MYC Mediates mRNA Cap Methylation of CanonicalWnt/b ......Published OnlineFirst November 29, 2016; DOI: 10.1158/1541-7786.MCR-16-0247 Herein, we report that MYC promotes mRNA cap methylation

Oncogenes and Tumor Suppressors

MYC Mediates mRNA Cap Methylation ofCanonicalWnt/b-CateninSignalingTranscriptsByRecruiting CDK7 and RNA MethyltransferaseValeriya Posternak, Matthew H. Ung, Chao Cheng, and Michael D. Cole

Abstract

MYC is a pleiotropic transcription factor that activates andrepresses a wide range of target genes and is frequentlyderegulated in human tumors. While much is known aboutthe role of MYC in transcriptional activation and repression,MYC can also regulate mRNA cap methylation through amechanism that has remained poorly understood. Here, it isreported that MYC enhances mRNA cap methylation of tran-scripts globally, specifically increasing mRNA cap methylationof genes involved in Wnt/b-catenin signaling. Elevated mRNAcap methylation of Wnt signaling transcripts in response toMYC leads to augmented translational capacity, elevated pro-

tein levels, and enhanced Wnt signaling activity. Mechanisticevidence indicates that MYC promotes recruitment of RNAmethyltransferase (RNMT) to Wnt signaling gene promotersby enhancing phosphorylation of serine 5 on the RNA poly-merase II carboxy-terminal domain, mediated in part throughan interaction between the TIP60 acetyltransferase complexand TFIIH.

Implications:MYC enhances mRNA cap methylation above andbeyond transcriptional induction. Mol Cancer Res; 15(2); 213–24.�2016 AACR.

IntroductionMYC is essential for normal cell growth, development, and

differentiation and plays an important role in cell metabolism,protein synthesis, and cell-cycle regulation (1, 2). When MYC isderegulated or constitutively activated via overexpression, ampli-fication, or chromosomal translocation, cells can be transformedand proliferate in the absence of extracellular mitogenic input(3). MYC overexpression is a hallmark of approximately 70% ofcancers (4), and inhibition of MYC has been shown to result intumor regression in a host- or cell-type–dependent manner (5).MYC is a pleiotropic transcription factor that induces genome-wide transcriptional amplification (6, 7) by interacting withits cofactor transactivation/transformation-associated protein(TRRAP; ref. 8) although there have beenmultiple interpretationsof the data regardingMYC's role in transcriptional gene activationand repression (9, 10). TRRAP is found in complex with eitherTIP60 or GCN5, histone acetyltransferases (HAT) that induce anopen chromatin conformation and transcription (11). Despitemany advances in the MYC field, functions of MYC independentof transactivation and repression of genes have not beencompletely evaluated.

Canonical Wnt signaling can promote transcription and ele-vated expression of MYC (12, 13). Conversely, MYC has beenshown to activate Wnt signaling by repressing the Wnt signalinginhibitors, DKK1 and SFRP1, indicating interplay between MYCand Wnt signaling (14). Canonical Wnt/b-catenin signalinghas been implicated in tissue homeostasis and development andregulates numerous cell processes via mitogenic stimulation,including cell differentiation, cell polarity, and motility(15, 16). Wnt signaling is associated with a number of diseases,including coronary artery disease, osteoporosis, diabetes, obesity,and cancer (16).

Recent studies have revealed that MYC possesses the ability topost-transcriptionally promote protein accumulation withoutaltering mRNA levels by stimulating mRNA cap methylation(17–19). This effect is independent ofMYC-induced transcriptionas mRNA cap methylation occurs in the absence of DNA and/orMAX binding. mRNA capping and methylation occur duringtranscriptional initiation (20, 21). Phosphorylation of serine-5(phospho-S5) on the carboxy-terminal domain (CTD) of RNApolymerase II (RNA Pol II) leads to the production of nascentmRNA and recruitment of RNA guanylyltransferase (RNGTT)and RNA (guanine-7-)-methyltransferase (RNMT; refs. 22–24).RNGTT adds an inverted guanosine cap and RNMT, in combina-tion with RNMT-activating mini protein (RAM), methylates thecap on mRNA (19, 25–27). mRNA cap methylation furtherstabilizes the nascent mRNA, protects from exonuclease attack,and is required for efficient cap-dependentmRNA translation andprotein production (28, 29). MYC was shown to promote mRNAcap methylation of select MYC transcriptional target genes andcooperate with RNMT to promote cell transformation (18, 19).Thus far, the extent to which MYC mediates mRNA cap methyl-ation is unknown. ThemechanismbehindMYC-mediatedmRNAcap methylation and its impact in the context of normal andtransformed cells are not well characterized.

Department of Molecular and Systems Biology, Geisel School of Medicine atDartmouth, Norris Cotton Cancer Center, Lebanon, New Hampshire.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Michael D. Cole, Geisel School of Medicine, NorrisCotton Cancer Center, One Medical Center Drive, Rubin 633 HB7936, Lebanon,NH 03756. Phone: 603-653-9975; Fax: 603-653-9952; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-16-0247

�2016 American Association for Cancer Research.

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Herein, we report that MYC promotes mRNA cap methylationand protein production of Wnt/b-catenin signaling transcriptsthrough recruitment of cyclin-dependent kinase 7 (CDK7) andconsequently RNMT to gene promoters. Interactions betweenMRG15, a component of the NuA4 TRRAP complex, and XPB,a component of TFIIH, are necessary for MYC-mediated CDK7recruitment. These findings demonstrate that MYC employs mul-tiple mechanisms, including mRNA capmethylation, to promoteprotein production, proliferation, and transformation.

Materials and MethodsCell lines, drug treatment, and Western blotting

For c-MYC induction experiments, the conditional Tet-MYCvector in P493-6 cells was repressed with 0.1 mg/mL tetracycline(Sigma) for 72 hours (30). P493-6 cells were kindly provided byChi Van Dang, University of Pennsylvania (Philadelphia, PA).MYC expression was induced for 1 or 24 hours by washing thecells three times with RPMI1640 medium containing 10% tetra-cycline system-approved FBS (Clontech). Immortalizedmamma-ry epithelial cells (IMEC) and MCF10A cells were cultured inDMEM:F12 50:50 medium supplemented with 10 ng/mL EGF,5 ng/mL insulin, 0.5 mg/mL hydrocortisone, and 5% FBS aspreviously described (31). MCF7, HeLa, HEK293T, and MDA-MB-231 cells were cultured inDMEM/10% FBS.MM1.S cells werepropagated in RPMI1640medium containing 15% FBS. For SNS-032 and THZ1 (APExBIO) treatments, MYC expression wasrepressed in P493-6 cells for 72 hours and restored throughtetracycline withdrawal for 0 or 23 hours before treatment. Cellswere treated with 100 nmol/L SNS-032 and 50 nmol/L or 250nmol/L THZ1 for 24 hours and 1 hour, respectively, followed by Fbuffer (10 mmol/L Tris pH 7.05, 50 mmol/L NaCl, 30 mmol/LNa4P2O7, 5 mmol/L ZnCl2, 50 mmol/L NaF, 10% Glycerol, 0.5%Triton X-100) lysis for immunoblotting or TRIzol RNA extractionfor mRNA analysis. Western blots were imaged using the Bio-RadMolecular Imager ChemiDoc XRSþ System and band intensitywas quantified using ImageJ.

Plasmids and RNA interferencePlasmid transfections (vector, HA-RNMT, FLAG-RAM, CBP-

MYC)were performed using LipoD293 InVitroDNATransfectionReagent per protocol (SignaGen). Cells were harvested for proteinor RNA 24 hours after transfection. Silencer Select Pre-designedsiRNAs were obtained from Life Technologies and transfectedusing RNAiMax (Invitrogen; 20 nmol/L). Cells were harvested forproteinor RNA48hours after transfection. Silencer Select negativecontrol #2 (Life Technologies) was used as a transfection control.

GST-eIF4E protein production and purificationE. coli optimized for efficient protein production [BL21 (De3);

Protein Express] were transformed with a pGEX-GST-eIF4E plas-mid. Bacteria were spun down at 8,300 � g for 15 minutes andlysed in 20 mL of LCB buffer (0.1 mol/L KCl, 20 mmol/L HEPES,0.2 mmol/L EDTA) plus 1% NP40, 1 mg/mL lysozyme, andprotease inhibitors. The lysate was kept on ice for 30 minutes,sonicated at 70% amplitude for 3minutes (1 second on, 3 secondoff), and spun at 27,000� g for 30 minutes. The supernatant wasrun over a column containing 0.3 mL immobilized 20/30-EDA-m7GTP agarose beads (Jena Bioscience) two times, followed bythree washes with Buffer A (20 mmol/L HEPES pH 7.5, 10%glycerol, 5 mmol/L 2-mercaptoethanol). GST-eIF4E was eluted

three times with 1 mmol/L m7GTP (Jena Bioscience) in Buffer A(600 mL each). The protein was dialyzed overnight in PBS plus 0.5mmol/L EDTA and 5% glycerol at 4�C.

GST-eIF4E immobilization and m7G-mRNA isolationGST-eIF4E (�7 mg) was added to a 1.5mLmicrocentrifuge tube

containing 0.5 mL F buffer. Magnetic glutathione (GSH) beads(�20 mL) preblocked in binding buffer (7mmol/LHEPES pH7.9,300 mmol/L NaCl, 0.08 mmol/L EDTA, 0.8 mmol/L DTT, 17%glycerol, 0.1% NP40, 1.1% polyvinyl alcohol, 400 mg/mL poly-uridylic acid, 17 mg/mL poly(deoxyguanylic-deoxycytidylic) acid)overnight were subsequently added to each tube and tubes wererotated for 2 hours at 4�C followed by two washes with F buffer(modified from McCracken, and colleagues; ref. 22). pppN-mRNA, GpppN-mRNA, and m7GpppN-mRNA were in vitro tran-scribed from pKS-EZH2 using theMaxiScript kit (Ambion). Maxi-Script mRNA (1 ng) or total RNA (1 mg) was added to each tubecontaining 0.3 mL binding buffer plus 1 mL RNasin Plus RNaseInhibitor (Promega) and rotated for 1.5 hours at 4�C to isolatem7G-mRNA. This rotationwas followed by twowashes withwashbuffer (20 mmol/L HEPES pH 7.9, 0.1 mol/L NaCl, 0.1 mmol/LEDTA, 1mmol/LDTT, 20%glycerol, 0.5%NP40)where the beadswere transferred to a new siliconized microcentrifuge tube inbetween the first and second wash. Following the second wash,mRNA bound to the beads was removed using TRIzol. mRNAwasprecipitated overnight in isopropanol (20 mg glycogen) at�20�Cand was resuspended in 30 mL RNase-free water. m7G-mRNAisolated from the pulldown was used as a substrate for RT-qPCRand signals were normalized to total input mRNA for eachcondition.

RNA sequencing and analysisRNA isolated from P493-6 cells at different time points fol-

lowing MYC induction (0 and 24 hours) was subjected to a m7G-mRNA pulldown using GST-eIF4E (1 mg RNA). Input RNA andmRNA isolated from the pulldown (four samples in total; twosamples from each time point, one input and one pulldown)weresubjected to 100-bp paired-end sequencing using the Illumina-HiSeq platform. Readswere aligned to the human genome (hg38)using STAR aligner version 2.3.0.1 (default setting), and readswere quantified using the human genome in Partek Flow. Totalmapped reads ranged from 13 to 17million per sample for inputs(total mRNA) and from 3 to 19 million per sample for m7G-mRNA pulldowns. Genes with

and cDNA was synthesized using the iScript cDNA Synthesis Kit(Bio-Rad). Two-step real-time PCR was performed using SYBRGreenMix on aC1000 Thermal cycler (Bio-Rad). Gene expressionwas normalized to cell number or total input mRNA andexpressed as fold change relative to control.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed acc-

ording to the Upstate ChIP Kit protocol (Millipore). Briefly, logphase cells were crosslinked with 1% formaldehyde at 37�Cand sonicated twice after lysis using a water bath sonicator(BioRuptor) on high intensity for 10 minutes at 30-second inter-vals. qPCR was used to measure enrichment and data are pre-sented as a proportion of bound DNA normalized to input foreach sample (i.e., % input) relative to an IgG control.

Polysome profilingLog phase cells were treated with 100 mg/mL cycloheximide for

1 minute, washed in PBS (100 mg/mL cycloheximide), and lysedin 200 mL lysis buffer (20mmol/L Tris pH7.5, 2.5mmol/LMgCl2,120 mmol/L KCl, 0.3% NP40, 100 mg/mL cycloheximide).Extracts were centrifuged at 19,400 � g for 15 minutes. Super-natants (100 mg total RNA) were layered over a 20-mL gradient of10% to 50% sucrose, 20 mmol/L Tris pH 7.5, 2.5 mmol/L MgCl2,60 mmol/L KCl, 100 mg/mL cycloheximide. Gradients were cen-trifuged at 166,880 � g for 4 hours. A Biocomp Gradient Masterwas used to collect 300-mL fractions and absorbance was mea-sured at 260nmol/L. RNAwas thenpurified frompooled fractionsby acid:phenol:chloroform (Ambion) extraction, and used as asubstrate for GST-eIF4E m7G-mRNA pulldown. RNA was ana-lyzed by RT-qPCR.

Protein synthesis assayP493-6 cells were labeled with 50 mmol/L Click-iT AHA

(L-azidohomoalanine; Invitrogen) for 2 hours at 37�C in methi-onine-free RPMI1640 medium at two time points for MYCinduction (0 hours, MYC off; 24 hours, MYC on). Manufacturer'sClick-iT labeling and protein detection protocols were followedwith minor modification. Cells were lysed in 100-mL lysis buffer(1% SDS in 50 mmol/L Tris-HCl, pH 8.0) for 15 minutes on ice,sonicated with a probe sonicator, and centrifuged at 19,400� g at4�C for 5minutes. Supernatant containing protein was saved, and60 mg of protein from each sample was added to the biotin alkynedetection reaction as specified in the protocol. A total of 30 mL(stock 200 mL) was run on an SDS-PAGE gel, and a Western blotanalysis was performed with probing using HRP-conjugatedstreptavidin (BioLegend) in 5% BSA for 1 hour. Intensity wasquantified using ImageJ.

TCF reporter assayFor T-cell factor (TCF) reporter assays, low-passage cells (105)

were plated onto 6-well plates. One day following plating, eachwell was transfected with 0.9 mg 14X Super TOP or 8X FOP FLASHplasmid (32) and 0.09 mg pRL-SV40 using LipoD293 In VitroDNA Transfection Reagent per protocol (SignaGen). Two daysafter transfection, cells were lysed, and luciferase and Renillaactivity were measured with Dual-Glo luciferase reagents (Pro-mega). Luciferase readings were normalized against Renilla read-ings, and TOP FLASH/FOP FLASH ratios were calculated.

Statistical analysisAll experimentswere repeated three times. Anunpaired Student

t test was performed with Welch correction to determine SD andstatistical significance. P �0.05 was considered statistically sig-nificant. Error bars represent SEM.

Further methodologic details are included in SupplementaryInformation.

The data discussed in this publication have been deposited inNCBI's Gene Expression Omnibus (Posternak and colleagues,2016) and are accessible through GEO Series accession numberGSE76614 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE76614).

ResultsMYC promotes mRNA cap methylation of essential Wntpathway transcripts

MYCwaspreviously shown topromotemRNAcapmethylationof a number of transcriptionally activated target genes in c-myc�/�

(HO 15.19) rat fibroblast cells expressing exogenous MYC(17, 18). To explore this activity further, we optimized andvalidated a capturing tool to isolate m7G-mRNA for use in atranscriptome-wide study (Supplementary Fig. S1). In our anal-ysis, we used P493-6 cells, a model of Burkitt lymphoma expres-sing a doxycycline-repressible MYC transgene. RNA was isolatedunder MYC-repressed and MYC-induced conditions for subse-quent analysis of both total mRNA and mRNA cap methylation.We captured m7G-mRNA using recombinant GST-eIF4E andperformed RNA-Seq on both input and eIF4E-bound mRNAs toidentify the MYC transcriptional targets as well as mRNAs withenhanced cap methylation over and above any transcriptionalinduction (Fig. 1A; see Materials and Methods). As previouslydescribed, induction ofMYC induced >1.2-fold transactivation ofapproximately 80% of the 13,500 genes analyzed (Supplemen-tary Fig. S2A), which is consistent with the recent publications(4, 6, 7, 14). Transcriptional induction was validated using Nano-String analysis (Supplementary Table S1). By comparing therelative induction of mRNA in the m7G fraction to the mRNAinputs, we determined the net effect on enhanced capmethylationfollowing 24 hours of MYC activation. Our analyses revealed thatroughly 30% of analyzed transcripts (�1,200) are induced>1.2-fold at the mRNA cap methylation level, over and abovethe transcriptional induction.

To validate the RNA-Seq data and explore mechanism, wefocused on transcripts that displayed a robust increase in mRNAcap methylation as these transcripts appeared most responsive toMYC for this modification. We found that MAX, a protein thatheterodimerizes with MYC (33), was upregulated by MYC at themRNA capmethylation and protein levels, above and beyond thesmall induction at the mRNA level (Supplementary Fig. S3).Furthermore, we noted that many key components in the canon-ical Wnt signaling pathway (14 transcripts) were among thefraction of genes with MYC-induced cap methylation, includingGSK3B, APC, LRP5, CTNNB1, and TCF7. We analyzed expressionof these five genes/proteins as they are direct mediators down-stream of Wnt-receptor signaling. MYC induction resulted inincreased total mRNA levels of all five Wnt signaling transcripts,as well as ACTB (a control transcript independent of Wntsignaling; Fig. 1B), indicating that these genes are MYC transcrip-tional targets.More importantly, all fiveWnt transcripts displayedaMYC-mediated increase inmRNA capmethylation, significantly

MYC Mediates mRNA Cap Methylation of Wnt Transcripts

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above and beyond the effect on transcription, whereas there wasno detectable increase in mRNA cap methylation of ACTB(Fig. 1C). This observation was validated for the same genes intwo additional MYC-transduced systems, myc-null rat fibroblastsand immortalized human mammary epithelial cells (IMECs), �exogenous MYC. In both systems, MYC overexpression led toenhanced total mRNA and Wnt-transcript–specific mRNA capmethylation (Supplementary Fig. S2B–S2E). We also analyzedthe protein levels of three of these genes (GSK3b, APC, andb-catenin) and found that the increase in mRNA cap methyl-ation corresponded to an increase in protein levels above theexpected effect due to enhanced transcription alone (Fig. 1D).Moreover, siRNA depletion of RNMT in IMEC:MYC cellsresulted in diminished protein levels of GSK3b and APC relativeto control siRNA (Supplementary Fig. S2F). Collectively, thesedata suggest that MYC post-transcriptionally induces an increase

in protein levels from genes involved in Wnt signaling. Toinvestigate whether or not MYC-mediated mRNA cap methyl-ation occurs in cancer, we tested multiple myeloma cancer celllines with varying MYC levels. mRNA cap methylation of Wntpathway transcripts was most elevated in the cell line that hadthe highest MYC level, MM1.S cells, relative to KMS11 cells (Fig.1E). We also found that mRNA cap methylation of Wnt signal-ing transcripts was enhanced in MM1.S cells compared withadditional cancer cell lines with lower MYC expression andcontrol, MCF-10A cells (Supplementary Fig. S2G).

MYC enhances translational capacity of Wnt pathway genes byinducing mRNA cap methylation

We next investigated the effect of MYC-mediated mRNA capmethylation on translation and protein synthesis. mRNA cappingand methylation are required for efficient binding of translation

Figure 1.

MYC-mediated global induction of mRNA cap methylation, including canonical Wnt/b-catenin signaling pathway transcripts. A, Unbiased clustering ofm7G-mRNA pulldowns from P493-6 cells normalized to cell number and changes at the level of total mRNA in response to MYC. Total of 3,294 genesanalyzed. Values transformed by log2 and mean centered (yellow, up; blue, down). B, Total mRNA levels of Wnt signaling transcripts in P493-6 cellsnormalized to cell number determined using RT-qPCR (n ¼ 3, biological). C,m7G-mRNA levels of Wnt signaling transcripts in P493-6 cells normalized to totalmRNA input (n ¼ 3, biological). D, Western blot analysis to detect protein levels of Wnt signaling factors in P493-6 cells. Quantification in right (n ¼ 3,biological). E, m7G-mRNA levels of Wnt signaling transcripts in KMS11 and MM1.S multiple myeloma cells normalized to total mRNA input. Western blotanalysis in right. Error bars indicate SEM and asterisks indicate significance (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

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initiation factors, and then m7G-mRNA is recruited to the 40Sribosomal subunit for translation (34, 35). Therefore, mRNA thatis m7G-capped in response to MYC is predicted to have increasedloading of ribosomes (i.e., polysomes) and an increased rate oftranslation. To assess polysome loading, mRNA bound to ribo-somes was extracted from P493-6 cells at 0, 1, and 24 hours post-MYC induction, and fractions were collected for analysis. Poly-some profiling revealed that P493-6 cells with maximal MYClevels 24 hours postinduction had a dramatic increase in theamount of mRNA in late polysome fractions (Fig. 2A), indicativeof a higher translational capacity. The gradual shift ofmRNA from

the monosomal 80S peak to late polysomal peaks correspondedwith the increase in MYC levels. Isolation and analysis ofm7G-mRNA from polysome fractions showed that m7G-Wnttranscripts were present in late polysome fractions when MYClevels were highest at 24 hours (Fig. 2B). Our results parallelpreviously published data from rat fibroblast cells in whichMYC-overexpressing cells had RNA profiles shifted to the latepolysome fractions (19). Our data suggest that the increase intranslation of Wnt pathway transcripts in P493-6 cells is in partdue to increased mRNA cap methylation and more efficientloading of Wnt signaling mRNA on ribosomes.

Figure 2.

MYC promotes translation and total protein synthesis by augmenting mRNA cap methylation of Wnt/b-catenin signaling pathway transcripts. A, Polysomeprofiling of ribosome-bound mRNA from P493-6 cells at three time points of MYC induction (0, 1, and 24 hours). Data shown are from onerepresentative sample. B,m7G-mRNA levels of Wnt signaling transcripts in each collected fraction (1 through 6) normalized to total mRNA input and fraction 1.(0 hours, top; 1 hour, middle; 24 hours, bottom; n ¼ 3, biological). C, Quantification of total protein synthesis detected with HRP-streptavidin (n ¼ 3,biological). D, Western blot detecting phosphorylation status and total LRP6 and b-catenin in P493-6 cells at 0 and 24 hours. E, Relative Wntsignaling activity measured in IMEC and IMEC:MYC cells using a TCF/LEF luciferase reporter assay (n ¼ 3, biological). Expressed as ratio (TOP Flash/FOPFlash). F, Relative Wnt signaling activity measured in IMEC and IMEC:MYC cells following RNMT depletion with siRNA using a TCF/LEF luciferasereporter assay (n ¼ 3, biological). Expressed as ratio (TOP Flash/FOP Flash). Error bars indicate SEM and asterisks indicate significance (� , P < 0.05).






To confirm that increased mRNA cap methylation andpolysome loading leads to a general increase in protein syn-thesis, a protein synthesis assay was performed. Increasedincorporation of an azide-tagged amino acid analogue wasdetected in P493-6 cells 24 hours after MYC was inducedcompared with baseline (Fig. 2C). This increased amino acidincorporation is indicative of enhanced total protein synthe-sis, partly as a result of increased mRNA cap methylation,polysome loading on mRNA, and augmented translationalcapacity.

To explore the net effect of MYC on Wnt signaling, we assayedphosphorylation of LRP6 and b-catenin in P493-6 cells. MYCinduction led to increased levels of both LRP6 and b-catenin andan even greater induction of p-LRP6 and reduction of p-b-catenin,consistent with increased Wnt signaling (Fig. 2D, SupplementaryFig. S4A and S4B). Similarly, transient transfection of TOP-Flash/FOP-Flash TCF/LEF reporter plasmids into IMECs showedincreased Wnt signaling with constitutive MYC overexpression(Fig. 2E). Conversely, depletion of RNA methyltransferase(RNMT) in MYC-overexpressing cells resulted in reduced Wntactivity (Fig. 2F), suggesting thatMYC-mediated induction ofWntsignaling activity is in part dependent on RNMT and mRNA capmethylation.

Depletion of RNMT or TRRAP complexes reduces mRNA capmethylation of Wnt signaling transcripts

The addition of a cap prevents degradation of a nascentmRNA (36, 37), whereas the addition of a methyl groupenables effective binding of eIF4E and recruitment of ribo-somes for translation (29). Although RNA methyltransferase(RNMT) has been shown to promote cap methylation of CyclinD1 mRNA in combination with MYC (19), the cappingenzyme, RNA guanylyltransferase (RNGTT), and its effect onmRNA cap methylation has not been studied. To assess a rolefor RNGTT and/or RNMT/RNMT-Activating Mini protein(RAM) complex in MYC-mediated mRNA cap methylation ofWnt transcripts, we depleted each protein in IMECs usingsiRNA. Depletion of RNMT or RAM resulted in a statisticallysignificant reduction in mRNA cap methylation of Wnt signal-ing transcripts (Fig. 3A), while depletion of RNGTT had noeffect (discussed in more detail below). The reduction in mRNAcap methylation of Wnt transcripts following depletion ofRNMT or RAM mimicked the results observed with MYCdepletion, suggesting that MYC mediates mRNA cap methyla-tion through modification or recruitment of RNMT and/orRAM. Conversely, increased mRNA cap methylation wasobserved when RNMT, RAM, or MYC was overexpressed inIMECs (Fig. 3B).

Previous data and results presented herein with respect to Wntpathway transcripts (Supplementary Fig. S2D) have shown thatMYC partially mediates mRNA cap methylation of specific tran-scripts viaMYCBox II (MBII), a conserved domainwithin allMYCproteins (18). Because transactivation is mediated by the MBII-dependent interaction of MYC with TRRAP and either TIP60 orGCN5 (8, 11), we tested whether MYC mediates mRNA capmethylation via TRRAP. Depletion of TRRAP and TIP60 resultedin reduced mRNA cap methylation of Wnt signaling transcripts(Fig. 3C), recapitulating the results detected with depletion ofMYC itself. Together, the data indicate that MYC induces mRNAcap methylation of Wnt signaling transcripts in part through itscofactor TRRAP, complexed with TIP60.

MYC promotes recruitment of RNMT to Wnt signaling genepromoters

To determine whether MYC plays a role in RNMT recruitmentto Wnt signaling promoters, ChIP was performed in HEK293FTcells expressing FLAG-tagged RNMT and RNGTT. MYC wassubsequently depleted using siRNA in the same cell lines. MYCdepletion resulted in a statistically significant reduction inrecruitment of RNMT to Wnt signaling promoters (GSK3B andAPC, Fig. 4A). MYC and TIP60 depletion in HEK293FT cellsoverexpressing RNMT, but not RNGTT, brought about a statis-tically significant reduction in mRNA cap methylation of allWnt signaling transcripts analyzed (Fig. 4B). This providesadditional evidence that RNMT, but not RNGTT, is the limitingfactor in MYC-mediated mRNA cap methylation.

To further validate a role for TIP60 in MYC-mediated mRNAcap methylation, we depleted TIP60 using siRNA in cells expres-sing FLAG-tagged RNMT or RNGTT. Consistent with MYC deple-tion, reducing TIP60 diminished recruitment of RNMT to Wntsignaling promoters, supporting a model that MYC facilitatesmRNA cap methylation via MBII and its cofactors, TRRAP andTIP60.

CDK7 is required for MYC-dependent mRNA cap methylationof Wnt signaling transcripts

CDK7phosphorylates S5 on theRNAPol II CTD,which recruitsboth RNGTT and RNMT to promoters (22–24). Hence, thismodification is a strong candidate to mediate MYC activity. Toexplore this, CDK7 and CDK9 (a kinase responsible for phos-phorylation of S2 on the CTD) were depleted in IMECs usingsiRNA, and m7G-mRNA was isolated for analysis. DepletingCDK7, but not CDK9, led to a statistically significant reductionin mRNA cap methylation of Wnt signaling transcripts (Fig. 5A).To confirm that this observation was due to CDK7 activity as partof the TFIIH basal transcription factor, another component spe-cific to TFIIH, XPD, was depleted using siRNA. XPD depletionmimicked CDK7 depletion, resulting in reduced mRNA capmethylation (Supplementary Fig. S5). Importantly, siRNA deple-tion of key factors involved in transcription did not result insignificant cell-cycle arrest, senescence or apoptosis (data notshown).

As an independent approach to study the role of CDK7 inMYC activities, P493-6 cells were treated with a pan-CDKinhibitor (SNS-032) or a CDK7-specific inhibitor (THZ1) withor without MYC induction. THZ1 treatment led to inhibition ofCDK7 (confirmed by RNA Pol II phospho-S5 reduction) andresulted in significantly reduced mRNA cap methylation of Wntsignaling transcripts even in the presence of maximal MYClevels 24 hours post-MYC induction (Fig. 5B). These datasupport the concept that MYCmediates mRNA cap methylationthrough CDK7.

To investigate whether MYC recruits CDK7 to Wnt signalingpromoters and thereby enhances RNA Pol II phospho-S5 levels,ChIP for CDK7 and phospho-S5 was performed. Occupancy ofCDK7 and phospho-S5 on GSK3B and APC promoters wasmeasured by qPCR using P493-6 cells. Interestingly, binding ofCDK7 to the GSK3B and APC promoters paralleled MYC bindingfrom the 0 to 24-hour time point, with a modest but statisticallysignificant increase at 1 hour of MYC induction (Fig. 5C). Con-sequently, phospho-S5 also accumulated significantly at both theGSK3B and APC promoters corresponding to increased MYClevels. No statistical change was observed between time points

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at the ACTB (b-actin) promoter, a negative control. A globalincrease in phospho-S5 levels has been observed in cell typesoverexpressing MYC (17). Accordingly, phospho-S5 was elevatedon a global scale in P493-6 cells, analogous to a global increase inMYC levels (Fig. 5D).

MYC recruits CDK7 to Wnt signaling gene promoters throughan interaction between MRG15 and XPB

Previous observations suggested that MYC may interact withCDK7 (17), but we were unable to confirm this in immunopre-cipitations from native cell extracts. We, therefore, consideredother mechanisms by which MYC could recruit CDK7. Onecandidate,MRG15, is a component of theNuA4 complex contain-

ing TRRAP/TIP60 and has recently been shown to interact withfactors containing a small conserved amino acid sequence, FxLP(38). Analysis of various components in the TFIIH complexrevealed that XPB, a factor in the core complex, contains a FxLPamino acid sequence. We asked whether there was an interactionbetween MRG15 and XPB which could bring MYC and CDK7 inproximity. Immunoprecipitation of XPB revealed an interactionbetween XPB, MRG15, MYC, TIP60, and CDK7 in HEK293T andIMEC cells (Fig. 6A, Supplementary Fig. S6). In support of aninteraction, independent immunoprecipitation of MRG15, MYC,TIP60, or CDK7 resulted in coimmunoprecipitation of the abovementioned factors within both the NuA4 and TFIIH complexes.Furthermore, depleting MRG15 using siRNA knockdown in

Figure 3.

MYC promotes mRNA cap methylation of canonical Wnt signaling pathway transcripts through TRRAP-containing cofactor complexes by utilizing RNMT.A, m7G-mRNA levels of Wnt signaling transcripts in IMEC cells following siRNA depletion of RNGTT, RNMT, RAM, or MYC (normalized to total mRNAinput). mRNA levels of each factor following siRNA knockdown in right (normalized to ACTB; n ¼ 3, biological). B, m7G-mRNA levels of Wnt signalingtranscripts in IMECs following transient transfection with HA-RNMT, FLAG-RAM, HA-RNMT and FLAG-RAM, or CBP-MYC (normalized to total mRNAinput). Protein levels of each factor following transfection in right (n ¼ 2, biological). C, m7G-mRNA levels of Wnt signaling transcripts in IMEC cells followingsiRNA depletion of TIP60 or TRRAP (normalized to total mRNA input). Protein levels of each factor following siRNA knockdown in right (n ¼ 3, biological).Error bars indicate SEM and asterisks indicate significance (� , P < 0.05; �� , P < 0.01; �� , P < 0.0001).






IMECs resulted in diminished phospho-S5 levels globally and atWnt signaling gene promoters, consistent with a reduction inphospho-S5 following TIP60 andMYC depletion (Fig. 6B and C).

DiscussionOur previous study found that MYC promotes an increase in

the protein levels of some targets in the absence of a change at themRNA level (17). Additional analysis revealed that MYC stimu-lates an increase in the protein levels of target genes above andbeyond the change in mRNA levels by enhancing mRNA capmethylation (19). Our study uses transcriptome-wide expressionanalysis to identify an important signaling pathway, the Wnt/b-catenin signaling pathway, affected by MYC at the posttran-scriptional level of mRNA cap methylation. Our findings providenovel evidence for a role of TRRAP complexes, specifically TIP60and MRG15, in MYC-mediated mRNA cap methylation throughtheir interaction with XPB and CDK7 in TFIIH.

To determine which transcripts are targeted by MYC for mRNAcap methylation transcriptome-wide, we performed RNA-Seq onm7G-mRNA pulldowns compared with RNA inputs from P493-6cells at two time points following MYC induction (0 and 24

hours). Roughly 30% of the transcripts analyzed were elevated atthe mRNA cap methylation level in response to MYC, over andabove any transcriptional induction, while the remaining fractionof the transcriptome exhibited only transcriptional inductionwith no net change in relative cap methylation. A smaller fractionof mRNAs exhibit transcriptional induction with an apparentreduction in the extent of cap methylation. We have focused thisstudy on increased cap methylation, and exploration of anyreduction in mRNA cap methylation will require further exper-imentation. It is notable that MAX was among the fraction oftranscripts targeted byMYC for increasedmRNA capmethylation,presumably providing more MAX protein to heterodimerize withincreased levels of MYC protein.

The Wnt/b-catenin signaling pathway is important in embry-onic development, normal cell proliferation, and differentiation;however, dysregulation of factors within the pathway due tomutations leads to aberrant activation of the pathway and carci-nogenesis (15, 16, 39). The Wnt/b-catenin signaling pathway isstimulated in the presence ofmitogens and results in transcriptionof MYC itself (13). Conversely, MYC has been shown to promoteactivation of canonical Wnt signaling transcripts by repressingWnt inhibitors DKK1 and SFRP1 (14). In our dataset, we found

Figure 4.

MYC and TIP60 promote RNMT recruitment to canonical Wnt signaling pathway gene promoters and mRNA cap methylation. A, FLAG ChIPs performed inHEK293FT cells overexpressing FLAG-tagged RNMT or RNGTT where MYC or TIP60 were depleted using siRNA. qPCR performed to detect levels ofeach factor at GSK3B and APC promoters (normalized to input, ACTBcontrol; n ¼ 3, biological). Error bars indicate SEM and asterisks indicate significance(� , P < 0.05; ��, P < 0.01; #, P < 0.001). B, m7G-mRNA levels of Wnt signaling transcripts in RNMT- or RNGTT-overexpressing cells following MYC orTIP60 depletion (normalized to total mRNA input; n ¼ 3, biological). siRNA depletion detected using Western blot analysis in right. Error bars indicate SEMand asterisks indicate significance (�/x, P < 0.05; ¶, P < 0.01; #, P < 0.001).

Posternak et al.





that MYC mediates mRNA cap methylation of canonical Wntsignaling transcripts and that these transcripts represent an impor-tant pathway upregulated by MYC using this post-transcriptionalmodification. At one level,MYCupregulates general translation asdemonstrated by an increase in mRNA ribosome loading andtotal protein synthesis. While an increase in translation and totalprotein production in response to MYC can be attributed to anumber of mechanisms (40–44), a greater amount of m7G-WntsignalingmRNAswere found in late polysome fractions followingMYC induction. This suggests that MYC increases translation and

protein synthesis of Wnt pathway transcripts in part by increasingmRNA cap methylation, which enhances the efficiency of ribo-some loading. Furthermore, MYC induction increases Wnt sig-naling activity. Regulation of Wnt/b-catenin signaling by MYCusing multiple mechanisms demonstrates how crucial MYC-induced Wnt signaling is in normal cell development andtumorigenesis.

RNMT and RAM are both required for methylation of themRNA cap (26, 45). We have demonstrated that RNMT andRAM, but not the capping enzyme, RNGTT, promote mRNA cap

Figure 5.

MYC mediates mRNA cap methylation of Wnt/b-catenin signaling pathway transcripts by recruiting CDK7 to gene promoters. A, m7G-mRNA levels of Wntpathway transcripts in IMECs following CDK7 or CDK9 siRNA depletion normalized to total mRNA input (n ¼ 3, biological). Depletion was confirmed usingWestern blot analysis in right. Error bars indicate SEM and asterisks indicate significance (�, P < 0.05; �� , P < 0.01). B,m7G-mRNA levels of Wnt pathway transcriptsin P493-6 cells at the 0-hour (MYC off) or 24-hour time point (MYC on) following treatment with either 100 nmol/L SNS-032 or 50/250 nmol/L THZ1.Normalized to total mRNA input (n ¼ 3, biological). Inhibition was confirmed using Western blot analysis in right. Error bars indicate SEM and asterisks indicatesignificance (x, P < 0.05; ¶, P < 0.01 relative to 0 hours DMSO and �, P < 0.05; #, P < 0.01 relative to 24 hours DMSO). C, ChIPs for MYC, CDK7, and phospho-S5RNA Pol II were performed in P493-6 cells at three time points of MYC activation (0, 1, and 24 hours). qPCR performed to detect levels of each factor at GSK3B andAPC promoters (normalized to input, ACTBcontrol; n ¼ 3, biological). D, Western blot detecting global phospho-S5 levels in P493-6 cells. Phospho-S5 levelsquantified in right (n ¼ 3, biological). Error bars indicate SEM and asterisks indicate significance (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






methylation of Wnt/b-catenin signaling pathway transcripts.Because methylation is not possible without prior guanylyla-tion by RNGTT, we presume that RNGTT is not rate limiting.Moreover, we find that MYC promotes RNMT recruitment toWnt signaling promoters and mRNA cap methylation of thesetranscripts. We presume this recruitment is indirect because wedo not observe a direct interaction between RNMT and MYC.This implies that RNMT is the limiting factor in mRNA capmethylation and suggests that MYC mediates mRNA cap meth-ylation of Wnt transcripts by inducing recruitment of RNMT totheir promoters. Furthermore, RNMT appears to be required forthe increase in MYC-induced Wnt signaling activity suggestingthat MYC-mediated mRNA cap methylation plays a critical rolein promoting Wnt signaling. RNMT is sufficient to transformmammary epithelial cells (19) and therefore appears to be abetter potential target of MYC regulation than RNGTT. Inde-pendently, MYC has been shown to promote mRNA cap meth-ylation of select transcripts, and this is partially MBII dependent(18). We show that MYC cofactor complexes, including TRRAPand TIP60, are required to stimulate mRNA cap methylation ofWnt signaling transcripts suggesting that MYC indirectly med-iates mRNA cap methylation via its cofactor complexes. Ourresults demonstrate that TRRAP cofactor complexes have dual

roles in transcriptional and post-transcriptional activation ofgenes in response to MYC.

CDK7 enhances RNGTT and RNMT recruitment to promotersby phosphorylating S5 on the RNA Pol II CTD (22, 46). Our datashow that CDK7, not CDK9, is required for mRNA cap methyl-ation of canonicalWnt pathway transcripts, and the active formofCDK7 is necessary for MYC-mediated mRNA cap methylation.Recent studies have demonstrated that CDK7 is critical for normalcell growth and, when deregulated, for carcinogenesis. CDK7inhibition using THZ1 has revealed integral roles for CDK7 inboth transcriptional pausing and mRNA cap methylation (24).These data have established that therapeutically targeting CDK7with THZ1, a CDK7-specific inhibitor, leads to tumor regressionand reduces levels of MYC in MYC overexpressing tumors (47,48). In our study, CDK7 recruitment and accumulation of phos-pho-S5 at both theGSK3B andAPCpromoters correlatewithMYClevels and MYC recruitment to the same promoters. We suggestthat MYC recruits CDK7 to promoters due to an interactionbetween the MYC/TRRAP/TIP60 complex and the TFIIH/CDK7/XPB module. We postulate that MRG15, a component ofthe NuA4/TRRAP complex, interacts with XPB, a core componentof the TFIIH complex, bringing MYC in close proximity to CDK7(Fig. 7). Consequently, MYC mediates phosphorylation of S5 on

Figure 6.

MYCmediates mRNA capmethylationof Wnt/b-catenin signaling pathwaytranscripts through an interactionbetween NuA4 and TFIIH. A,Immunoprecipitation of XPB, MYC,TIP60, CDK7, or MRG15 from HEK293Tcells overexpressing MYC. Co-immunoprecipitated proteinsdetected using Western blot analysis.B, ChIP for phospho-S5 RNA Pol II wasperformed in IMECs where TIP60,MRG15, or MYC were depleted usingsiRNA. qPCR performed to detectlevels of each factor atGSK3B andAPCpromoters (normalized to input, ACTBcontrol; n ¼ 3, biological). C, Westernblot analysis to detect globalphospho-S5 levels following depletionof TIP60, MRG15, or MYC. Error barsindicate SEM and asterisks indicatesignificance (� , P < 0.05; �� , P < 0.01;�� , P < 0.001).

Posternak et al.





the CTD of RNA Pol II through CDK7, which leads to therecruitment of RNGTT and RNMT resulting in augmented mRNAcapping and methylation.

One aspect of our proposed mechanism that remains unclearis why particular promoters are responsive to MYC-mediatedmRNA cap methylation because we were unable to find acommon motif. In addition, further experiments are necessaryto confirm a direct interaction between MRG15 and XPB, toverify that members within NuA4 and TFIIH exist in one largecomplex, and to determine the specific motifs required for thisinteraction. Nevertheless, our study presents a novel set oftranscripts involved in the Wnt/b-catenin signaling pathwaythat are affected by MYC at the level of mRNA cap methylation,above and beyond changes at the transcriptional level ofmRNA.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: V. Posternak, M.D. ColeDevelopment of methodology: V. PosternakAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Posternak

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Posternak, M.H. Ung, C. Cheng, M.D. ColeWriting, review, and/or revision of the manuscript: V. Posternak, M.D. ColeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. PosternakStudy supervision: V. Posternak, M.D. Cole

AcknowledgmentsThe authors thank Joanna Hamilton, Xiangjun Xiao, and Carol Ringelberg

for help with RNA-Seq data analysis, and members of the Cole lab for helpfuldiscussions and feedback. Thisworkwas supported by a grant from theNationalCancer Institute (CA080320; MDC).

Grant SupportThis study was supported by a grant from the National Cancer Institute

(CA055248; to M.D. Cole). C. Cheng is supported by the NIH Centers ofBiomedical Research Excellence (COBRE; GM103534), the Dartmouth Clinicaland Translational Science Institute (UL1TR001086) and theNational Center forAdvancing Translational Sciences (KL2TR001088).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 21, 2016; revised October 26, 2016; accepted November 4,2016; published OnlineFirst November 29, 2016.

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Model for MYC-mediated mRNA capmethylation of Wnt/b-cateninsignaling pathway transcripts. MYCinteracts with NuA4, composed ofTRRAP, TIP60, and MRG15. MRG15interacts with XPB, a component ofTFIIH, bringing CDK7 in proximity topromoters and RNA Pol II. CDK7phosphorylates S5 on the RNA Pol IICTD, which leads to the recruitment ofRNGTT and RNMT/RAM. RNGTT capsnascent mRNA and RNMT mediatesmRNA cap methylation.






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-CateninβMYC Mediates mRNA Cap Methylation of Canonical Wnt/

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MYC Mediates mRNA Cap Methylation of CanonicalWnt/b ......Published OnlineFirst November 29, 2016; DOI: 10.1158/1541-7786.MCR-16-0247 Herein, we report that MYC promotes mRNA cap methylation