~E (indicated by the red marker in Figure 5C) has been mutated and expression in RD cells. eliminated as a site of bHLH binding. Results are indicated as the means Additional file 5: Figure S3. Differential effects on miRNA expression ± SEM from three independent experiments. Control indicates the results by the forced MD~E dimer. (A) miRNA northern blot for miR-199a* (also from transfection with empty vector. *: P<0.05. known as miR-199a-5p) from RD cells either transduced with a control or Additional file 15: Figure S11. MD~E expression results in greater MD~E virus. (B) miRNA northern blot for miR-29b as in panel A. myotube formation than MD~E2/5 expression. (A) Light microscopy Additional file 6: Figure S4. miR-206 affects alveolar subtype RMS cells images of RD cells transduced with either control virus (Control) or virus and miR-133b does not share its effects. (A) Immunostains for MHC in expressing either the MD~E or MD~E2/5 forced protein dimers and RhJT cells transfected with either a pre-miR-206 or control construct. allowed to differentiate for 24 h. Arrows indicate representative cells that DAPI stains all nuclei. (B) Stains as in A after transfection of RD cells with have appeared to form myotubes. (B) Western blot for MyoD and alpha- pre-miR-133b or a control construct. (C) qPCR for CKM in RD cells treated tubulin, as a loading control, from cells treated as in (A). The size of the as in B. qPCR data was normalized to TIMM17b and control set to 1, with bands detected with the MyoD antibody in MD~E and MD~E2/5 lanes bars representing the mean ± SEM of three independent experiments. *: are as expected given the approximate calculated size of the forced P<0.05. dimers. Additional file 7: Figure S5. MyoD activity increases ZNF238 expression during myogenic conversion. 10T1/2 fibroblast cells expressing an Abbreviations estradiol-inducible version of MyoD were induced to undergo bHLH: Basic helix-loop-helix; ChIP: Chromatin immunoprecipitation; ChIP- myogenesis by addition of beta-estradiol to the culture medium. RNA seq: Chromatin immunoprecipitation coupled to high-throughput was taken at the indicated times under indicated conditions and qPCR sequencing; RMS: Rhabdomyosarcoma. performed to quantitate the relative levels of ZNF238. All bars indicate the mean ± SEM of at least three independent experiments. Time 0 was Competing interests set to 1, and TIMM17b served as the internal control. *: P<0.05. The authors declare no potential conflicts of interest. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 13 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Acknowledgements 14. Cohen MM: Perspectives on RUNX genes: an update. Am J Med Genet A KLM was supported by a Developmental Biology Predoctoral Training Grant 2009, 149A:2629–2646. (T32HD007183). ZY was supported by the NIH Interdisciplinary Training Grant 15. Zhu X, Yeadon JE, Burden SJ: AML1 is expressed in skeletal muscle and is in Cancer Research (T32CA080416). SJT was supported by NIH NIAMS regulated by innervation. 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J Biol Chem 2009, 284:31921–31927. doi:10.1186/2044-5040-2-7 Cite this article as: MacQuarrie et al.: miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells. Skeletal Muscle 2012 2:7. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state. Methods: Potential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results: Modulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions: These findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation). Keywords: Rhabdomyosarcoma, RUNX1, ZNF238, miR-206, MSC, myogenesis Background differentiation [6]. However, in RMS the ability of MyoD Rhabdomyosarcoma (RMS) is a soft tissue sarcoma char- to induce differentiation is impaired [7]. acterized by expression of myogenic regulatory factors, Our recent study indicated that rhabdomyosarcomas especially MyoD, and other skeletal muscle genes [1-3]. represent an arrested progress through a normal transi- MyoD is capable of converting multiple cell types into tional state that is regulated by the formation of hetero- terminally differentiated skeletal muscle [4,5] and nor- dimers between MyoD and the full-length E-proteins mally acts as a nodal point in differentiation to integrate [8]. MyoD binds DNA as a heterodimer with an E-pro- multiple signals to balance regulative growth and cell tein (E2A, E2-2, or HEB) [9]. Normal myoblasts and RMS express a transcriptionally less active splice form of E2A as well as the bHLH (basic helix-loop-helix) proteins ID and Musculin (MSC), both of which * Correspondence: stapscot@fhcrc.org compete with the full-length E-proteins for heterodi- Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 merization with MyoD. The demonstration that a Fairview Ave N, C3-168, Seattle, WA 98109, USA Department of Neurology, University of Washington, Seattle, WA 98105, USA forced heterodimer of MyoD and a full-length E-protein Full list of author information is available at the end of the article © 2012 MacQuarrie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 2 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 suppressed multiple inhibitory mechanisms and all conditions. Transient transfections of pre-microRNA induced differentiation in the RD and other rhabdo- constructs were performed using 25 μMofpre-miR con- myosarcomas suggested that a central integrating structs (Ambion, Grand Island, USA) and siPORT NeoFX mechanism might regulate the switch from regulative (Ambion) according to the manufacturer’sdirections. growth to differentiation [8]. If a central integrating mechanism does exist, then mul- qPCR and RT-PCR tiple pathways should regulate its activity and multiple fac- All qPCR was performed using SybrGreen from Bio-Rad tors should be able to induce differentiation in RMS. In this on an Applied Biosystems 7900HT. Relative expression manuscript we demonstrate that modulation of multiple levels were calculated using cDNA dilution standard different myogenic factors can induce differentiation in curves or delta-delta Ct calculations. All values are rhabdomyosarcoma cells and that their activity is inte- reported as the mean ± SEM of at least three independent grated, at least in part, through the activation of miR-206, biological experiments and significance tested with t-tests. which acts as a genetic switch to transition the cell from a All RT-PCRs were performed simultaneously with minus proliferative growth phase to differentiation. These findings reverse transcriptase controls to check the absence of sig- suggest that multiple components of differentiation path- nal. Primers used for amplification are listed in (Additional ways that converge on miR-206 might be targeted for dif- file 1: Table S1). ferentiation therapies in at least some rhabdomyosarcomas. Methods microRNA microarrays Plasmid construction RNA was isolated using acid-phenol purification from Coding sequences of RUNX1 and ZNF238 were ampli- two biologically independent sets of RD cells transduced fied by PCR from human myotube cDNA, and cloned with either MD ~ E or empty vector pCLBabe retro- into pRRLSIN.cPPT.PGK/GFP.WPRE, pCLBabe, and viruses and differentiated for 24 h after puromycin selec- pCS2. The miR-206 lentivirus was purchased from Open tion. miRNAs were labeled using Exiqon’s miRCURY Biosystems. Lentiviral supernatant was produced by the labeling kit, and then competitively hybridized to in- FHCRC core viral facility, and viruses from pCLBabe house spotted miRNA arrays (FHCRC core facility). Cut- plasmids packaged using BBS-mediated calcium precipi- offs for significant changes were a FDR <0.05 and a tation into Phoenix cells. MD ~ E2/5 was cloned into the fold-change >2. Data are available under GEO accession pCLBabe backbone. For the miR-206 promoter lucifer- number GSE35921. ase reporter, a piece of approximately 2.5 kb of DNA up- stream of human miR-206 was amplified using primers microRNA northerns located in (Additional file 1: Table S1). microRNA northern blots were performed as described previously [10]. Probe sequences used are listed in (Add- Cell culture, transient transfections, and luciferase assays itional file 1: Table S1). RD cells were obtained from ATCC (American Type Cul- ture Collection) in approximately 1990, and all analyses have been performed on cells that originated from low pas- Expression microarrays sage number frozen aliquots. RhJT cells were obtained from RNA was isolated using the RNeasy mini kit (Qiagen) PJ Houghton in 1990 and, as with RD cells, all experiments from RD cells infected with either RUNX1-, ZNF238-, have used cells from original low passage number frozen miR-206-, or GFP-expressing lentiviruses and allowed to cells. RD and RhJT cells were maintained in DMEM with differentiate for 72 h. Each condition was performed 10% bovine calf serum and 1% Pen-Strep (Gibco, Grand Is- with three independent biological replicates. RNA was land, USA). Low-serum differentiation media consisted of hybridized to Illumina Human HT-12 v4 BeadChips. DMEM with 1% horse serum, 1% Pen-Strep and 10 μg/mL Analysis was performed in R/Bioconductor using the insulin and transferrin. Transient transfections for lucifer- lumi and limma packages with annotations found in the ase assays were performed using Superfect according to lumiHumanAll.db package. P values were adjusted to ac- manufacturer’s directions with a total of 3 ug of plasmid count for multiple testing using Benjamini and Hochberg’s DNA in each well (Qiagen, Valencia, USA). Luciferase method, and cutoffs for significant changes were a FDR assays used the Dual-Luciferase Assay kit (Promega, Madi- <0.05 and a fold-change >2. GO category enrichment son, USA) according to manufacturer’s directions. All tests were performed using the conditional algorithm of results were corrected to co-transfected Renilla-pCS2 and the GOstats package and a gene ‘universe’ of any gene are reported as the mean ± SEM of at least three independ- with a GO annotation that was called as ‘present’ in at ent experiments, with significance calculated using a t-test, least one of the 12 array datasets. Data are available and each experiment having three biological replicates of under GEO accession number GSE35921. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 3 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 ChIPs (Chromatin immunoprecipitations) confirmed that both RUNX1 and ZNF238 increased with ChIPs were performed as has been described previously differentiation induced in the RD RMS line by the forced [11]. Antibodies used were as follows: RUNX1 (Abcam, MD ~ E dimer and that myotubes formed from MyoD- ab23980), MyoD [12], MSC (Santa Cruz, sc-9556X), induced myogenesis in normal fibroblasts had higher ex- Acetylated Histone H4 (Upstate 06-866). Primers used pression levels of both factors compared to RD cells for amplification are listed in (Additional file 1: Table S1). (Figure 1A and 1B). Therefore, we hypothesized that RUNX1 and/or ZNF238 might cooperate with MyoD to EdU and BrdU labeling, western blots, and cell stains drive muscle gene expression and tested each factor for After 24 h in low-serum differentiation media, cells were its ability to induce differentiation in RD cells. shifted to differentiation media supplemented with EdU Lentiviral-mediated expression of each factor in RD at a final concentration of 50 μM (Invitrogen) and incu- cells (Additional file 2: Figure S1) induced muscle differ- bated for a further 24 h. Cells were then fixed and entiation, as measured by morphology and expression of stained according to the manufacturer’s protocols using myosin heavy chain (MHC) (Figure 1C), muscle-specific the Click-iT kit, and total nuclei and EdU positive nuclei creatine kinase (CKM) (Figure 1D), and withdrawal from counted by hand. the cell cycle based on EdU incorporation (Figure 1E). RD cells were labeled with BrdU at a final concentra- As in normal myogenesis, expression of ZNF238 tion of 30 μg/mL in differentiation media for 24 h. Cells decreased both ID2 and ID3 (Figure 1F). This differenti- were treated with hydrochloric acid before incubation ation is not restricted to the embryonal RMS RD cell with anti-BrdU antibody (Invitrogen A21300), and fluor- line; the RhJT alveolar line expressing RUNX1 showed escent secondary antibody (Jackson Immunoresearch). similar results (Additional file 3: Figure S2). Nuclei were detected with DAPI, and cells counted by hand. miR-206 expression correlates with factor-induced Western blots were performed on whole cell differentiation in RMS cells lysates collected in Laemelli buffer containing 10% We previously demonstrated that the expression of the beta-mercaptoethanol. All blots were blocked in 3% milk MD ~ E dimer in RD cells down-regulated multiple myo- (w/v) in 0.5% Tween-20-containing PBS before incubation genic inhibitors [8], consistent with induction of a with primary antibody (MHC: MF-20, MyoD: 5.8A, microRNA. MicroRNA microarrays from MD ~ E trans- RUNX1: Abcam, ab23980), a HRP-conjugated secondary duced RD cells identified several microRNAs that chan- antibody (Jackson, West Grove, USA), and chemilumines- ged expression and miR-206, a microRNA that has been cent detection (Amersham, Pittsburgh, USA). shown to induce myogenic differentiation in normal Cells were fixed with 2% paraformaldehyde for cells and RMS [19,20], was the most consistently 6 min at room temperature before permeabilization increased (Additional file 4: Table S2) and was confirmed with Triton X-100. Myosin heavy chain was detected by miRNA Northern blotting (Figure 2A, upper panel), with the MF-20 antibody, and nuclei detected with as was miR-133b, a miRNA from the same primary tran- DAPI. script (Figure 2A, second panel). RT-PCR for the pre- sumptive primary transcript also showed a substantial Electrophoretic mobility shift assays increase (Figure 2B). Northern blotting confirmed that Electrophoretic mobility shift assays were performed as the forced dimer decreased miR-199a* expression, as sug- described previously [13]. Probe sequences are listed in gested by the array results (Additional file 5: Figure S3A), (Additional file 1: Table S1). and that miR-29b, previously implicated in RMS differ- entiation, was expressed at low levels and did not Results change in response to MD ~ E expression (Additional RUNX1 and ZNF238 differentiate rhabdomyosarcoma cells file 5: Figure S3B) [21]. Furthermore, RD cells differen- RUNX1 is a runt-related transcription factor with a well- tiated through RUNX1 and ZNF238 expression showed characterized hematopoietic role [14] that is expressed an increase in mature miR-206 in both cases (Figure 2C), in muscle cells [15], functions in denervated muscle along with an increase in primary transcript (data not [16], and is regulated by the MyoD network [8,17], but shown). miR-206 levels in the myogenic C2C12 cell line has an uncharacterized role in developing muscle. showed that miR-206 expression in proliferative and dif- ZNF238 (aka RP58) is induced by MyoD and directly ferentiated RMS resembled the changes as C2C12 cells down-regulates the inhibitory Id factors [18]. MyoD shift from beginning myogenesis (90% confluency) to ChIP-seq (chromatin immunoprecipitation coupled to myotubes (DM) (Figure 2D). high-throughput sequencing) found an association be- In agreement with previous reports demonstrating that tween the RUNX1 and ZNF238 binding motifs and miR-206 alone is sufficient to differentiate RMS cells MyoD bound sites in muscle cells [11] and qPCR [20,22,23], transient transfection of pre-miR-206 constructs MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 4 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 1 Expression of RUNX1 or ZNF238 leads to terminal differentiation of RMS cells. (A) qPCR for RUNX1 was performed in RD cells either infected with a control virus, or the forced MyoD ~ E dimer (MD ~ E) as well as control (0 h) human fibroblasts and fibroblasts differentiated into myotubes (96 h). (B) RT-PCR for the two isoforms of ZNF238 in RD cells and fibroblasts as in 1A. (C) Myosin heavy chain (MHC) immunostains in RD cells either not infected (no infection), infected with a control GFP-expressing lentivirus (GFP control) or RUNX1 or ZNF238 expressing lentivirus. All cells were infected at approximately equivalent MOIs, and cells were allowed to differentiate for 72 h in low-serum media before staining. GFP was detected directly, without the use of an antibody. (D) qPCR for muscle-specific creatine kinase (CKM) in RD cells infected with either ZNF238 or RUNX1 viruses compared to cells with control retroviruses. (E) After 24 h of differentiation, RD cells were pulsed for a further 24 h with EdU-containing differentiation media, before fixation and quantification of the percentage of EdU-positive cells. (F) qPCR for ID2 and ID3 in control and ZNF238-expressing RD cells. All qPCR data are normalized to TIMM17b expression, and the level in control cells is set to 1. All -4 bar graphs represent the mean ± SEM of at least three independent experiments. *: P< 0.05; **: P< 0.01; ***: P< 0.001; ****: P< 1×10 into RD cells resulted in myotube formation (Figure 2E), data not shown). As would be expected from prior reports an increase in CKM expression (Figure 2F), and evidence of its effect on myogenic cells [24], introduction of miR- that cells undergoing morphological change withdrew 133b did not lead to RMS differentiation as judged by ei- from the cell cycle (Figure 2G, H), with similar results in ther morphology or gene expression (Additional file 6: the alveolar RhJT cells (Additional file 6: Figure S4A and Figure S4B, C). MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 5 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 MyoD directly regulates miR-206 [25] and a putative MyoD to enhance the expression of miR-206. ZNF238 RUNX1 binding site exists near the MyoD binding site. did not activate the miR-206 reporter (data not shown), RUNX1 alone showed minor activation of a miR-206 suggesting that the reporter does not have the elements luciferase reporter, while RUNX1 combined with MyoD or context for ZNF238 regulation. showed synergistic activation (Figure 2I, black bars), RUNX1 also induced the expression of ZNF238 in RD which was dependent on the integrity of the RUNX1 cells and ChIP identified RUNX1 binding close to the binding site (Figure 2I, grey bars). ChIP experiments in TSS of ZNF238, suggesting it functions directly to acti- RD cells confirmed that RUNX1 binds the miR-206 pro- vate ZNF238 (Figure 3A and 3B). Consistent with moter (Figure 2J). Therefore, RUNX1 cooperates with this model, ID2 and ID3 expression were decreased Figure 2 (See legend on next page.) MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 6 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 (See figure on previous page.) Figure 2 MyoD ~ E, RUNX1, and ZNF238 increase miR-206. (A) microRNA northern blots to detect the mature form of the indicated microRNAs in RD cells infected with either empty (control) retrovirus, or retrovirus expressing MyoD ~ E (MD ~ E). (B) RT-PCR using primers located in the pre- and pri-miR-206 sequence to detect the primary miR-206 transcript. TIMM17b is the internal control. (C) microRNA northerns as in 2A, in RD cells expressing a transcription factor as indicated. (D) microRNA northerns in C2C12 cells at various stages of differentiation ranging from undifferentiated myoblasts (50% GM), through beginning differentiation (90% GM) to myotubes (DM). (E) Immunostains for MHC in RD cells transiently transfected with either a pre-miR-206 RNA construct, or a negative control construct. Nuclei were stained with DAPI. (F) qPCR for CKM in RD cells treated as in E. (G) RD cells treated as in E were pulsed with BrdU for 24 h and then stained and counted to determine the extent of co-localization of MHC + myotubes and BrdU + nuclei. (H) RD cells transiently transfected as in E were pulsed for 24 h with BrdU-containing differentiation media before fixation and quantification of the percentage of BrdU positive cells. The percent reduction of BrdU + nuclei almost exactly equals the percent of cells found to be MHC + (not shown). (I) Luciferase activity in RD cells using a miR-206 promoter driven reporter and transiently transfected factors as indicated. ‘206 RUNX mutant’ indicates that the reporter has had a putative RUNX1 binding site mutated to prevent RUNX1 binding. Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co- transfected renilla plasmid. (J) RUNX1 ChIP assays, both with normal PCR and qPCR results, at the miR-206 promoter and a control locus before (Control) and after (RUNX1) infection of the cells with empty or RUNX1-expressing retrovirus. All PCRs in the imaged gel (upper) were performed for the same number of cycles. qPCR results (lower) represent the mean ± SEM of two independent ChIP experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. All other graphs in this figure represent the mean ± SEM of at least three independent experiments. * : P< 0.05; ** : P< 0.01; *** : P< 0.001. in RD cells expressing RUNX1 (Figure 3C). In con- Together, this shows that MyoD functions in nested trast, ZNF238 did not upregulate RUNX1 expression, feed-forward circuits with RUNX1 and ZNF238 to but we confirmed the prior report [18] that MyoD activate miR-206 expression and induce differenti- activates ZNF238 (Additional file 7: Figure S5). ationinthe RD cells. Figure 3 ZNF238 is a downstream target of MyoD and RUNX1. (A) qPCR for RUNX1 and ZNF238 in RD cells transduced with virus expressing the converse factor. (B) RUNX1 ChIP results at the intron of ZNF238 and a control locus before (Control) and after (RUNX1) infection of the cells with empty or RUNX1-expressing retrovirus. (C) qPCR for ID genes in control and RUNX1-expressing RD cells. PCRs in the imaged gel in 3B were performed for the same number of cycles. The graph in 3B represents qPCR data showing the mean ± SEM of two independent experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. qPCRs in 3A and 3C are represented as the mean ± SEM of at least three independent experiments. *: P< 0.05; **: P< 0.01. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 7 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 RUNX1, ZNF238, and miR-206 activate a common We have previously shown that the inhibitory bHLH differentiation program factor Musculin (MSC) inhibits MyoD activity in RD Gene expression arrays were performed on RD cells cells and other RMS [8]. Furthermore, since miR-206 transduced individually with RUNX1-, ZNF238-, and has been shown to inhibit MSC expression [19], we miR-206 expressing viruses. GO analysis ranked by hypothesized that the opposing activities of MyoD and P values identified multiple muscle-related categories MSC might constitute an on-off switch at the miR-206 induced by each factor, with four of the ten most signifi- promoter. ChIP showed that MSC was bound to the same cant categories shared between all factors (Additional region as MyoD in undifferentiated RD cells (Figure 5A), file 8: Table S3). In agreement with our deduced epistatic and MSC occupancy diminished in RD cells that under- relationship, the number of genes significantly regulated went RUNX1- (Figure 5B) or MD ~ E-mediated differenti- (fold change> 2, FDR <0.05) by each factor became se- ation (Additional file 11: Figure S7). High-throughput quentially reduced from RUNX1 (734) to ZNF238 (616) sequencing of the MyoD and MSC ChIP material to miR-206 (355) and showed substantial overlap (ChIP-seq) from undifferentiated RD cells with analysis (Figure 4A) and correlation (Figure 4B). Loosening the restricted to sequences mapping to the miR-206 promoter fold-change threshold showed that nearly all genes regu- revealed distinct MyoD and MSC peaks over two adjacent lated by miR-206 similarly changed expression in re- E-boxes (Figure 5C), indicating a MyoD bound E-box adja- sponse to RUNX1 and/or ZNF238 (Figure 4C, top). cent to a MSC bound E-box. Similarly, a large portion of the ZNF238 genes were also Electrophoretic mobility shift assaysdemonstrated that regulated by RUNX1 (Figure 4C, bottom). the E-box associated with the MyoD peakhad a higher RUNX1, ZNF238, and miR-206 affected the expression affinity for MyoD compared to the E-box underthe MSC of a variety of transcription factors and signaling path- peak, but that both E-boxes can be bound by either- ways involved in myogenesis (Additional file 9: Table S4), MyoD or MSC in vitro (Additional file 12: Figure S8). and a subset of the changes were confirmed by RT-PCR Whenthe E-protein is in excess, gel shift assays show that (Figure 4D). The MRF myogenin, a MyoD target [26], MyoD:Eheterodimers can compete with MSC:E heterodi- increased in response to all three of the factors. MEF2C mers to bindthe E-box under the MyoD ChIP-seq peak and MEF2D, cooperative factors for MyoD activity [17], (Figure 5D, leftpanel, lane 1) and that decreasing the were up-regulated. Down-regulation of positive regula- amount of MSC shiftsthe binding progressively toward tors of the cell cycle (MYCN, RCOR2, E2F2)and various MyoD:E protein heterodimers(lanes 2–5). Even with a rela- members of the HES/HEY family (HEY1, HES6, HEYL, tive excess of E-protein,MSC:E heterodimers outcompete HES1) was observed as well. It has previously been MyoD:E heterodimers onthe E-box under the MSC demonstrated that interference with HES1 contributes ChIP-seq peak andMyoD:E bindingto this site occurred to RMS proliferation [27], and the HES/HEY family is only when MSC levels were decreased (Figure 5D, right known to be Notch responsive [28], a signaling pathway panel, lanes 1–5). Therefore the relative affinitiesof MSC:E with myogenic inhibitory effects [29-31]. Among miR- and MyoD:E heterodimers for the two E-boxesin the 206’s most strongly down-regulated targets were two miR-206 regulatory regions likely account for theob- members of the Notch signaling pathway, DLL3 and served endogenous binding (Figure 5C) and suggest that NOTCH3. decreasinglevels of MSC would result in occupancy of both E-boxes by MyoD. Co-transfection experiments showed that MyoD:E12 The miR-206 promoter integrates multiple inputs to heterodimers robustly activated the miR-206 reporter switch from inhibition by MSC to activation by MyoD containing both E-boxes and this was prevented by We have previously shown that MyoD binds and acti- MSC (Figure 5E, black bars). MSC also repressed ac- vates the expression of the miR-206 gene in normal tivation by the MD ~ E dimer, suggesting the effect of myogenesis [25], whereas our current data indicate that MSC is due to DNA binding, not interference with the miR-206 promoter integrates multiple inputs to the formation of MyoD:E dimers (Additional file 13: modulate its activation by MyoD. Somewhat to our sur- Figure S9). Mutation of the MSC binding site made prise, ChIP in RD cells showed that MyoD was bound to the reporter insensitive to activation by MyoD and the miR-206 promoter in undifferentiated RD cells that E12 (Figure 5E, grey bars). This suggests that MSC is repressing miR-206 by physically occluding an E-box expressed only low levels of miR-206 (Additional file 10: Figure S6A) and regional enrichment for acetylated H4 that MyoD needs to occupy for full activation. The histones indicated active regional histone acetyltransfer- MyoD and MSC ChIP-seq data also supports this conclusion. Compared to the MyoD peak in RD cells, ase activity (Additional file 10: Figure S6B). Therefore, a factor, or factors, was preventing efficient transcriptional there is a broadening of the MyoD peak in myotubes activation by the bound MyoD. that appears to widen to include E-boxes located MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 8 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 4 RUNX1, ZNF238, and miR-206 function through common mechanisms. (A) 3-way Venn diagram representing the overlap between significantly regulated (fold-change >2, FDR <0.05, overlap considers only changes in same direction) gene targets in RD cells differentiated either through RUNX1, ZNF238, or miR-206 expression relative to GFP-infected controls. The table indicates the breakdown of upregulated versus down-regulated genes for each portion of the Venn diagram. (B) Scatter plots showing pairwise comparisons of gene expression. All values are plotted as the log of the fold-change relative to GFP-infected controls, as indicated along the x- and y-axes. Correlation is listed for each comparison in the matrix. (C) (upper panel) Bar graph demonstrating that the majority of the 95 genes listed as being ‘uniquely’ regulated by miR-206 in 4A are also regulated by RUNX1 and/or ZNF238, but at lower levels of expression change. FDR was kept constant (<0.05) in this analysis, and to be included as a ‘shared’ target, the change had to occur in the same direction (either up- or down-regulated) in RUNX1 and/or ZNF238 as in miR-206. (bottom panel) Analysis as in the top panel for genes in the ZNF238 unique and ZNF238:miR-206 intersection groups relative to RUNX1 changes. (D) RT-PCR for select gene targets from Additional file 9: Table S4. TIMM17b serves as the internal control. more proximally to the start of the miR-206 tran- activation domain by splicing exon 2 directly to exon 5 script (Figure 5C, bottom panel), suggesting that in that we called E2A-2/5. To determine whether the E2A- differentiated muscle, MyoD occupies additional positions. 2/5 splice form contributed to the bHLH balance at the Consistent with a model in which miR-206 activity miR-206 promoter, we tested the response of RD cells to requires multiple E-boxes to be bound by MyoD for full expression of a forced protein dimer consisting of MyoD activation, mutation of the MyoD-binding E-box also and the E12-2/5 splice form of E12 (MD ~ E2/5). MD ~ resulted in a dramatic reduction in the ability of the re- E2/5 expressing cells formed substantially fewer myo- porter to be activated by MyoD and E12 (Additional file tubes (Additional file 15: Figure S11A) while expressing 14: Figure S10). roughly equivalent amounts of dimer (Additional file 15: Figure S11B) compared to cells expressing the full-length An alternative splice form of E2A modulates MyoD MD ~ E dimer. MD ~ E expressing cells expressed sub- activation of miR-206 stantially more CKM (Figure 5F), RUNX1 (Figure 5G), We previously described a developmentally regulated and miR-206 (Figure 5H) than MD ~ E2/5 cells. However, splice form of E2A that removes a portion of the first in all cases, MD ~ E2/5 expressing cells still expressed MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 9 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Figure 5 (See legend on next page.) MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 10 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 (See figure on previous page.) Figure 5 MSC interferes with the ability of MyoD to positively regulate the microRNA miR-206 by blockading a necessary MyoD-binding site. (A) Site-specific ChIP for MSC in the miR-206 promoter and at a control locus. (B) MSC ChIP as in (A) in RD cells infected either with an empty retrovirus (Control), or RD cells differentiated through expression of RUNX1 (RUNX1). (C) Screenshot from the human UCSC Genome Browser of the region that corresponds to the miR-206 promoter. Mapped reads from ChIP-seq for MyoD in RD and HFF cells are indicated, with the number on the left-hand y-axis indicating the number of reads mapped at the peak of occupancy. The location of E-boxes are indicated at the bottom of the panel by the black rectangles. Vertical lines are drawn through the apparent highest points of occupancy for MyoD (red) and MSC (green) in RD cells. (D) Electrophoretic mobility shift assays using in vitro transcribed and translated proteins as indicated and probes that correspond to either the E-box located at the peak of MyoD binding as indicated by the red mark in 5C, or the E-box located at the peak of MSC binding, indicated by the green mark. The position of MyoD:E and MSC:E heterodimers are indicated by the arrows. The lane marked 2x E12 indicates protein mixtures that included double the amount of E12 compared to other lanes, and the triangles indicate decreasing amounts of either MSC or MyoD in the mixtures as other proteins were maintained at constant levels and total protein amounts were balanced with translation of empty CS2. (E) Luciferase assays in RD cells with constructs as indicated below the figure using either the miR-206 promoter luciferase reporter (206) or one in which the E-box that the peak of MSC occupancy is located over has been mutated (206 MSC-binding Ebox mutant). Control indicates transfection with an empty plasmid. All luciferase assays were normalized to the results from a co-transfected renilla plasmid. (F) qPCR for CKM from RD cells transduced with either an empty virus (control), or one expressing either the MD ~ E or MD ~ E2/5 forced dimer. Cells were differentiated for 24 h before collection of RNA for use in qPCR. (G) qPCR for RUNX1 from the RD cells assayed in E. (H) qPCR for pri-miR-206 from the RD cells assayed in E and F. For all ChIPs, relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified with no antibody. The control locus is located at hemoglobin beta, a silent gene in myogenic cells. Corrected relative enrichment (5B) is the ratio of enrichment at miR-206 to enrichment at the control locus. All graphs represent the mean ± SEM of at least three independent experiments. All qPCR of gene expression was corrected to TIMM17b and control cells set to 1. *: P <0.05; ** : P< 0.01; *** : P< 0.001; † : P = 0.058. more of the MyoD targets than control cells, suggesting induction of this inhibitory factor serves two purposes: that, while the specific E-protein partner is important for (1) to down-regulate genes that inhibit myogenesis; and full activation of miR-206, the inhibitory effect of MSC is (2) to interfere with MyoD binding at genes it might crucial. regulate in myoblasts. Despite differences in their direct targets, RUNX1 and ZNF238 both increase miR-206 Discussion transcription and lead to a terminally differentiated state. We have previously proposed that RMS represent an For RUNX1 we demonstrated a direct binding of the arrested phase of normal development at the transition miR-206 promoter, whereas the mechanism for ZNF238 point between regulative growth and terminal differenti- remains speculative, possibly through its suppression of ation, a transition regulated in part through the balance ID gene expression, which would increase the propor- of repressive and activating bHLH protein dimers [8]. tion of productive MyoD:E-protein heterodimers. While We showed that increasing the abundance of MyoD:E- in vitro in nature, our data on the relative ability of protein heterodimers tipped the balance to differentiation MyoD:E and MSC:E to shift the regulatory E-box and proposed that this heterodimer might target an un- sequences that control miR-206 expression (Figure 5D known central integrating function since multiple myo- and Additional file 12: Figure S8) suggest that even rela- genic repressors were down-regulated. One prediction of tively small changes in the availability of E-protein part- this earlier model was that modulating the abundance of ners could make a dramatic difference in the expression any of the factors that impinge on the integrating func- of miR-206. tion might be sufficient to induce differentiation in RMS. MSC, a bHLH that inhibits myogenesis [32], sup- Our present study supports and extends this model by presses the activation of miR-206 by binding an E-box demonstrating that miR-206 integrates the activity of required for induction by MyoD. A requirement for multiple proliferative and myogenic factors and acts as a more than one MyoD-bound E-box to drive full target switch that transitions the RMS from growth to activation has been described before [33-37], and it is differentiation. currently unclear whether MSC is simply preventing To test whether different myogenic co-factors can in- MyoD binding or recruiting repressive factors to the duce differentiation in RMS, we chose one transcrip- locus. The fact that the miR-206 locus has acetylated H4 tional activator and one repressor, RUNX1 and ZNF238, even when not robustly expressed suggests that MSC respectively. RUNX1 enhances MyoD activation at a var- may have a simple obstructionist role at this locus. Fu- ture work will be necessary to determine the relative iety of MyoD targets, including ZNF238 and possibly MYOG and the MEF genes. In contrast, ZNF238 down- roles of MSC and MyoD at miR-206 and other myogenic regulates multiple members of the inhibitory HES and targets. Data reported in this manuscript, when combined with HEY protein family and factors that drive proliferation. Our data on ZNF238 regulation, motif analysis, and gene previous data from us and others [8,11,18,19] suggests a targets in this and previous work [11] suggest that the specific model for the regulation of miR-206 that involves MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 11 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 nested positive and negative feed-forward and feed-back output of oscillating circuits and acts as a genetic switch to loops to create a molecular switch for regulating the tran- transition the cell from a proliferative growth phase to sition from growth to differentiation in myogenic cells differentiation. (Figure 6). In replicating myoblasts, a MyoD:E-protein het- Hematological malignancies are often categorized based erodimer binds an E-box site in the regulatory regions of on an arrested transition between stages of cellular differen- ID2 and ID3 [11] creating the potential for an oscillating tiation. Our work suggests that the same might apply to circuit: any increase in MyoD activity would increase ID RMS and possibly other solid tumors. bHLH factors control expression, which would dampen MyoD activity by de- cell fate and differentiation in multiple cell types and a bal- creasing the availability of the E-protein dimer partner. ance among bHLH dimer partners and other co-factors However, if MyoD:E-protein heterodimers pass a threshold might establish similar ‘tipping points’ at critical genes that of activity sufficient to initiate a feed-forward circuit acti- regulate the transition from regulative growth to differenti- vating RUNX1 and ZNF238, then ZNF238 shuts off ID ation. Our emerging model of multiple pathways (some production by occluding the MyoD binding sites and functioning as oscillating circuits) integrated by switch- thereby relieving the negative-feedback regulation of points for differentiation has significant implications for MyoD. The increased MyoD and RUNX1 activity can then drug therapies to induce differentiation. Different cell types more effectively compete with MSC on the miR-206 regu- may not exhibit identical convergence of pathways. There- latory regions and the increased miR-206 levels feed back fore, combining multiple drugs that each has a small to inhibit MSC, and likely other growth promoting factors effect on different components might induce differen- [8,19,20,24,38], thereby locking the cell into a committed tiation in the target cells while exhibiting low toxicity differentiation program. Therefore, miR-206 integrates the and few off-target effects. Figure 6 miR-206 integrates the output of oscillating circuits and acts as a genetic switch to transition from growth to differentiation. The experimental data support a network model composed of coupled oscillators with miR-206 functioning as a switch regulating the transition from one stable state to another. In myoblasts, MyoD, E-proteins, and ID proteins compose the first oscillating circuit: (1) MyoD:E heterodimers bind an E-box in the regulatory regions of the ID2 and ID3 genes and drive ID transcription; (2) the ID protein competitively forms dimers with the E-protein, limiting the production of active MyoD:E-protein heterodimers; (3) the decline in active MyoD:E-heterodimers results in decreased ID production; and (4) the decreased ID permits an increase in active MyoD:E-protein heterodimers and more ID production. The second oscillating circuit is composed of MyoD, E-proteins, MSC, and miR-206: (1) MyoD:E-protein and MSC:E-protein heterodimers compete for binding at the E-box in the miR-206 regulatory region, which oscillates between MyoD-activated and MSC-repressed states; (2) limiting amounts of E- protein prevent full activation by MyoD; and (3) low levels of miR-206 prevent full suppression by MSC. These circuits are coupled by their shared response to the concentration of active MyoD:E-protein heterodimers. The oscillating circuits bifurcate to a new determined state when the concentration and/or activity of the MyoD:E-proteins become sufficient to activate the expression of RUNX1 and ZNF238 in a feed-forward circuit that blocks the expression of the ID genes and permits the accumulation of active MyoD:E-protein complexes. The increase of MyoD:E-protein heterodimers together with RUNX produces higher miR-206 expression, and the increased miR-206 suppresses MSC and other inhibitors of differentiation. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 12 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Conclusions Our results in a rhabdomyosarcoma cell line provide evi- Additional file 8: Table S3. Top GO categories of genes regulated by dence for feed-forward and feed-back regulatory circuits RUNX1, ZNF238, and miR-206. in myogenic cells that regulate miR-206 expression as a Additional file 9: Table S4. Select potential regulators of myogenesis affected by RUNX1, ZNF238, and miR-206. genetic switch to transition a muscle cell from growth to Additional file 10: Figure S6. The miR-206 promoter in RD cells is differentiation. One circuit is composed of MyoD, bound by MyoD and has acetylated histone H4. (A) ChIP for MyoD in RD RUNX1, ZNF238, and ID2/3 and might function, at least cells in differentiation media shows MyoD enrichment upstream of miR- in part, to regulate the availability of E-proteins to form 206, compared to a control locus at a non-expressed gene (hemoglobin beta). (B) Site-specific ChIPs in RD cells for acetylated histone H4, at heterodimers with MyoD; whereas the second circuit hemoglobin beta (control), miR-206, and the myogenin promoter composed of MyoD, RUNX1, and MSC regulates the ex- (MYOG). ChIP results are represented as the mean ± SEM of at least three pression of miR-206. These have characteristics of independent experiments. Relative enrichment is calculated as the ratio of the % of input amplified with antibody to the % of input amplified coupled oscillatory circuits that can dampen the activity of with no antibody. *: P<0.05; **: P<0.01. MyoD during expansionary growth, or, under differenti- Additional file 11: Figure S7. MSC occupancy in the miR-206 ation promoting conditions, can be switched off and promoter diminishes with MD~E differentiation. ChIP for MSC in the miR- thereby enhance MyoD activity and drive differentiation. 206 promoter in RD cells either transduced with empty virus (Control), or differentiated through the expression of the forced MD~E protein dimer Modulating the abundance of multiple different compo- (MD~E). Values are the means ± standard deviation of two independent nents of these coupled circuits can drive differentiation, experiments. Corrected relative enrichment equals the relative suggesting that multiple targets within these circuits enrichment at miR-206/the relative enrichment at the control locus. Relative enrichment is calculated as the ratio of the % of input amplified might be candidates for targeting differentiation-inducing with antibody to the % of input amplified with no antibody. therapeutics. Additional file 12: Figure S8. In vitro assessment of MyoD and MSC binding in the miR-206 promoter. Electrophoretic mobility shift assays were performed using in vitro translated proteins as indicated and probes Additional files that represent the DNA sequence under either the E-box occupied most prominently by MyoD in RD cells as assessed by ChIP-seq results (MyoD- Additional file 1: Table S1. Primer and oligonucleotide sequences. binding E-box) or the E-box occupied most prominently by MSC (MSC- binding E-box). Bound complexes were competed with cold competitor Additional file 2: Figure S1. RD cells infected with ZNF238 and RUNX1 probes prepared at the indicated excess. viruses increase expression of the appropriate factor. (A) RT-PCR for ZNF238 in RD cells infected with either a control virus or the ZNF238- Additional file 13: Figure S9. MSC inhibits the activation of the miR- containing virus. TIMM17b is used as a loading control. (B) Western blot 206 reporter by the forced MD~E dimer. Luciferase assay results in RD using whole cell lysates for RUNX1 in control and RUNX1 virus infected cells using the miR-206 promoter reporter with constant amounts of RD cells. The blot was then stripped and reprobed for alpha-tubulin as a MyoD and E12 introduced individually or as the forced dimer, in the loading control. Bands were confirmed to be of the correct size through presence of varying amounts of co-transfected MSC. - indicates no MSC a protein size ladder (not shown). was added, 1x indicates that the MSC transfected was equal by mass to the amount of MyoD or MD~E, and 0.1x indicates that the MSC Additional file 3: Figure S2. RUNX1 differentiates alveolar subtype RMS transfected was equal to 1/10th that amount. Results are indicated as the cells. (A) Western blots on whole cell lysates from RhJT cells infected with means ± SEM from three independent experiments. Control indicates the either a RUNX1-expressing or control virus. MHC is myosin heavy chain, a results from transfection with empty vector. marker of myogenesis, and alpha-tubulin is the loading control. Blots were serially stripped and reprobed, and bands confirmed to be of the Additional file 14: Figure S10. Strong miR-206 activation is dependent correct size. (B) RT-PCR for CKM (muscle specific creatine kinase) on cells on multiple E-boxes. Luciferase assay results in RD cells with transient treated as in A. TIMM17b is the internal control. transfection as indicated using the miR-206 promoter and a reporter in which the E-box exhibiting the peak of MyoD occupancy in RD cells Additional file 4: Table S2. miRNA changes in response to MD~E (indicated by the red marker in Figure 5C) has been mutated and expression in RD cells. eliminated as a site of bHLH binding. Results are indicated as the means Additional file 5: Figure S3. Differential effects on miRNA expression ± SEM from three independent experiments. Control indicates the results by the forced MD~E dimer. (A) miRNA northern blot for miR-199a* (also from transfection with empty vector. *: P<0.05. known as miR-199a-5p) from RD cells either transduced with a control or Additional file 15: Figure S11. MD~E expression results in greater MD~E virus. (B) miRNA northern blot for miR-29b as in panel A. myotube formation than MD~E2/5 expression. (A) Light microscopy Additional file 6: Figure S4. miR-206 affects alveolar subtype RMS cells images of RD cells transduced with either control virus (Control) or virus and miR-133b does not share its effects. (A) Immunostains for MHC in expressing either the MD~E or MD~E2/5 forced protein dimers and RhJT cells transfected with either a pre-miR-206 or control construct. allowed to differentiate for 24 h. Arrows indicate representative cells that DAPI stains all nuclei. (B) Stains as in A after transfection of RD cells with have appeared to form myotubes. (B) Western blot for MyoD and alpha- pre-miR-133b or a control construct. (C) qPCR for CKM in RD cells treated tubulin, as a loading control, from cells treated as in (A). The size of the as in B. qPCR data was normalized to TIMM17b and control set to 1, with bands detected with the MyoD antibody in MD~E and MD~E2/5 lanes bars representing the mean ± SEM of three independent experiments. *: are as expected given the approximate calculated size of the forced P<0.05. dimers. Additional file 7: Figure S5. MyoD activity increases ZNF238 expression during myogenic conversion. 10T1/2 fibroblast cells expressing an Abbreviations estradiol-inducible version of MyoD were induced to undergo bHLH: Basic helix-loop-helix; ChIP: Chromatin immunoprecipitation; ChIP- myogenesis by addition of beta-estradiol to the culture medium. RNA seq: Chromatin immunoprecipitation coupled to high-throughput was taken at the indicated times under indicated conditions and qPCR sequencing; RMS: Rhabdomyosarcoma. performed to quantitate the relative levels of ZNF238. All bars indicate the mean ± SEM of at least three independent experiments. Time 0 was Competing interests set to 1, and TIMM17b served as the internal control. *: P<0.05. The authors declare no potential conflicts of interest. MacQuarrie et al. Skeletal Muscle 2012, 2:7 Page 13 of 14 http://www.skeletalmusclejournal.com/content/2/1/7 Acknowledgements 14. Cohen MM: Perspectives on RUNX genes: an update. Am J Med Genet A KLM was supported by a Developmental Biology Predoctoral Training Grant 2009, 149A:2629–2646. (T32HD007183). ZY was supported by the NIH Interdisciplinary Training Grant 15. Zhu X, Yeadon JE, Burden SJ: AML1 is expressed in skeletal muscle and is in Cancer Research (T32CA080416). SJT was supported by NIH NIAMS regulated by innervation. 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J Biol Chem 2009, 284:31921–31927. doi:10.1186/2044-5040-2-7 Cite this article as: MacQuarrie et al.: miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells. Skeletal Muscle 2012 2:7. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Apr 29, 2012

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