Downloaded from http://www.jbc.org/ at Centro Nacional de Investigaciones Cardiovasculares (CNIC) on July 8, 2020 cro 2001 promoter, an , ‡ MNT , ¶ , and ‡ ). MYC can also interact 1 ), which alternatively can (2020) 295(7) 2001–2017 ). Whereas MYC–MAX, upon 6, 7 , Rosa Blanco ) serving as the link between ‡ 2, 3 8, 9 , Peter Hurlin § J. Biol. Chem. MLX–MONDO. protein MLX ( , M. Dolores Delgado § , Ignacio Varela MLXIP (MONDOA) and MLXIPL (MONDOB) ‡4 and bind E-boxes ( E-boxes, acts primarily as a transcriptional activator, is a basic helix-loop-helix leucine zipper (bHLH-LZ) ). MNT can bind not only to MAX but also to the Division of Basic Sciences, Fred Hutchinson Cancer , Patrick Carroll 10 § ‡ 4, 5 MNT is expressed constitutively in proliferating and quies- MNT bHLH-LZ, basic helix-loop-helix leucine zipper;tor MAX, X; MYC-associated fac- qPCR, quantitativeGSEA, PCR; Gene RNA-seq, Set RNA Enrichment sequencing;reads FL, Analysis; per kilobase full-length; ChIP-seq, pair ChIP-sequencing; per million. RPKM, The abbreviations used are: MNT, MAX network transcriptional repressor; binding to interact with proteins. Therefore, MNT participates bothMLX-centered in the networks MAX- ( and protein, and it is the mostily, divergent member which of the also MXDMNT fam- includes forms MXD1, heterodimers MXI1, withdomain MAX MXD3, and binds through to and E-box the DNA MXD4. bHLH-LZ most sequences. prevalent MYC human is oncoproteins one ( of the the typical effect of MNT–MAXsion is ( the transcriptional repres- cent cells, and the protein levels do not show major fluctuations with MAX MAX–MYC and 10 MAX-like HLH MNT–MAX dimers bind andeffect repress that depends the on one of theIn two MAX-deficient E-boxes cells, on MNT this was promoter. overexpresseduted and to redistrib- the cytoplasm. Interestingly, MNTproliferation was required even for cell in theMAX-deficient absence cells, of MNT MAX.homodimers. RNA-sequencing We experiments binds revealed that show MNT to thatregulates the in MLX, expression of but several genesMAX, even with also in many the of forms these absence genes of beingulation and involved DNA in repair. cell Of cycle note, reg- MNT–MNT homodimersulated reg- the transcription of some genesation. involved The in cell tight prolifer- regulationwithout of MAX suggest MNT a major and role for its MNT in functionality cell proliferation. even . Published, Papers in Press, January 9, 2020, DOI 10.1074/jbc.RA119.010389 , Ester Molina , Robert N. Eisenman ‡7 8 ʈ , Andrea Quintanilla ‡1,3 Shriners Hospitals for Children Research Center, and Department of Cell, ¶ [email protected] oncogene modula- , Julia Aresti ‡6 . MYC , Judit Liaño-Pons ‡1,2 , Montse Sanchez-Céspedes ʈ , Gabriel Bretones ‡5 Figs. S1–S9 and Tables S1–S8 ‡9 Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, and Department of ‡ ARTICLE Genes and Cancer Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute–IDIBELL, ʈ Javier León Octavio A. Romero tute, University of Groningen, 9712 CP Groningen, Netherlands. Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain. Medicina, Instituto UniversitarioOviedo, de 33003 Oncología Oviedo, Asturias, (IUOPA), Spain. Universidad de ics and Molecular Medicine,United University Kingdom. of Edinburgh, EH4 2XU Scotland, Catarina (UFSC), 88040-900 Florianópolis, Brazil. address: Sorrell Lab, Dept. ofter, Cell Dallas, Biology, TX UT 75390. Southwestern Medical Cen- Investigación, Spanish Government (to J. L. andFEDER M. D. Program D.), funded from in part the by Grant CA57138/CA European from NCI Union, (to R. National N.for E.), Institutes and Children grants of from (to Shriners P. Health Hospitals J. H.).interest The with authors the declare contents that of theysibility this have of article. no the The conflicts authors content and of is doesof solely not the necessarily the National represent respon- Institutes the of official Health. views Archive with the accession number PRJEB23604. The MAX network transcriptional repressor (MNT) is an To whom correspondence should be addressed. E-mail: Present address: Groningen Biomolecular Sciences and Biotechnology Insti- Present address: Josep Carreras Leukemia Research Institute, Campus ICO- Present address: Dept. de Bioquímica y Biología Molecular, Facultad de Recipient of an F.P.U. fellowship fromPresent the address: Spanish Edinburgh Government. Cancer Research UK Centre, Institute of Genet- Present address: Dept. of Biochemistry, Universidade Federal de Santa Both authors contributed equally to thisRecipient work. of an F.P.U. fellowship from the Spanish Government. Present Developmental and Cancer Biology, Knight Cancer Institute,the Oregon Health and Science University, Portland, Oregon 97239, and Research Center, Seattle, Washington 98109, the X 7 8 9 6 4 5 1 2 3 This work was supported by Grant SAF2017-88026-R from Agencia Estatal de The RNA-seq and ChIP-seq data have been deposited to the European Nucleotide This article contains Edited by Eric R. Fearon From the (MAX)-deficient cells M. Carmen Lafita-Navarro Fabiana Ourique and supports proliferation in MYC-associated factor X Received for publication, July 28, 2019, and in revised form, December 16, 2019 The MNT transcription factor autoregulates its expression X tor, but little is knownMNT about localizes its to regulation. the We nucleus show of here MAX-expressing cells that and that MXD family transcription factor(bHLH) of family. the basic MNT helix-loop-helix dimerizesregulator, MYC-associated factor with X (MAX), another and down-regulates transcriptional genes by binding to E-boxes. MAX alsooncogenic dimerizes bHLH with transcription MYC, factor. an Upon E-boxMYC–MAX binding, dimer the activates gene expression. MNT also bindsthe to MAX dimerization proteinand MLX MNT–MAX (MLX), dimers and co-exist. However, MNT–MLX have all been attributed MNT to functions MNT–MAX dimers, and nothe functions of MNT–MLX dimer have beenrole has described. been linked MNT’s to biological its function as a 08908 Barcelona, Spain Molecular Biology, Universidad de Cantabria, 39005 Santander, Spain, the © 2020 Lafita-Navarro et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. MNT functions in the absence of MAX when quiescent cells are mitotically stimulated (5, 10, 11). For this purpose, we transfected UR61 cells with a construct MntϪ/Ϫ mice are not viable (10, 12), whereas Mxd1Ϫ/Ϫ, carrying human MAX cDNA driven by the metallothionein ϩ Mxi1Ϫ/Ϫ, and Mxd3Ϫ/Ϫ mice survive, suggesting that MNT promoter, which is activated by Zn2 cations (24). Several function is not redundant with that of the other MXD proteins clones were isolated, and two of them with robust MAX induc- (13–15). Moreover, MNT is the only MXD-related protein in tion were mixed, and the resulting cells were termed URMax34. invertebrates (9). We also generated a cell line transfected with the empty vector, Consistent with MNT functioning as a MYC transcriptional termed URMT, which is a pool of five transfected clones. The 2ϩ antagonist, enforced MNT expression inhibits cell proliferation induction of MAX in response to Zn in URMax34 cells was and impairs MYC-dependent transformation (5, 11). The defi- confirmed by immunoblot (Fig. 1B). We examined the effect of ciency or down-regulation of MNT in fibroblasts leads to MAX induction on MNT levels in URMax34 cells, and we increased proliferation (i.e. similarly to MYC overexpression) found that MNT was down-regulated upon MAX induction by Zn2ϩ (Fig. 1B). As expected, the treatment of URMT cells with and partially rescues the proliferative arrest caused by MYC Downloaded from 2ϩ deficiency (10, 11, 16, 17). MNT ablation in vivo leads to breast Zn did not change the MNT levels (Fig. S1A). To confirm this and T-cell tumors (10, 12, 17), and according to the Cancer result and rule out effects potentially related to the generation Genome Atlas, about 10% of human tumors show deletion of a of stably transfected clones (as the URMax34 system), UR61 MNT allele (1). cells were transiently transfected with a MAX expression vec- tor, and the results showed a decrease in MNT protein levels in Partial or total MNT deficiency in mouse models impairs http://www.jbc.org/ MAX-transfected cells (Fig. 1C). It is noteworthy that the MYC-dependent tumorigenesis (18, 19), and MNT knockout in down-regulation of MNT provoked by the re-expression of some cell models inhibits proliferation and promotes apoptosis MAX in these cells also occurred at the mRNA levels, as deter- (10, 16, 17). Thus, MYC and MNT proteins co-exist in prolif- mined by RT-qPCR (Fig. 1D). We then analyzed the effect of erating cells, and depending on the model, MNT acts as a MYC MAX-enforced expression in three human small-cell lung can- antagonist or a cooperator of MYC. cer cell lines deficient in MAX (21). MAX-enforced expression at Centro Nacional de Investigaciones Cardiovasculares (CNIC) on July 8, 2020 However, there is scarce information about MNT transcrip- was achieved by lentiviral transduction, and the levels of MNT tional regulation, and it is unknown whether MNT exerts func- were examined by immunoblot in Lu134, Lu165, and H1417 tions without MAX. In this work, we studied possible MAX- cell lines (Fig. 1E). In all cell lines, MAX expression resulted in independent functions of MNT using UR61 cells as the main lower MNT protein levels. The MNT mRNA levels were also model. These rat pheochromocytoma cells do not express down-regulated in Lu165 and in H1417 cells upon MAX a functional MAX protein but a truncated form (termed expression (Fig. 1F). PC12 MAX ) that lacks the second helix and leucine zipper region We next sought to confirm this in a different cell type with of the bHLH-LZ domain, which are the regions responsible for endogenous MAX. A MAX expression vector was transfected dimerization with MYC and MNT (20).