miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells
© MacQuarrie et al.; licensee BioMed Central Ltd. 2012
Received: 18 January 2012
Accepted: 29 April 2012
Published: 29 April 2012
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.
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.
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.
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).
KeywordsRhabdomyosarcoma RUNX1 ZNF238 miR-206 MSC myogenesis
Chromatin immunoprecipitation coupled to high-throughput sequencing
Rhabdomyosarcoma (RMS) is a soft tissue sarcoma characterized by expression of myogenic regulatory factors, especially MyoD, and other skeletal muscle genes [1–3]. MyoD is capable of converting multiple cell types into terminally differentiated skeletal muscle [4, 5] and normally acts as a nodal point in differentiation to integrate multiple signals to balance regulative growth and cell differentiation . However, in RMS the ability of MyoD to induce differentiation is impaired .
Our recent study indicated that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the formation of heterodimers between MyoD and the full-length E-proteins . MyoD binds DNA as a heterodimer with an E-protein (E2A, E2-2, or HEB) . 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 compete with the full-length E-proteins for heterodimerization with MyoD. The demonstration that a forced heterodimer of MyoD and a full-length E-protein suppressed multiple inhibitory mechanisms and induced differentiation in the RD and other rhabdomyosarcomas suggested that a central integrating mechanism might regulate the switch from regulative growth to differentiation .
If a central integrating mechanism does exist, then multiple pathways should regulate its activity and multiple factors should be able to induce differentiation in RMS. In this manuscript we demonstrate that modulation of multiple different myogenic factors can induce differentiation in rhabdomyosarcoma cells and that 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. These findings suggest that multiple components of differentiation pathways that converge on miR-206 might be targeted for differentiation therapies in at least some rhabdomyosarcomas.
Coding sequences of RUNX1 and ZNF238 were amplified by PCR from human myotube cDNA, and cloned into pRRLSIN.cPPT.PGK/GFP.WPRE, pCLBabe, and pCS2. The miR-206 lentivirus was purchased from Open Biosystems. Lentiviral supernatant was produced by the FHCRC core viral facility, and viruses from pCLBabe plasmids packaged using BBS-mediated calcium precipitation into Phoenix cells. MD ~ E2/5 was cloned into the pCLBabe backbone. For the miR-206 promoter luciferase reporter, a piece of approximately 2.5 kb of DNA upstream of human miR-206 was amplified using primers located in ( Additional file 1: Table S1).
Cell culture, transient transfections, and luciferase assays
RD cells were obtained from ATCC (American Type Culture Collection) in approximately 1990, and all analyses have been performed on cells that originated from low passage number frozen aliquots. RhJT cells were obtained from PJ Houghton in 1990 and, as with RD cells, all experiments have used cells from original low passage number frozen cells. RD and RhJT cells were maintained in DMEM with 10% bovine calf serum and 1% Pen-Strep (Gibco, Grand Island, USA). Low-serum differentiation media consisted of DMEM with 1% horse serum, 1% Pen-Strep and 10 μg/mL insulin and transferrin. Transient transfections for luciferase assays were performed using Superfect according to manufacturer’s directions with a total of 3 ug of plasmid DNA in each well (Qiagen, Valencia, USA). Luciferase assays used the Dual-Luciferase Assay kit (Promega, Madison, USA) according to manufacturer’s directions. All results were corrected to co-transfected Renilla-pCS2 and are reported as the mean ± SEM of at least three independent experiments, with significance calculated using a t-test, and each experiment having three biological replicates of all conditions. Transient transfections of pre-microRNA constructs were performed using 25 μM of pre-miR constructs (Ambion, Grand Island, USA) and siPORT NeoFX (Ambion) according to the manufacturer’s directions.
qPCR and RT-PCR
All qPCR was performed using SybrGreen from Bio-Rad on an Applied Biosystems 7900HT. Relative expression levels were calculated using cDNA dilution standard curves or delta-delta Ct calculations. All values are reported as the mean ± SEM of at least three independent biological experiments and significance tested with t-tests. All RT-PCRs were performed simultaneously with minus reverse transcriptase controls to check the absence of signal. Primers used for amplification are listed in ( Additional file 1: Table S1).
RNA was isolated using acid-phenol purification from two biologically independent sets of RD cells transduced with either MD ~ E or empty vector pCLBabe retroviruses and differentiated for 24 h after puromycin selection. miRNAs were labeled using Exiqon’s miRCURY labeling kit, and then competitively hybridized to in-house spotted miRNA arrays (FHCRC core facility). Cutoffs for significant changes were a FDR <0.05 and a fold-change >2. Data are available under GEO accession number GSE35921.
RNA was isolated using the RNeasy mini kit (Qiagen) from RD cells infected with either RUNX1-, ZNF238-, miR-206-, or GFP-expressing lentiviruses and allowed to differentiate for 72 h. Each condition was performed with three independent biological replicates. RNA was hybridized to Illumina Human HT-12 v4 BeadChips. Analysis was performed in R/Bioconductor using the lumi and limma packages with annotations found in the lumiHumanAll.db package. P values were adjusted to account for multiple testing using Benjamini and Hochberg’s method, and cutoffs for significant changes were a FDR <0.05 and a fold-change >2. GO category enrichment tests were performed using the conditional algorithm of the GOstats package and a gene ‘universe’ of any gene with a GO annotation that was called as ‘present’ in at least one of the 12 array datasets. Data are available under GEO accession number GSE35921.
ChIPs (Chromatin immunoprecipitations)
ChIPs were performed as has been described previously . Antibodies used were as follows: RUNX1 (Abcam, ab23980), MyoD , MSC (Santa Cruz, sc-9556X), Acetylated Histone H4 (Upstate 06-866). Primers used for amplification are listed in ( Additional file 1: Table S1).
EdU and BrdU labeling, western blots, and cell stains
After 24 h in low-serum differentiation media, cells were shifted to differentiation media supplemented with EdU at a final concentration of 50 μM (Invitrogen) and incubated for a further 24 h. Cells were then fixed and stained according to the manufacturer’s protocols using the Click-iT kit, and total nuclei and EdU positive nuclei counted by hand.
RD cells were labeled with BrdU at a final concentration of 30 μg/mL in differentiation media for 24 h. Cells were treated with hydrochloric acid before incubation with anti-BrdU antibody (Invitrogen A21300), and fluorescent secondary antibody (Jackson Immunoresearch). Nuclei were detected with DAPI, and cells counted by hand.
Western blots were performed on whole cell lysates collected in Laemelli buffer containing 10% beta-mercaptoethanol. All blots were blocked in 3% milk (w/v) in 0.5% Tween-20-containing PBS before incubation with primary antibody (MHC: MF-20, MyoD: 5.8A, RUNX1: Abcam, ab23980), a HRP-conjugated secondary antibody (Jackson, West Grove, USA), and chemiluminescent detection (Amersham, Pittsburgh, USA).
Cells were fixed with 2% paraformaldehyde for 6 min at room temperature before permeabilization with Triton X-100. Myosin heavy chain was detected with the MF-20 antibody, and nuclei detected with DAPI.
Electrophoretic mobility shift assays
RUNX1 and ZNF238 differentiate rhabdomyosarcoma cells
Lentiviral-mediated expression of each factor in RD cells ( Additional file 2: Figure S1) induced muscle differentiation, as measured by morphology and expression of myosin heavy chain (MHC) (Figure 1C), muscle-specific creatine kinase (CKM) (Figure 1D), and withdrawal from the cell cycle based on EdU incorporation (Figure 1E). As in normal myogenesis, expression of ZNF238 decreased both ID2 and ID3 (Figure 1F). This differentiation is not restricted to the embryonal RMS RD cell line; the RhJT alveolar line expressing RUNX1 showed similar results ( Additional file 3: Figure S2).
miR-206 expression correlates with factor-induced differentiation in RMS cells
In agreement with previous reports demonstrating that miR-206 alone is sufficient to differentiate RMS cells [20, 22, 23], transient transfection of pre-miR-206 constructs into RD cells resulted in myotube formation (Figure 2E), an increase in CKM expression (Figure 2F), and evidence that cells undergoing morphological change withdrew from the cell cycle (Figure 2G, H), with similar results in the alveolar RhJT cells ( Additional file 6: Figure S4A and data not shown). As would be expected from prior reports of its effect on myogenic cells , introduction of miR-133b did not lead to RMS differentiation as judged by either morphology or gene expression ( Additional file 6: Figure S4B, C).
MyoD directly regulates miR-206  and a putative RUNX1 binding site exists near the MyoD binding site. RUNX1 alone showed minor activation of a miR-206 luciferase reporter, while RUNX1 combined with MyoD showed synergistic activation (Figure 2I, black bars), which was dependent on the integrity of the RUNX1 binding site (Figure 2I, grey bars). ChIP experiments in RD cells confirmed that RUNX1 binds the miR-206 promoter (Figure 2J). Therefore, RUNX1 cooperates with MyoD to enhance the expression of miR-206. ZNF238 did not activate the miR-206 reporter (data not shown), suggesting that the reporter does not have the elements or context for ZNF238 regulation.
RUNX1, ZNF238, and miR-206 activate a common differentiation program
RUNX1, ZNF238, and miR-206 affected the expression of a variety of transcription factors and signaling pathways involved in myogenesis ( Additional file 9: Table S4), and a subset of the changes were confirmed by RT-PCR (Figure 4D). The MRF myogenin, a MyoD target , increased in response to all three of the factors. MEF2C and MEF2D, cooperative factors for MyoD activity , were up-regulated. Down-regulation of positive regulators of the cell cycle (MYCN RCOR2 E2F2) and various members of the HES/HEY family (HEY1, HES6, HEYL HES1) was observed as well. It has previously been demonstrated that interference with HES1 contributes to RMS proliferation , and the HES/HEY family is known to be Notch responsive , a signaling pathway with myogenic inhibitory effects [29–31]. Among miR-206’s most strongly down-regulated targets were two members of the Notch signaling pathway, DLL3 and NOTCH3.
The miR-206 promoter integrates multiple inputs to switch from inhibition by MSC to activation by MyoD
We have previously shown that MyoD binds and activates the expression of the miR-206 gene in normal myogenesis , whereas our current data indicate that the miR-206 promoter integrates multiple inputs to modulate its activation by MyoD. Somewhat to our surprise, ChIP in RD cells showed that MyoD was bound to the miR-206 promoter in undifferentiated RD cells that expressed only low levels of miR-206 ( Additional file 10: Figure S6A) and regional enrichment for acetylated H4 histones indicated active regional histone acetyltransferase activity ( Additional file 10: Figure S6B). Therefore, a factor, or factors, was preventing efficient transcriptional activation by the bound MyoD.
Electrophoretic mobility shift assaysdemonstrated that the E-box associated with the MyoD peakhad a higher affinity for MyoD compared to the E-box underthe MSC peak, but that both E-boxes can be bound by eitherMyoD or MSC in vitro (Additional file 12: Figure S8). Whenthe E-protein is in excess, gel shift assays show that MyoD:Eheterodimers can compete with MSC:E heterodimers to bindthe E-box under the MyoD ChIP-seq peak (Figure 5D, leftpanel, lane 1) and that decreasing the amount of MSC shiftsthe binding progressively toward MyoD:E protein heterodimers(lanes 2–5). Even with a relative excess of E-protein,MSC:E heterodimers outcompete MyoD:E heterodimers onthe E-box under the MSC ChIP-seq peak andMyoD:E bindingto this site occurred only when MSC levels were decreased (Figure 5D, right panel, lanes 1–5). Therefore the relative affinitiesof MSC:E and MyoD:E heterodimers for the two E-boxesin the miR-206 regulatory regions likely account for theobserved endogenous binding (Figure 5C) and suggest that decreasinglevels of MSC would result in occupancy of both E-boxes by MyoD.
Co-transfection experiments showed that MyoD:E12 heterodimers robustly activated the miR-206 reporter containing both E-boxes and this was prevented by MSC (Figure 5E, black bars). MSC also repressed activation by the MD ~ E dimer, suggesting the effect of MSC is due to DNA binding, not interference with the formation of MyoD:E dimers ( Additional file 13: Figure S9). Mutation of the MSC binding site made the reporter insensitive to activation by MyoD and E12 (Figure 5E, grey bars). This suggests that MSC is repressing miR-206 by physically occluding an E-box that MyoD needs to occupy for full activation. The MyoD and MSC ChIP-seq data also supports this conclusion. Compared to the MyoD peak in RD cells, there is a broadening of the MyoD peak in myotubes that appears to widen to include E-boxes located more proximally to the start of the miR-206 transcript (Figure 5C, bottom panel), suggesting that in differentiated muscle, MyoD occupies additional positions. Consistent with a model in which miR-206 activity requires multiple E-boxes to be bound by MyoD for full activation, mutation of the MyoD-binding E-box also resulted in a dramatic reduction in the ability of the reporter to be activated by MyoD and E12 ( Additional file 14: Figure S10).
An alternative splice form of E2A modulates MyoD activation of miR-206
We previously described a developmentally regulated splice form of E2A that removes a portion of the first activation domain by splicing exon 2 directly to exon 5 that we called E2A-2/5. To determine whether the E2A-2/5 splice form contributed to the bHLH balance at the miR-206 promoter, we tested the response of RD cells to expression of a forced protein dimer consisting of MyoD and the E12-2/5 splice form of E12 (MD ~ E2/5). MD ~ E2/5 expressing cells formed substantially fewer myotubes ( Additional file 15: Figure S11A) while expressing roughly equivalent amounts of dimer ( Additional file 15: Figure S11B) compared to cells expressing the full-length MD ~ E dimer. MD ~ E expressing cells expressed substantially more CKM (Figure 5F), RUNX1 (Figure 5G), and miR-206 (Figure 5H) than MD ~ E2/5 cells. However, in all cases, MD ~ E2/5 expressing cells still expressed more of the MyoD targets than control cells, suggesting that, while the specific E-protein partner is important for full activation of miR-206, the inhibitory effect of MSC is crucial.
We have previously proposed that RMS represent an arrested phase of normal development at the transition point between regulative growth and terminal differentiation, a transition regulated in part through the balance of repressive and activating bHLH protein dimers . We showed that increasing the abundance of MyoD:E-protein heterodimers tipped the balance to differentiation and proposed that this heterodimer might target an unknown central integrating function since multiple myogenic repressors were down-regulated. One prediction of this earlier model was that modulating the abundance of any of the factors that impinge on the integrating function might be sufficient to induce differentiation in RMS. Our present study supports and extends this model by demonstrating that miR-206 integrates the activity of multiple proliferative and myogenic factors and acts as a switch that transitions the RMS from growth to differentiation.
To test whether different myogenic co-factors can induce differentiation in RMS, we chose one transcriptional activator and one repressor, RUNX1 and ZNF238, respectively. RUNX1 enhances MyoD activation at a variety of MyoD targets, including ZNF238 and possibly MYOG and the MEF genes. In contrast, ZNF238 down-regulates multiple members of the inhibitory HES and HEY protein family and factors that drive proliferation. Our data on ZNF238 regulation, motif analysis, and gene targets in this and previous work  suggest that the induction of this inhibitory factor serves two purposes: (1) to down-regulate genes that inhibit myogenesis; and (2) to interfere with MyoD binding at genes it might regulate in myoblasts. Despite differences in their direct targets, RUNX1 and ZNF238 both increase miR-206 transcription and lead to a terminally differentiated state. For RUNX1 we demonstrated a direct binding of the miR-206 promoter, whereas the mechanism for ZNF238 remains speculative, possibly through its suppression of ID gene expression, which would increase the proportion of productive MyoD:E-protein heterodimers. While in vitro in nature, our data on the relative ability of MyoD:E and MSC:E to shift the regulatory E-box sequences that control miR-206 expression (Figure 5D and Additional file 12: Figure S8) suggest that even relatively small changes in the availability of E-protein partners could make a dramatic difference in the expression of miR-206.
MSC, a bHLH that inhibits myogenesis , suppresses the activation of miR-206 by binding an E-box required for induction by MyoD. A requirement for more than one MyoD-bound E-box to drive full target activation has been described before [33–37], and it is currently unclear whether MSC is simply preventing MyoD binding or recruiting repressive factors to the locus. The fact that the miR-206 locus has acetylated H4 even when not robustly expressed suggests that MSC may have a simple obstructionist role at this locus. Future work will be necessary to determine the relative roles of MSC and MyoD at miR-206 and other myogenic targets.
Hematological malignancies are often categorized based on an arrested transition between stages of cellular differentiation. Our work suggests that the same might apply to RMS and possibly other solid tumors. bHLH factors control cell fate and differentiation in multiple cell types and a balance among bHLH dimer partners and other co-factors might establish similar ‘tipping points’ at critical genes that regulate the transition from regulative growth to differentiation. Our emerging model of multiple pathways (some functioning as oscillating circuits) integrated by switch-points for differentiation has significant implications for drug therapies to induce differentiation. Different cell types may not exhibit identical convergence of pathways. Therefore, combining multiple drugs that each has a small effect on different components might induce differentiation in the target cells while exhibiting low toxicity and few off-target effects.
Our results in a rhabdomyosarcoma cell line provide evidence for feed-forward and feed-back regulatory circuits in myogenic cells that regulate miR-206 expression as a genetic switch to transition a muscle cell from growth to differentiation. One circuit is composed of MyoD, RUNX1, ZNF238, and ID2/3 and might function, at least in part, to regulate the availability of E-proteins to form heterodimers with MyoD; whereas the second circuit composed of MyoD, RUNX1, and MSC regulates the expression of miR-206. These have characteristics of coupled oscillatory circuits that can dampen the activity of MyoD during expansionary growth, or, under differentiation promoting conditions, can be switched off and thereby enhance MyoD activity and drive differentiation. Modulating the abundance of multiple different components of these coupled circuits can drive differentiation, suggesting that multiple targets within these circuits might be candidates for targeting differentiation-inducing therapeutics.
KLM was supported by a Developmental Biology Predoctoral Training Grant (T32HD007183). ZY was supported by the NIH Interdisciplinary Training Grant in Cancer Research (T32CA080416). SJT was supported by NIH NIAMS (R01AR045113).
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