Research Article
26 December 2020

The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

ABSTRACT

Mycis a transcription factor which is dependent on its DNA binding domainfor transcriptional regulation of target genes. Here, we report thesurprising finding that Myc mutants devoid of direct DNA bindingactivity and Myc target gene regulation can rescue a substantialfraction of the growth defect inmyc−/−fibroblasts. Expression of the Myc transactivation domain alone inducesa transcription-independent elevation of the RNA polymerase II (Pol II)C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) andCDK9 and a global increase in CTD phosphorylation. The Myctransactivation domain binds to the transcription initiation sites ofthese promoters and stimulates TFIIH binding in an MBII-dependentmanner. Expression of the Myc transactivation domain increases CDK mRNAcap methylation, polysome loading, and the rate oftranslation. We find that some traditional Myctranscriptional target genes are also regulated by this Myc-driventranslation mechanism. We propose that Myc transactivationdomain-driven RNA Pol II CTD phosphorylation has broad effects on bothtranscription and mRNAmetabolism.
Members of the Myc family are champion oncogenes(12,45). N-Myc orc-Myc expression is frequently elevated in cancers of diverse origins,and transgenic mice with elevated Myc expression develop tumors in manyorgans. In normal cellular physiology, Myc induction is an integratorof extracellular signals which drive cell proliferation. Growth factorsinduce Myc expression, and this induction is necessary for cellproliferation (4,19). During oncogenesis,Myc expression becomes elevated independently of growth factors andsurvival signals, permitting unrestrained cell proliferation.
Mycforms a heterodimer with Max and operates as a transcription factorwhich both activates and represses genes. Numerous microarray analyseshave revealed that Myc is a weak but pleiotropic transcription factor,activating or repressing around 5 to 10% of all genes about 1.5- to2-fold (16). A number ofMyc cofactors facilitate this role. Myc binds to the TRRAP complex,recruiting histone acetylation activity to promoters, which promotestranscription by opening up chromatin structure(34,35). Myc has also beenfound to bind and act in conjunction with other transcriptionalregulators (15). Inaddition to driving transactivation, Myc has also been found to drivetranscription elongation via the recruitment of P-TEFb (positivetranscription elongation factor b)(18). Full Myctranscriptional activity is associated with rapid turnover of theprotein, and Skp2, HectH9, and Fbw7 mediate this process(1,24,56-58).Regardless of the cofactor involved, transactivation ultimately mustincrease RNA polymerase II (Pol II) activity, but exactly how Myc doesthis remains unclear.
Myc/Max was first characterized as asequence-specific transcription factor that binds to a consensus site,CACGTG(6). This sequence hasbeen validated as the binding site required for stimulatedtransactivation of many target genes(http://www.myc-cancer-gene.org).However, the necessity for Myc to function by binding to the Mycconsensus sequence has been called into question by in vivo Myc bindingstudies which have placed Myc at promoters without consensus bindingsites (10,20,26,30,44). Currently, itremains unclear whether Myc functions at all of these nonconsensussites and indeed whether there is always a consequence to Myc bindingnear a promoter. Myc has not been found to regulate transcription atmany of the promoters it binds to, but the possibility remains that Mychas some transcription-independent role at thesesites.
RNA polymerase II activity is governed by acycle of phosphorylation and dephosphorylation of the largesubunit C-terminal domain (CTD)(5,29,52). HypophosphorylatedRNA Pol II is recruited to initiation complexes, and transcriptioninitiation is associated with TFIIH-driven CTD serine-5 (S5)phosphorylation (42).After 20 to 50 nucleotides have been transcribed, RNA becomes capped bythe addition of an inverted 7-methylguanosine triphosphate in a seriesof reactions which are most efficient when cotranscriptional(13,46). Capping requiresthree sequential enzymatic activities. RNA triphosphatase removes theterminal phosphate from the first nucleotide, and guanylyltransferaseadds an inverted guanylyl to the mRNA. In mammals, both of theseenzymes are present in the same polypeptide, which is recruited andactivated by the S5-P CTD(11,33,55,59). The third step is7-methylation of the guanosine, and the methyltransferase is alsorecruited by the S5-P CTD(25,48).Guanosine addition stabilizes the mRNA against degradation, but themRNA becomes competent to be translated only when methylated(50,51). Since the cappingenzymes are recruited and activated by the phosphorylated CTD,transcription and mRNA processing are intricately linked andcodependent.
We initiated a study of Myc biological activity thatis independent of direct DNA binding and transcriptional activation orrepression. We present the striking finding that Myc can promote aglobal elevation in phosphorylation of the RNA Pol II CTD which has apotentially broad impact on transcription, mRNA processing, andtranslation.

MATERIALS ANDMETHODS

Cell culture, transfection,retroviral infection, siRNA transfection, and assays.

Rat-1fibroblasts (TGR cells), ratc-myc null fibroblasts (HO15.19 cells), Tet-21/N cells, andretroviral producer PhoeNX cells were cultured in Dulbecco modifiedEagle medium-10% fetal calf serum. Immortalized mammaryepithelial cells (IMECs) were cultured as previously described(17). Retroviralinfection of HO15.19 and IMECs was performed using PhoeNX cells.Tet-21/N cells were maintained in 0.2 μg/ml doxycycline for 2days to repress N-myc expression. For growth curves,104 cells from log-phase cultures were plated in six-wellplates. Every 24 h, cells were trypsinized and counted usinga hemocytometer. Cells expressing cyclin-dependent kinase 7 (CDK7)dominant negative (DN) or the vector control were counted 2days following infection. A 10-cm plate of TGR cells was transfectedwith 24 μl 1.2 nM small interfering RNA (siRNA; Dharmacon)(scrambled control, Myc siRNA#1 or Myc siRNA#2; sequences availableupon request). After 24 h, cells were processed asappropriate.

Microarrays.

RNA was harvested from twoindependent cultures of myc null rat fibroblasts (HO15.19)expressing the vector control, MycWT, MycΔMBII, and MycBM usingan RNeasy kit (QIAGEN). RNA integrity and concentration were verifiedby gel electrophoresis. Arrays were performed as described previously(14). Only genes with asignal >20% above background and with at least 70% good dataacross the arrays were considered for further analysis. The data werecentered to the average of the two vector-only arrays and filtered foraverage changes in signal for either MycWT ormutants.

Immunoprecipitations.

To detect Myc/Max interactions inmyc−/− cells,a 15-cm plate of cells was lysed in 1 ml F buffer(53) and extractsincubated with anti-FLAG antibody-conjugated beads (Sigma) for3 h at 4°C. Immunoblot analyses were performed onimmunoprecipitated protein using anti-N-Myc or anti-Max polyclonalantibody (Santa Cruz). To detect Myc/CDK7 interactions in 293 cells, a10-cm plate of 293 cells was transfected with 1 μg expressionvector for N-MycWT or N-MycΔMBII and 1 μg expressionvector for CDK7 or the relevant vector control. After 2 days, nucleiwere prepared by hypotonic lysis and extracted with Dignam C buffer.Extracts were mixed 1:1 with F buffer, and 25% of each extract was usedfor an immunoprecipitation (IP) using polyclonal anti-CDK7 oranti-N-Myc antibody (Santa Cruz). Immunoblot analyses were performed onimmunoprecipitated protein using monoclonal anti-CDK7 (Santa Cruz) orN-myc (Upstate) antibody. For the CDK-activating kinase (CAK) assay,CDK7 kinase activity was measured as described previously(32) using recombinantCDK2 (Santa Cruz) as a substrate. To detect endogenous Myc/CDK7interactions in 293 cells, nuclei from 4- by 15-cm plates wereextracted with Dignam C buffer. Cell extracts were mixed 1:1 with Fbuffer and precleared for 1 h using anti-mouse antibodies.The nuclear extract was divided, and immunoprecipitation was carriedout with either anti-c-Myc antibodies (C33; Santa Cruz) or controlmonoclonal antibodies. Western blot analyses were performed asdescribed above.

Chromatinimmunoprecipitation.

TheUpstate chromatin immunoprecipitation (ChIP) assay kit was used. IPswere carried out using polyclonal anti-FLAG antibody (Sigma) andpolyclonal antibodies raised against N-Myc, MAT1, p62, or controlantibody (Santa Cruz). PCR was carried out on the resultant DNA samplesand 1% of the input using 32P end-labeled primer pairs(sequences available upon request). PCR products were resolved on 5%Tris-borate-EDTA-polyacrylamide gel electrophoresis (PAGE),visualized by a phosphorimager, and quantitated using ImageQuantsoftware. For each reaction, at least three independent ChIPs wereperformed and the PCRs were monitored to be well within the linearrange.

RT-PCR.

RNA was extracted from log-phasecells by using the RNeasy kit (QIAGEN) and normalized. Reversetranscription (RT)-PCR was carried out using the Platinumquantitative RT-PCR system (Invitrogen). The annealing temperature was55°C, and the number of cycles to meet the linear range of thereaction for all primers pairs was determined (typically between 18 and22). The primer sequences are available upon request. Products werequantitated as described above. The mean expression for two independentRNA samples was calculated, with error bars indicating standarddeviations.

Polysome preparation.

Polysomes were prepared from log-phase cells by cell extract centrifugation over 30% sucrose as described previously(9). RNA was extractedusing TRIzol from the monosome/hnRNA fraction at the sucrose bufferinterface and from the polysome pellet. The distribution of specificmRNAs in each fraction was determined by performingRT-PCR.

Anti-methyl cap IP.

RNA was extracted by TRIzol followedby phenol-chloroform, and 2 μg was suspended in 100 μlbinding buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 1 mg/mltRNA [baker's yeast; Roche], 0.1 mg/ml bovine serum albumin).Anti-2,2,7-methylguanosine antibody-bound beads or antibodycontrol-bound beads (Calbiochem) were blocked with binding buffer. RNAwas rotated for 1 h at 4°C with 20 μlanti-2,2,7-methylguanosine antibody-bound beads or 40 μlantibody control-bound beads to control for nonspecific binding. Beadsand flowthrough were separated, and the volume wasincreased to 200 μl by adding binding buffer. Samples wereextracted with phenol-chloroform, precipitated with ethanol, andresuspended in 50 μl H2O. Two microliters of RNA wasused for each 20-μl RT-PCR volume, and RT-PCR was performed asdescribed above except that 24 to 27 cycles wereused.

Immunoblots.

Two days following seeding,subconfluent cells were lysed in a modified radioimmunoprecipitationassay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 130 mM NaCl, 1% NP-40,0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 20 mM NaF, 1μg/ml of leupeptin, pepstatin, and aprotinin, 1 mMdithiothreitol, and phosphatase inhibitor cocktails I and II [Sigma]).Protein concentration was determined using a modified Lowry proteinassay kit (Pierce). Equivalent amounts of protein were subjected toSDS-PAGE, transferred to a polyvinylidene difluoride membrane, andimmunoblotted using antibodies raised against the proteins indicated inthe figures. To detect RNA Pol II, nuclei were isolated by Douncehomogenization of the cell pellet in Dignam A buffer. Nuclear proteinswere extracted by resuspension in Dignam C or F buffer. Equivalentamounts of protein were immunoblotted for RNA Pol II (Santa Cruz) andanti-CTD phospho-S5 (H14) and anti-CTD phospho-S2 (H5)(Covance).

EMSA.

An electrophoretic mobility shiftyassay (EMSA) was performed as previously described(53). Complexes wereidentified by incubating 0.2 μg of the relevant polyclonalantibody with the reaction mixes to assay for supershift or loss of theband (not shown). Complexes were separated on a 5% acrylamide gel andvisualized by a phosphorimager.

In vivocell labeling experiments.

For each IP, a 10-cm plate ofmyc+/+ fibroblasts ormyc−/−fibroblasts expressing MycWT, MycBM, MycΔC, or the vectorcontrol was incubated for 30 min in 4 ml Dulbecco modified Eagle mediumwithout cysteine and methionine (Sigma) and in 10% dialyzed fetal calfserum. Promix (10 μl; Amersham) was added for 0.5, 1, or1.5 h as indicated. Chase was performed by washing the cellsthree times and incubating them in normal growth medium for 2, 4, or6 h as indicated. RIPA extracts were made and normalized forprotein content. Samples were precleared by 1 h of incubationwith 25 μl normal rabbit immunoglobulin G agarose. Polyclonalcyclin T1 or CDK9 antibody (1 μg) was incubated with theextracts overnight, and then 25 μl protein A/G-Sepharose wasadded for 1 h. Beads were washed in RIPA buffer and eluted insample buffer. Immunoprecipitated protein and cell extracts wereresolved on 8% SDS-PAGE. Bands were visualized using a Stormphosphorimager and quantitated using ImageQuant software. Eachexperiment was performed on at least two independent occasions. Theexperiment whose results are shown is representative, and error barsindicate the standard deviations for duplicates. Total labelincorporation was calculated by trichloroacetic acid (TCA)precipitation of cell extracts, followed by label detection using ascintillationcounter.

RESULTS

Designand characterization of Myc mutants defective for DNAbinding.

We are interested inthe biological activity of the Myc transactivation domain and wanted toseparate the function of this domain from the downstream effects of Myctarget genes. Such analysis can reveal novel Myc activities as well asprovide insight into wild-type Myc function. To this end, we createdtwo mutants of murine N-Myc, called MycBM and MycΔC, whichcould not bind to DNA directly (Fig.1A). In MycBM, the C-terminal, basic region which binds tothe DNA phosphate backbone is mutated into an acidic region, i.e.,amino acids 381 to 384, RQRR, are mutated to ADAA. MycΔC has adeletion of the entire DNA binding and Max dimerization domain (aminoacids 372 to 454) and a nuclear localization signal added at the Cterminus. These two DNA binding mutants were designed to becomplementary. MycBM retains the Max binding domain whereasMycΔC does not. Equivalent cellular responses to the expressionof both mutants are unlikely to be due to Max titration. To furtherinvestigate the role of the N terminus of Myc, an additional deletionof amino acids 103 to 119, encompassing the Myc homology box II domain(MBII), was made in the full-length Myc and MycBM proteins, creatingMycΔMBII and MycBMΔMBII, respectively. MBII is highlyconserved in evolution and is necessary for nearly all reportedN-terminal interactions and functions, including oncogenictransformation(54).
We confirmedthat MycBM and MycΔC did not bind to the Myc consensus sites orregulate Myc target genes and that they bound to Max as predicted. Toassess Max interaction, Myc proteins were immunoprecipitated via theirFLAG tag and immunoblotted for Myc and Max (Fig.1B). As predicted, MycWT,MycBM, and their respective ΔMBII mutants were able to bind toendogenous Max, whereas MycΔC could not. Since MycBM retainsmuch of the DNA binding domain, we wanted to confirm that it could notbind to Myc consensus sites. An EMSA was performed using transientlyexpressed proteins and the Myc/Max consensus binding site,CACGTG, as a probe. MycWT, MycΔMBII, andMycBM were expressed at equivalent levels (Fig.1C, upper panel). In theEMSA, Myc/Max and MycΔMBII/Max heterodimers could bind to aCACGTG probe and were detected as acharacteristic band that migrated faster than the endogenous upstreamstimulatory factor-DNA complex. MycBM did not havedetectable DNA binding activity, despite the fact that it dimerizedavidly with Max (Fig.1B). The presence of bothMyc and Max in the DNA binding complexes was confirmed by a supershiftusing anti-Max and anti-Myc antibodies (data not shown).
Toconfirm that MycBM was not regulating Myc target genes, thetranscription of many established Myc-activated and -repressed geneswas measured (Fig. 1D andE). Quantitative RT-PCR was performed on RNA extracted frompools of myc null cells expressing MycWT, MycΔMBII,and MycBM. MycWT was found to increase transcription of theestablished Myc-activated CAD, HSP60, and nucleolin genes two- tothreefold, but MycBM did not increase the transcription of these genesabove background levels (Fig.1D). Transcription ofestablished Myc-repressed genes, the GADD45 and endogenousc-myc (reported by the neomycin gene driven by thec-myc promoter in myc null cells) genes, wererepressed about fivefold by MycWT, but MycBM failed to repress eithertarget. Microarray analysis concurred that MycBM does not activate Myctarget genes. Previously, Myc target genes in the ratfibroblast system had been established using microarrays andauxiliary techniques(43). We found 21previously reported Myc target genes to be upregulated by theexpression of MycWT in our experiments (Fig.1E). In contrast, theexpression of MycBM does not upregulate any of these genes, and thevector control values and MycBM values are not significantly different,giving a P value of 0.4 using the t test. Anequivalent microarray analysis of the transcriptional response toMycΔC was carried out and showed that MycΔC also doesnot upregulate Myc target genes (not shown).
Recent findings haveshown that Myc is a regulator of rRNA synthesis, and it is hypothesizedthat this has a role in mediating the Myc growth phenotype(3,21,22). We found that theexpression of MycWT increased total RNA content per cell (the majorityof which is rRNA) about 1.5-fold above that of vector control cells(not shown). As predicted from the observation that MycBM cannotactivate target genes, the expression of MycBM did not elevate cellularRNA content.

Myc induces morphologicalchanges independently of target gene expression.

To investigate the activity of the MycN terminus independent of target genes, MycWT and the DNA bindingmutants were stably expressed in the myc null rat fibroblastcell line HO15.19 (31).The myc−/−background allows Myc mutant phenotypes to be revealed rather thanmasked by endogenous Myc. Equivalent expression levels of the Mycproteins were verified by immunoblotting (Fig.1B).myc−/−fibroblasts are broad, flat, and nonrefractile. As previously reported,the expression of MycWT reverted themyc−/− cellmorphology to become comparable to that of Rat1 fibroblasts, themyc+/+ line from which themyc−/− cellswere derived(31). TheMycWT-reconstituted cells appear smaller (although their volume isunchanged), darker, and refractile (Fig.2A). MycWT cells also grew to a higher density than the vector controlmyc−/− cells(not shown). Surprisingly, the expression of MycBM partially rescuedthe morphology ofmyc−/− cells.MycBM cells appeared darker, smaller, and more refractile than vectorcontrol cells (Fig. 2A).They were also able to grow to a higher density than vector cells (notshown). This phenotype was immediately apparent upon the expansion ofpolyclonal cell populations and reproduced in three independentinfections. The morphological changes driven by MycBM werecompletely dependent on an intact N terminus because MycBMΔMBIIcells had the same morphology as vector control cells (Fig.2A). Therefore, the Nterminus of Myc can partially rescuemyc−/− cellmorphology independently of direct DNA binding and target geneexpression.

The proliferation defect inmyc−/− cellscan be partially rescued by Myc DNA binding mutants.

Cell proliferation is dependent on Mycexpression, and therefore, we investigated whether MycBM andMycΔC could rescue the proliferation defect ofmyc−/− cells.The doubling times of the cell lines were measured by countingequivalently seeded cells on consecutive days (Fig.2B). In line with previouspublications,myc−/− cellshad a doubling time of 40 h and MycWT-expressing cells had adoubling time of 19 h(31). The expression ofboth MycBM and MycΔC resulted an intermediate doubling time of28 h. Thus, a significant proportion of the growth defect inmyc−/− cellscan be restored by Myc proteins that cannot regulate target genetranscription. Consistent with morphological changes, thisproliferation rescue was dependent on an intact N terminus, since theMycBMΔMBII cells had a doubling time of 36 h, closeto that of the vector control. Moreover, this proliferative activity isindependent of Max titration, since MycBM can bind to Max whereasMycΔC cannot. The ability of Myc DNA binding mutants to rescuethe myc−/−cell proliferation defect was confirmed by cell cycle analysis usingpropidium iodide staining and fluorescence-activated cell sorteranalysis (not shown). MycBM and MycΔC, unlike MycWT, do notrescue the growth defect in serum-starved cells and do not induceapoptosis or cell transformation (notshown).

Myc increases total cellular CAKactivity.

Having demonstrateda potent biological activity for the Myc N terminus, we were interestedin resolving the mechanism of this activity and its relationship towild-type Myc function. Myc-induced cell proliferation is correlatedwith an increase in cell cycle CDK activity(32). CDK complexes areactivated by CAK, which phosphorylates the CDK T loop(23,37). Since we had foundthat the expression of MycBM and MycΔC acceleratedproliferation in a target gene-independent manner, we investigatedwhether total cellular CAK activity is increased in response to theexpression of MycWT and mutants by using a CAK assay (Fig.3A). The CAK assay only indicates the level of CAK activity in a cell lineand does not distinguish increases in the specific activity of CAK fromincreases in the concentration of CAK itself, which are addressedbelow. CAK was immunoprecipitated from cell extracts by using ananti-CDK7 antibody and incubated with CDK2 as a substrate (Fig.3A). In the representativeassay whose results are shown, CAK activity in MycWT cell extracts wasfound to be 1.9-fold over that of the vector control, as reportedpreviously (32). CAKactivity was also elevated in MycBM cell extracts (1.7-fold over thatof the vector control). Increased CAK activity was dependent on anintact N terminus because extracts from MycBMΔMBII cells havethe same level of kinase activity as vector control cells (0.8-fold).In correlation with increased total cellular CAK activity, we foundelevated levels of phospho-CDC2 and phospho-CDK2 in cells expressingMycWT, MycBM, and MycΔC compared to those in the vector controland MycBMΔMBII (not shown).
To demonstrate that CAK has acritical role in cell proliferation and T-loop phosphorylation in vivo,we expressed a catalytically inactive CDK7 mutant, CDK7D155N, in themyc+/+ fibroblast cellline TGR (28). Expressionof this dominant interfering CDK7 inhibited both CDC2 and CDK2phosphorylation levels and CDK2 expression (Fig.3B). The cellproliferation rate was also inhibited by CDK7D155N (Fig.3C).

Myc increases CAK expression independently of Myc target genes.

CAK is a complex of CDK7,cyclin H, and MAT1. We investigated whether the increased CAK activityfound in Myc-expressing cells could be a result of increased CAKcomponent protein expression (Fig.4A). To investigate whether CAK component expression was upregulated inresponse to endogenous Myc expression, we compared the parentalmyc+/+ fibroblasts withmyc−/−fibroblasts and we used two independent Myc siRNAs to knockdown endogenous Myc expression inmyc+/+ fibroblasts. Bothcyclin H and MAT1 expression levels were reduced inmyc−/−fibroblasts compared to those inmyc+/+ cells andmyc+/+ cells transfectedwith both Myc siRNAs but not the control siRNA. Next, weinvestigated CAK component expression in response to exogenous MycWTand the DNA binding mutants. In agreement with previous reports, wefound a substantial increase in CDK7, cyclin H, and MAT1 proteinexpression in response to exogenous MycWT(32). Interestingly, wealso found a comparable increase in CAK protein expression in cellsexpressing MycBM and MycΔC. These effects were dependent on anintact N terminus because cells expressing MycΔMBII andMycBMΔMBII did not express increased levels of CDK7, cyclin H,and MAT1. Thus, the increased cellular CAK activity in MycWT-,MycBM-, and MycΔC-expressing cells can be wellcorrelated with elevated protein expression, and it is unlikely thatthe specific activity of CAK itself increases.
Wewanted to determine how CAK components were being upregulated inresponse to Myc. CDK7 and MAT1 have never been reported as Myc targetgenes, and cyclin H is reported as a Myc-repressedgene(http://www.myc-cancer-gene.org)(49). However, we wantedto determine whether the transcription of these genes is activated inour specific cell system. Although the MycBM and MycΔC proteinslack DNA binding activity and activation of traditional Myc targetgenes, it was possible that they could upregulate the transcription ofmembers of the CAK complex indirectly. We measured mRNA levels by acarefully controlled quantitative RT-PCR, carried out in the linearrange on independent RNA samples. We found that CDK7, cyclin H, or MAT1mRNA was not induced in response to modulation of endogenous Mycexpression, both when comparingmyc+/+ withmyc−/−fibroblasts and in response to both Myc siRNAs (Fig.4B). We also looked at CAKcomponent expression in response to exogenous MycWT and DNA bindingmutants. We found that CDK7, cyclin H, or MAT1 mRNA was not induced incells expressing MycBM or MycΔC compared to that in the vectorcontrol (Fig. 4B). Theseresults were confirmed by microarray analysis (not shown). There wasalso no induction of CDK7 or cyclin H by MycWT. MAT1 mRNA was elevatedin response to exogenous MycWT (and not in response toendogenous Myc levels). We conclude that endogenous Myc and thetranscription-defective mutants MycBM and MycΔC lead toelevated CAK expression through a posttranscriptionalmechanism.
We were interested in whether CAK protein expressionwas Myc responsive in other cell systems. We investigated CAKexpression in cells from other lineages and in response to c-Myc aswell as N-Myc. We examined CAK subunit expression in Tet-21/N cells, aneuroblastoma cell line that expresses N-Myc under doxycyclineregulation (27), and inIMECs, a low-passage immortalized mammary epithelial line, engineeredto stably express exogenous c-MycWT or c-MycΔMBII(17). We found increasedexpression of CDK7, cyclin H, and MAT1 proteins in response to elevatedN-Myc expression in the neuroblastoma cell line and in response toelevated c-MycWT but not c-MycΔMBII in the IMECs (Fig.4A). In agreement with thefibroblast data, Myc-driven upregulation of these proteins was found tobe entirely posttranscriptional. CDK7, cyclin H, or MAT1 mRNA was notMyc regulated in either cell line (Fig.4B). For the mammaryepithelial cell line, this result was also confirmed by microarrayanalysis of RNA extracted from log-phase cells (not shown).
Insummary, Myc can increase the expression of CAK proteins by aposttranscriptional mechanism in a variety of cell lines, in responseto exogenous and endogenous Myc levels, and in response to both c-Mycand N-Myc.

Myc expression elevatescellular TFIIH and P-TEFb activity by a posttranscriptionalmechanism.

The CAK componentsCDK7, cyclin H, and MAT1 are also components of the basal transcriptionfactor TFIIH. CDK7 is the TFIIH kinase which phosphorylates the RNA PolII CTD S5 (52). Since wehad found elevated CAK expression in response to Myc, we investigatedwhether this also led to increased TFIIH kinase activity. The proteinexpression level of RNA Pol II large subunit was unalteredin response to modulation of Myc expression in rat fibroblasts, theTet-21/N neuroblastoma, and IMEC lines (Fig.5, bottom panels). However, Myc expression was ratelimiting for RNA Pol II CTD S5 phosphorylation, as detected byphospho-specific antibodies (Fig.5A). S5 phosphorylationwas reduced inmyc−/−fibroblasts compared to that inmyc+/+ fibroblasts and wasalso reduced in myc+/+fibroblasts in which endogenous Myc was knocked down by two siRNAs. CTDS5 phosphorylation was also elevated in response to the expression ofexogenous MycWT in fibroblasts, neuroblastoma cells, and epithelialcell lines (Fig. 5A toC). Furthermore, S5 phosphorylation was increased inresponse to the expression of the transcription-defective mutants MycBMand MycΔC but not MycBMΔMBII (Fig.5A). Therefore,Myc-induced CTD S5 phosphorylation is dependent on the Myc N terminusalone but is also induced by MycWT. It is important to stress that theelevated CTD phosphorylation found in Myc-expressing cells issufficiently large to be detected in the total nuclear pool of RNA PolII. Thus, TFIIH is Myc regulated and rate limiting for CTDphosphorylation.
The S2 position of the RNA Pol II CTD is alsophosphorylated, predominantly by P-TEFb. P-TEFb consists of CDK9 andseveral cyclins, most commonly cyclin T1. We found that Myc expressionupregulates the subunits of P-TEFb (Fig.5D). Endogenous Mycexpression in fibroblasts elevated CDK9 and cyclin T1 proteinexpression levels since expression was higher in controlmyc+/+ cells than inmyc+/+ cells with Mycknocked down or inmyc−/− cells.Overexpression of MycWT inmyc−/−fibroblasts, the Tet21/N neuroblastoma line, and the IMEC line alsoincreases expression of CDK9 and cyclin T1 (Fig.5D). Again, elevatedexpression was dependent on the Myc N terminus alone since MycBM andMycΔC but not MycBMΔMBII induced increased CDK9 andcyclin T1 protein expression levels. As with TFIIH, the elevation inCDK9 and cyclin T1 proteins was not mediated by elevated mRNA levels(Fig. 5E).
TheMyc-dependent increase in P-TEFb expression correlated with RNA Pol IIS2 CTD phosphorylation. S2 phosphorylation was reduced uponthe loss of endogenous Myc expression and increased upon the expressionof MycWT, MycBM, and MycΔC in rat fibroblasts. S2phosphorylation was also elevated in neuroblastomas with high N-Myclevels and in IMECs overexpressing MycWT or MycΔC (Fig.5). Again, we stress thatthe elevated S2 phosphorylation was found in the total nuclear pool ofRNA Pol II.
In summary, both S5 and S2 phosphorylation levels inthe RNA Pol II CTD are increased in response to exogenous andendogenous Myc expression. Mutant analysis demonstrated that this isdependent on an intact N terminus but independent of direct Myc DNAbinding and target gene expression.

Mycincreases the translation rate of cyclin T1 and CDK9.

The findings above demonstrate that Myccan increase the expression of a subset of CDKs and cyclins by aposttranscriptional mechanism and independently of Myc target geneexpression. Next, we investigated whether these proteins were beingupregulated as a result of an increased translation rate or a decreaseddegradation rate. We used pulse-chase experiments to compare thetranslation rates and degradation rates of the P-TEFb complex proteinsCDK9 and cyclin T1. We measured these rates in the parentalmyc+/+ fibroblasts andmyc−/−fibroblasts expressing exogenous MycWT, MycΔMBII, MycBM,MycΔC, and the vector control. The decision to investigateP-TEFb rather than CAK and TFIIH was technical, i.e., in pulse-chaseexperiments, the simple P-TEFb heterodimer was more amenable to theresolution of individual protein components than the multisubunitTFIIH.
Cells were pulse-labeled with [35S]methionineand [35S]cysteine for 0.5, 1, or 1.5 h, followedby a chase with cold amino acids for 2, 4, or 6 h. Cell extracts from each time point were normalized forprotein content. To measure total cellular label incorporation for eachtime point, protein was precipitated using TCA and the counts werequantitated (Fig.6A). Label incorporation during the pulse wasapproximately 1.5-fold higher inmyc+/+ cells than inmyc−/− cells(P > 0.1). Label incorporation intomyc−/− cellsexpressing MycWT, MycBM, MycΔC, and the vector control wasstatistically indistinguishable (P < 0.1). Label lossduring the chase was lowest formyc−/− cellsand highest for myc+/+cells andmyc−/− cellsexpressing MycWT, but the values were statistically indistinguishable(P > 0.1). To measure label incorporation into CDK9and cyclin T1 at each time point, these proteins wereimmunoprecipitated and resolved by SDS-PAGE. Label incorporation wasdetected and quantitated using a phosphorimager (Fig.6B and C). During thepulse, the rate of label incorporation into cyclin T1 was found to beapproximately 4.5-fold higher inmyc+/+ cells than inmyc−/− cellsand approximately 2.5-fold higher inmyc−/− cellsexpressing MycWT, MycBM, or MycΔC than in cells expressing thevector control (P > 0.1). Similarly, the rate of labelincorporation into CDK9 during the pulse was found to be approximately5.5-fold higher in myc+/+cells than inmyc−/− cellsand approximately 3-fold higher inmyc−/− cellsexpressing MycWT, MycBM, or MycΔC than in cells expressing thevector control (P > 0.1). Label incorporation intocyclin T1 and CDK9 in cells expressing MycΔMBII wasindistinguishable from incorporation in vector control cells (see Fig.S1 in the supplemental material). During the chase, label losses fromcyclin T1 were similar for all cell lines but actually slowest formyc−/− cellscompared to those for all other cell lines tested, i.e., cyclin T1 wasmost stable inmyc−/− cells.Total label loss from CDK9 was statistically indistinguishable for allcell lines (P < 0.1). Since cyclin T1 degradation isactually slowest inmyc−/− cellsand CDK9 degradation rates are equivalent in all cell lines tested, theincreased labeled amino acid incorporation during the pulse in cellsexpressing endogenous Myc, exogenous MycWT, MycBM, and MycΔCcan be concluded to be a reflection of an increased translation rate.In addition, the increased translation rate is dependent on MBII, sincelabel incorporation in cyclin T1 and CDK9 is indistinguishable forMycΔMBII and the vector control.
We demonstrated abovethat Myc upregulates CAK subunits by a posttranscriptional mechanism aswith P-TEFb. One or more CAK components may also be upregulated at thelevel of translation; however, due to the complexity of CAK and TFIIH,it was not possible to investigate the translation rates of thesecomplexes by pulse-chase experiments.

Mycincreases polysome loading of cyclin T1, CDK9, and specific Myc targetgenes.

Efficiently translatedmRNAs are found to be associated with polysomes rather than monosomes.Since we had shown that CDK9 and cyclin T1 are translated more rapidlyin response to Myc, we expected to see a correlative increase inpolysome loading. We used centrifugation over a sucrose bed to separatemonosomes/hnRNA from polysomes in the fibroblast cell lines(9). Initially, wequantitated 18S rRNA distribution as a measure of ribosome distribution(Fig. 6D). For all celllines, the 18S rRNA was found predominantly in the monosome fraction.Vector controlmyc−/− cellshad a slight decrease in the proportion of 18S rRNA found in thepolysome fraction compared to the Myc-expressing cell lines. mRNAextracted from polysome and monosome fractions was used as a substratefor RT-PCR to look at the distribution of specific messages (Fig.6D). Inmyc−/− cells,less than 50% of the cyclin T1 mRNA was associated with polysomes,whereas in myc+/+ cellsand in myc−/−cells expressing MycWT, MycBR, and MycΔC, approximately 90% ofthe cyclin T1 mRNA was associated with polysomes. The same trend wasfound with CDK9. Inmyc−/− cells,less than 10% of the CDK9 mRNA was associated with polysomes, whereasin all Myc-expressing cell lines, approximately 50% of the CDK9 mRNAwas associated with polysomes. The increased association of cyclin T1and CDK9 with polysomes in response to exogenous and endogenous MycWTand in response to Myc DNA binding mutants is consistent with theincreased translation rate of these mRNAs in Myc-expressingcells.
The data above document a novel, target gene-independentactivity of Myc; however, we also wanted to explore the possibilitythat Myc could influence polysome loading of traditional Myc targetgenes, i.e., increase both the transcription and the translation ofsome genes. We investigated the polysome loading of three genes whichhave been established as Myc target genes in rat fibroblasts, theHSP60, RUVBL1, and nucleolin genes(43). We confirmed thatthe transcription of all three of these genes was upregulated inresponse to MycWT but not the Myc DNA binding mutants (Fig.1D and data not shown).For all three mRNAs, polysome loading was increased inmyc+/+ cells compared tothat inmyc−/− cellsand was increased in response to exogenous MycWT. For HSP60mRNA, the increase in polysome loading was mild, increasing from 35% inmyc−/− cellsto 50% in myc+/+ cells,and there was no increased polysome loading in response to the DNAbinding mutants. However, for nucleolin and RUVBL1, the effect was moresignificant, and polysome loading was increased in response to the MycDNA binding mutants as well as the wild-type protein. This differentialeffect of Myc and Myc mutants is discussed later. Polysome loading forGAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA was notsignificantly Myc dependent. Inmyc−/− cellsexpressing the vector control and Myc proteins, about 40% of GAPDH mRNAwas found to be polysome associated. The parentalmyc+/+ line had a mildincrease of up to 60% GAPDH mRNA that was polysomeassociated.
Since the percentage of polysome loading of nucleolinand RUVBL1 is increased in response to MycWT and Myc DNA bindingmutants, the prediction is that the expression of nucleolin and RUVBL1protein will be increased in response to both MycWT and the Myc DNAbinding mutants. We performed Western blot analyses on our panel of ratfibroblast cell lines by using anti-RUVBL1 and antinucleolin antibodies(Fig. 6E). As predicted,we saw increased expression of both proteins in response to endogenousand exogenous MycWT and in response to MycBM and MycΔC comparedto that inmyc−/− cells.The increase in protein expression was larger in response to MycWT thanin response to the Myc DNA binding mutants. This is consistent with thefact that these are also Myc transcriptional target genes, i.e.,nucleolin and RUVBL1 mRNAs are elevated in response to MycWT but notthe DNA binding mutants (Fig.6E). Therefore, theincreased expression of these proteins in response to MycWT is likely aresult of increased transcription and increased polysome loading andtranslation. The increase in expression is lower in cells expressingMycBM and MycΔC, probably because it is a result of increasedtranslation alone.

Myc increases mRNA capmethylation.

Next, weinvestigated the mechanism by which Myc increases mRNA polysome loadingand translation. We found that the nucleocytoplasmic distribution ofall the mRNAs discussed above was unaltered by the expression of MycWTor Myc mutants (not shown), and therefore, Myc does not regulatepolysome loading and translation of these mRNAs by regulating theirnuclear export. We also monitored the expression of a comprehensivepanel of translation initiation and elongation factors and found nodifferences between MycBM, MycΔC, and control cells (data notshown).
Translation of mRNA is dependent on the addition of the7-methylguanosine triphosphate 5′ cap, which occurs mostefficiently cotranscriptionally(5,40). Guanylylationstabilizes mRNA, and methylation permits mRNA translation.(50). Since we had founda group of genes that were equivalently expressed at the mRNA level butthat were not equivalently translated, we hypothesized that capmethylation might be regulated.
To measure the proportion ofmethylated CDK9 and cyclin T1 mRNA, we used ananti-2,2,7-methylguanosine antibody which also binds to7-methylguanosine (7). RNAisolated from fibroblasts and epithelial cells expressing MycWT,MycΔC, and the vector control was offered as a substrate forimmunoprecipitations using the anti-methyl cap antibody. The RNA boundto the anti-methyl cap antibody was isolated, and RT-PCR was used tocalculate the antibody-bound fraction (and therefore methylation) ofeach mRNA relative to that of the input mRNA (Fig.6F). We found that theproportion of methylated CDK9 and cyclin T1 mRNA was higher in MycWT orMycΔC cells than in the vector control in both fibroblasts andmammary epithelial cells. We also found that nucleolin but not HSP60mRNA cap methylation was significantly increased in response to MycWTand MycΔC. This is consistent with nucleolin but not HSP60 mRNApolysome loading being well regulated by Myc (Fig.6D). GAPDH methylation wasnot regulated by Myc levels for either cell line, which correlates witha lack of stimulation of polysome loading in these cells. Nonspecificbinding of mRNA to control antibody beads was uniform and relativelylow, <10% of input RNA. We propose that the mechanism by whichMyc increases the translation rate of specific messages is byincreasing mRNA methyl cap formation for selectgenes.

The Myc N terminus is recruited totranscription start sites.

mRNA capping occurs most efficientlycotranscriptionally, and cap methyltransferase is recruited by theTFIIH-phosphorylated RNA Pol II CTD(25,48). Therefore, weinvestigated whether Myc increases TFIIH recruitment to specific genes,in correlation with the increased capping, polysome loading, andtranslation of these genes. We investigated the in vivo binding ofTFIIH to the cyclin T1, cyclin H, and nucleolin transcriptionalinitiation sites (the CDK9 promoter proved to be refractory to PCR).Using ChIP, we found enhanced recruitment of MAT1 and p62 (subunits ofTFIIH) to the cyclin H, cyclin T1, and nucleolin transcriptioninitiation sites in MycWT-, MycBM-and MycΔC-expressingcells compared to that in the vector control (Fig.7A). In correlation with the fact that MycΔMBII isdefective in increasing CDK9 and cyclin T1 translation and expression,enhanced recruitment of TFIIH to transcription initiation sites was notfound in cells expressing MycΔMBII (see Fig. S2 in thesupplemental material). There was also no Myc-dependentrecruitment of TFIIH to the GAPDH transcription initiation sites, whichalso did not have increased cap methylation and polysomeloading.
We had demonstrated that Myc expression increases TFIIHrecruitment and mRNA methylation and that this is an activity of the Nterminus of Myc independent of the DNA binding domain. We wanted todetermine whether Myc could have a direct role in this process by beingrecruited to the transcription initiation sites via the N terminus. Weinvestigated Myc binding by ChIP using anti-FLAG and anti-Mycantibodies. To confirm that MycΔC was not bindingnonspecifically to DNA, we established that MycWT but not MycΔCwas recruited to Myc/Max consensus sites in the established HSP60 andnucleolin target genes (Fig.7B). This confirms thatthe DNA binding domain is necessary for direct Myc binding to E-box DNAin vivo. In contrast, we found that MycΔMBII, MycBM, andMycΔC were recruited to the transcription initiation sites ofcyclin H, cyclin T1, and nucleolin (Fig.7C and see Fig. S2 in thesupplemental material). Therefore, the Myc N terminus can be recruitedto these transcriptional initiation sites indirectly and independentlyof the DNA binding domain. The fact that MycΔMBII is recruitedto transcription initiation sites but that MycΔMBII-expressingcells do not have enriched TFIIH recruitment to these sites suggeststhat Myc-dependent enhanced TFIIH recruitment is dependent on priorMycWT binding. As a negative control, Myc was not found to be recruitedto GAPDH initiation sites (Fig.7C).
Consistentwith the model that MBII recruits TFIIH to the transcription initiationsites of certain genes, we found that Myc could be coimmunoprecipitatedwith CDK7, the kinase subunit of TFIIH (Fig.7C). Furthermore, MycWTbut not MycΔMBII could be coimmunoprecipitated with CDK7 byusing an anti-CDK7 antibody, and CDK7 could be coimmunoprecipitatedwith MycWT but not MycΔMBII by using an anti-Myc antibody.Endogenous CDK7 could also be immunoprecipitated with endogenous Mycprotein from 293 cells (Fig.7D).

DISCUSSION

Mycincreases CTD kinase activity.

Here, we report that Myc induces theexpression of both CDK7 and CDK9 kinases, which phosphorylate the RNAPol II CTD as components of TFIIH and P-TEFb, respectively, and thatthere is a concomitant increase in the phosphorylation of theirrespective substrates, S5 and S2, in the CTD. All of the activities wedemonstrate for MycWT are manifested by the Myc transactivation domainalone and thus are within the repertoire of normal Myc function butindependent of target gene activation. We show that these kinaseactivities are induced in response to c-Myc and N-Myc in fibroblasts,epithelial cells, and neuroblastoma cells. We also demonstrate that Myccan promote TFIIH recruitment, cap methylation, and translation of asubset of genes. Myc can be recruited to transcription initiation sitesvia the N terminus alone. Myc can also bind CDK7 and promote TFIIHrecruitment in an MBII-dependent manner and therefore may have a directrole in this process. The fact that Myc can significantly increase thelevel of CTD phosphorylation in the total pool of RNA Pol II, ratherthan at specific promoters, is evidence of the potent effect of the MycN terminus. RNA Pol II phosphorylation governs many aspects oftranscription and cotranscriptional processing(5,29,52), and the finding thatMyc can substantially increase CTD phosphorylation is suggestive of aglobal role for Myc in these processes.
It is interesting thatMyc can directly enhance the recruitment of TFIIH to both the cyclin Hand cyclin T1 promoters (Fig.7). Myc inducesposttranscriptional elevation in these cyclin protein levels as well asother CDK components. The increased levels of these CDK/cyclincomplexes could synergize with Myc-induced recruitment in a positivefeedback loop to further enhance cellular CTD kinaselevels.

Myc activates CAK and promotesproliferation independently of target gene expression.

Myc-driven cell proliferation isessential for its role in development, normal cell physiology, and celltransformation (8,41). One of the mostsurprising findings of this study is that the N terminus of Myc hassufficient biological activity to actually rescue a large fraction ofthe proliferation defect of myc null fibroblasts. AlthoughMycΔC and MycBM do not regulate the transcription of Myc targetgenes, they have sufficient activity to rescue the cell doubling timefrom 40 h to 26 h. MycΔC and MycBM wereused as complementary mutants since MycΔC cannot bind to Max,whereas MycBM can. Since both mutants have an increased proliferationrate, we can rule out disturbance of the Max network as beingresponsible for this Myc N-terminal activity. In addition, theN-terminal MycBMΔMBII mutant cannot induce cell proliferationdespite being able to bind to Max equivalently to MycBM, thuslocalizing the proliferative activity to a critical N-terminaldomain.
Cell proliferation is driven by a cycle of CDK activationand degradation. CAK phosphorylates other CDKs, increasing the activityand stability of cell cycle CDK/cyclin complexes(23). Previously, MycWThas been shown to increase cellular CAK activity(32). In this paper, weshow that MycWT and DNA binding mutants increase the expression andactivity of CAK independently of target gene expression. In addition,we show that CDK7 is rate limiting for CDK phosphorylation and cellproliferation. Since CAK is also a component of TFIIH, it is notpossible to deduce whether inhibition of CDK7 reduces cellproliferation because it inhibits TFIIH-induced RNA Pol IIphosphorylation or CAK-induced CDK phosphorylation. Indeed, Myc mayinduce proliferation via CDK7 by activating both TFIIH and CAK.
Aprevious report which addressed the mechanism by which Myc drivesproliferation demonstrated that DNA binding and dimerization with Maxare necessary for Myc to maintain cells in S phase following serumwithdrawal (2), andindeed, our non-DNA binding mutants behave similarly underserum-starved conditions (unpublished data). We do not believe thatthis previous study conflicts with our own because our studies usedlog-phase cells, i.e., subconfluent cultures grown in serum. These twostudies reveal redundant mechanisms by which Myc can driveproliferation, both dependent and independent of generegulation.

Myc regulation of mRNA capmethylation.

In this paper,we report the first example of the regulation of mammalian mRNA capmethylation. Yeast mutant analysis and biochemical assays havedemonstrated that mRNA guanylylation is necessary for stabilizing mRNA,and methylation of the guanylyl group is necessary to permittranslation of the mRNA(40,50,51). Since these twosteps are carried out by separate enzymes and the addition of guanylyland methyl groups occurs with a time delay in vitro, it has beenproposed that differential regulation of these enzymes may exist(38,51). Here, we show thatMyc can increase the methylation of specific messages and that this isa property of the Myc N terminus alone. Myc is recruited totranscription initiation sites accompanied by TFIIH in anMBII-dependent manner. The TFIIH-phosphorylated CTD has beenreported to recruit methyltransferase, but it remains possible that Mycmay stimulate the methyltransferase either via TFIIH or more directly.It is interesting to draw comparisons between our study of Myc and arecent study of GAL4(39). A GAL4 mutant wasdemonstrated to increase CTD phosphorylation and translation ratherthan transcription of target genes, and we propose that methylation ofthese target gene mRNAs may be responsible for the increased GAL4target translation rate.
TFIIH recruitment is also associatedwith increased transcription and mRNA guanylylation, both of whichresult in increased mRNA abundance. However, in our system, although wesee Myc-dependent recruitment of TFIIH to some promoters, we do not seean increase in mRNA abundance for these genes. There are manyexplanations that could account for this observation, the simplestbeing that transcription, capping, and cap methylation may be promoterdependent and differentially sensitive to the TFIIH-phosphorylatedCTD.

MycWT upregulates the transcriptionand translation of a subset of Myc target genes.

We found that two traditional Myctarget genes, the nucleolin and RUVBL1 genes, had increased capmethylation and polysome loading in response to MycWT and the DNAbinding mutants. Consistent with increased polysome loading, we foundthat nucleolin and RUVBL1 protein expression levels are also elevatedin response to the expression of MycWT, MycBM, andMycΔC. The increase in protein expression is largerin response to MycWT than to the DNA binding mutants, consistent withthese genes having both increased transcription and increasedtranslation in response to MycWT. Thus, Myc may increase the proteinconcentration of a large subset of genes by upregulating both theirtranscription and their translation. Another Myc target gene, the HSP60gene, did not exhibit a robust increase in cap methylation and polysomeloading in response to the Myc DNA binding mutants, revealing that therecruitment of Myc to an E box is not sufficient to stimulate capmethylation. A small increase in HSP60 polysome loading was observed inresponse to MycWT, and this may be a reflection of the increasedexpression of eukaryotic initiation factors 4E and 4G induced by MycWT(but not Myc DNA binding mutants)(47). Indeed, MycWTexpression has been reported previously to increase the polysomeloading of specific mRNAs(36). More global studieswill be required to determine which target genes are cap methylated inresponse to Myc.

Summary.

Myc is a potent and pleiotropiceffector of cellular growth and a well-established transcriptionfactor. Here, we report that Myc activity extends beyondtranscriptional regulation of target genes to a general role inpromoting global phosphorylation of the RNA Pol II CTD. This activityis likely to have broad-ranging effects on transcription and mRNAmetabolism, such as increased cap methylation on some mRNAs. Thecombination of this global activity with the transactivation ofnumerous target genes may account for the prominent role of Myc as anoncogene.
FIG. 1.
FIG. 1. DNAbinding-deficient Myc mutants. (A) A diagram of murine N-Mycmutants. MycΔMBII has a deletion of amino acids 103 to 119.MycBM has amino acids 381 to 384 mutated from RQRR to ADAA.MycBMΔMBII has both previous mutations. MycΔC istruncated at amino acid 370 and has a nuclear localization signal (NLS)at the C terminus. DNA binding in EMSA and Max binding incoimmunoprecipitation are summarized. (B) Myc proteins wereimmunoprecipitated from reconstitutedmyc−/− cellsusing anti-FLAG antibody and immunoblotted for N-Myc and Max (upperpanels). Cell extracts were immunoblotted for Max (lower panel).(C) EMSA was performed on extracts from 293 cells expressingthe indicated Myc proteins, using a Myc/Max consensus binding siteprobe. Extracts were also immunoblotted with anti-FLAG to detect theMyc proteins (upper panel). (D) RNA extracted frommyc−/− celllines expressing the indicated Myc protein was used for RT-PCR usingprimers specific for the Myc-activated CAD, HSP60, and nucleolin (NUCL)genes, the Myc-repressed neomycin (Neo) (under the control of thec-myc promoter) and GADD45 genes, and GAPDH as a control.(E) The same RNA was hybridized to rat oligonucleotidemicroarrays (see Materials and Methods). The graph shows the relativeexpression levels of Myc target genes in response to MycWT,MycΔMBII, and MycBM compared to that of the vector control.When the expression levels of the samples were compared to those of thevector control, the t test returned the following Pvalues: MycWT, 1.4 × 10−7; MycΔMBII,0.09; and MycBM, 0.5. WB, Western blot; USF, upstream stimulatoryfactor.
FIG. 2.
FIG. 2. DNAbinding-deficient Myc mutants induce cell proliferation andmorphological change. The results of an analysis ofmyc−/− cellsexpressing MycWT and mutants are shown. (A) Phase-contrastmicrographs of log-phase cells. (B) Growth curves. The datashown represent the means for at least three experiments, and errorbars show the standarddeviations.
FIG. 3.
FIG. 3. TheDNA binding-deficient Myc mutant elevates total cellular CAK activity.(A) A CAK assay was performed on extracts frommyc−/−fibroblasts expressing the indicated Myc protein. Proteinimmunoprecipitated with anti-CDK7 or control antibody was incubatedwith recombinant CDK2 and [32P]ATP, and reaction productswere run on a gel, visualized by a phosphorimager (left panel), andquantitated (right panel). The results of a representative experimentare shown. (B) Cell extracts were prepared frommyc+/+ fibroblasts (TGR)expressing the vector control and CDK7 DN as indicated. Extracts wereimmunoblotted using antibodies raised against phospho-CDC2 (CDC2-P),CDC2, phospho-CDK2 (CDK2-P), CDK2, and γ-tubulin (Tub).(C) Growth curves formyc+/+ fibroblasts (TGR)expressing the vector control and CDK7 DN. The data shown represent themeans for two experiments, and error bars show the standarddeviations.
FIG. 4.
FIG. 4. Mycinduces CAK expression by a posttranscriptional mechanism.(A) Cell extracts were prepared frommyc+/+ ormyc−/−fibroblasts expressing the indicated Myc protein from several cellsystems: myc+/+fibroblasts transfected for 24 h with control siRNA or twoindependent Myc siRNAs, a neuroblastoma cell line (Tet-21/N) expressingdoxycycline-off N-Myc cultured in 0 and 0.2 μg/ml doxycycline(Dox) for 2 days, and IMECs expressing c-MycWT orc-MycΔMBII. Immunoblot analyses were performed oncell extracts using antibodies raised against CAK components, CDK7,cyclin H, and MAT1, and γ-tubulin (Tub). Immunoblot analyseswere also performed using anti-c-Myc antibodies onmyc+/+ cell extracts andIMEC extracts and using anti-N-Myc antibodies on neuroblastoma cellextracts. (B) RNA was extracted from two independentlog-phase samples. RT-PCR was performed in the linear range usingprimers specific for CDK7, cyclin H, MAT1, and GAPDH. Mean relativeexpression levels were calculated, and error bars indicate the standarddeviations.
FIG. 5.
FIG. 5. Mycinduces RNA Pol II phosphorylation. Nuclear extracts were prepared fromthe following log-phase cells: (A) myc+/+ fibroblasts,myc−/−fibroblasts expressing the indicated Myc protein,myc+/+ fibroblaststransfected with control siRNA or two independent Myc siRNAs,(B) a neuroblastoma cell line (Tet-21/N) expressing N-Myccultured in 0 and 0.2 μg/ml doxycycline (Dox) for 2 days, and(C) IMECs expressing the indicated Myc protein. Immunoblotanalyses were performed using monoclonal antibodies (mAb) raisedagainst RNA Pol II CTD phospho-S5 (H14) and phospho-S2 (H5) and the RNAPol II large subunit. (D) Immunoblot analyses were performedon cell extracts using antibodies raised against P-TEFb components,CDK9 and cyclin T1, and γ-tubulin (Tub). (E) RT-PCRwas performed in the linear range using primers specific for CDK9 andcyclin T1 on RNA from two independent samples. Mean relative expressionlevels were calculated, and error bars indicate the standarddeviations.
FIG. 6.
FIG. 6. Mycelevates cyclin T1 and CDK9 translation rate, polysome loading, andmRNA cap methylation. Log-phasemyc+/+ fibroblasts andmyc−/−fibroblasts expressing MycWT, MycBM, MycΔC, or the vectorcontrol were labeled (“pulse”) with[35S]methionine/cysteine for 0.5, 1.0, or 1.5 h.Subsequently, cells were washed and incubated in regular growth medium(“chase”) for 6 h. Cell extracts wereprepared and normalized for protein content. (A) Protein wasprecipitated from cell extracts using TCA and the counts detected usinga scintillation counter. The left panel represents label incorporation(Incorp.) during “pulse,” and the right panelrepresents label loss during “chase.” (B) Cyclin T1 and CDK9 were immunoprecipitated from cell extracts, resolvedon SDS-PAGE, and visualized by a phosphorimager. (C) Meanquantitation of duplicates from a representative experiment is shown.Error bars show the standard deviations. The upper panels indicaterelative label incorporation into cyclin T1 and CDK9 during“pulse,” and the lower panels indicate label lossduring “chase.” (D) Using the same cell linesas described above, polysomes were separated from monosomes/hnRNA bycentrifugation through a sucrose bed. The proportion of 18S rRNA in thepolysome fraction and monosome fraction was calculated. mRNA from eachfraction was used as substrate for RT-PCR for the mRNA indicated. Theproportion of each mRNA in polysome and monosome fractions for eachcell line is depicted. The results of a representative experiment areshown. Mean values of duplicates are shown, and error bars indicate thestandard deviations. (E) Protein extracts made from the samepanel of cell lines were used to perform Western blot analyses, probingwith anti-RUVBL1 and anti-nucleolin (NUCL) antibodies. RT-PCR wasperformed on independently isolated RNA samples from the same cells byusing primers specific for RUVBL1 and nucleolin. (F) Capmethylation was assessed for RNA extracted frommyc−/−fibroblasts expressing the vector control, MycWT, and MycΔC andfrom immortalized mammary epithelial cells expressing the vectorcontrol and MycWT. RNA was subjected to immunoprecipitation usinganti-2,2,7-methylguanosine antibody. RNA was purified and used as atemplate for RT-PCR using primers specific for the genes indicated. Thequantity of immunoprecipitated RNA is expressed as a fraction of inputRNA. The results of representative experiments areshown.
FIG. 7.
FIG. 7. Myc promotes TFIIH binding and binds to the transcription initiation sites via the N terminus. ChIP was performed onmyc−/−fibroblasts expressing MycWT, MycBM, MycΔC, or the vectorcontrol. (A) Complexes were immunoprecipitated withantibodies raised against the TFIIH subunits MAT1 and p62 or controlantibody. Coprecipitated DNA was used as a template for PCR usingprimers specific for cyclin T1, cyclin H, nucleolin (NUCL), and GAPDHtranscriptional start sites. PCR products were labeled, run on a gel,and visualized by a phosphorimager (upper panels). PCR products werequantitated and normalized to input PCR, and the relative (Rel.) signalis depicted. The mean values for at least two independent experimentsare shown in the graphs. Error bars show the standard deviations.(B) Complexes were immunoprecipitated with anti-FLAG (Myc) orcontrol antibody. PCR was carried out using primers specific for HSP60and nucleolin E boxes. (C) Complexes were immunoprecipitatedwith antibodies raised against the FLAG (Myc) tag and Myc or controlantibody. PCR was carried out using primers specific for cyclin T1,cyclin H, nucleolin, and GAPDH transcription start sites. (D) 293 cells were transfected with MycWT, MycΔMBII, and CDK7 asindicated. Immunoprecipitation was carried out with cell extracts usinganti-CDK7 (left panel) or anti-Myc (middle panel). Immunoprecipitatedproteins and cell extracts (right panel) were subjected to Westernblotting using anti-CDK7 and anti-Myc antibodies. (E) Immunoprecipitation was carried out using anti-c-Myc antibodies orcontrol antibodies on untransfected 293 cells. Immunoprecipitatedprotein and cell extracts were subjected to Western blotting usinganti-CDK7 and anti-Mycantibodies.

Acknowledgments

We thank Larry Myers, Gary LeRoy, William Rigby, Janice Ascano, and members of the Cole lab for helpful discussions.
This work was supported by grants from theNational Cancer Institute to M.D.C.

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Molecular and Cellular Biology
Volume 27Number 615 March 2007
Pages: 2059 - 2073
PubMed: 17242204

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Received: 26 September 2006
Revision received: 17 November 2006
Accepted: 5 January 2007
Published online: 26 December 2020

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Victoria H. Cowling
Departments of Pharmacology and Genetics, Dartmouth Medical School, One Medical Center Drive, Lebanon, New Hampshire 03756
Michael D. Cole [email protected]
Departments of Pharmacology and Genetics, Dartmouth Medical School, One Medical Center Drive, Lebanon, New Hampshire 03756

Notes

Publishedahead of print on 22 January 2007.
Supplemental material for this article may be found athttp://mcb.asm.org/.

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