The MNT transcription factor autoregulates its expression and supports proliferation in MYC-associated factor X (MAX)-deficient cells

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.This work was supported by Grant SAF2017-88026-R from Agencia Estatal de Investigación, Spanish Government (to J. L. and M. D. D.), funded in part by FEDER Program from the European Union, National Institutes of Health Grant CA57138/CA from NCI (to R. N. E.), and grants from Shriners Hospitals for Children (to P. J. H.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

CD21/CR2 as a possible Myc target gene in hematopoietic cell lines

c-Myc is a transcription factor which belongs to the family of basic-helix/loop/helix-leucine zipper proteins. It is found deregulated in more than 50% of human tumors, including Burkitt lymphoma, a human B-cell tumor, where Myc is chromosomally translocated. There is an African epidemic version of Burkitt lymphoma associated with the infection of Epstein-Barr virus (EBV). CD21/CR2 is the co-receptor of B-cell receptor (BCR) that serves as receptor for this virus. We explored whether CR2 could be a potential Myc target gene, since previous results in our laboratory showed that CR2 and Myc were correlated. We have used several hematopoietic cell lines (Raji and K562) in which we have measured CR2 mRNA levels by qPCR upon different conditions of Myc expression. Our results indicate that Myc overexpression in K562 cells results in CR2 overexpression and this effect is reproduced in the absence of protein synthesis. Moreover, in Burkitt cells, inhibition of Myc through the drug JQ1 (which impairs Myc transcription) resulted in CR2 mRNA decrease. The bioinformatic analysis of the ChIP-seq data of the ENCODE project revealed that Myc is bound to the CR2 promoter. Altogether the data strongly suggest that CR2 is a novel Myc target gene and this may explain the association of Burkitt lymphoma in EBV infection.Máster en Biología Molecular y Biomedicin

The MYC oncogen as a transcriptional activator of the Epstein-Barr virus receptor (CR2/CD21)

RESUMEN: MYC es un oncogen que se encuentra desregulado en más de la mitad de los tumores humanos. La desregulación de MYC es frecuente en enfermedades hematopoyéticas y especialmente frecuente en linfoma de Burkitt (BL) donde se encuentra traslocado en prácticamente todos los casos. El BL también está asociado a la infección por virus del Epstein-Barr (EBV). El EBV infecta las células a través de su receptor el CR2. En este trabajo se realizaron diferentes ensayos de ganancia y pérdida de función de MYC en los cuales se demostró que los niveles de MYC se correlacionaban con los obtenidos por CR2. Además, se realizaron inmunoprecipitaciones de MYC demostrando la unión de MYC al promotor de CR2. Finalmente, se infectaron células de BL EBV negativas con virus de EBV, bajo diferentes condiciones de expresión de MYC y se observó que bajos niveles de MYC se correlacionaban con peores eficiencias de infección.ABSTRACT: MYC is an oncogene that is found deregulated in at least a half of human cancers. MYC translocation is frequent in hematopoietic malignancies such as Burkitt lymphoma (BL) which is frequently associated with the Epstein-Barr virus (EBV). EBV infects lymphocytes and epithelial cells through complement receptor CR2/CD21 (CR2 herein after). We have shown that MYC induces CR2 mRNA and protein expression using different cellular models. Consistently, the inhibition of MYC via lentiviral infection with short-hairpin MYC vectors results in a decrease in CR2 mRNA and protein levels. Chromatin immunoprecipitation (ChlP) was performed in Raji (BL cell line) cells confirming the data from ENCODE project. Finally, we exposed BL negative cell line with different MYC levels conditions to EBV showing that low levels of MYC correlated with less EBV infection efficiency. This open the novel hypothesis that MYC translocation in BL could favor EBV infection.Esta Tesis ha sido realizada en el Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC) perteneciente a la Universidad de Cantabria (UC) y al Consejo Superior de Investigaciones Científicas (CSIC). La financiación para la realización de esta Tesis doctoral ha sido proporcionada por el Ministerio de Educación y Ciencia (SAF2014-53526-R) (MINECO/FEDER,UE) (SAF2017-88026-R) (AEI/FEDER,UE). He disfrutado de varios contratos de investigación financiados por los proyectos anteriormente mencionados, la Universidad de Cantabria y SODERCAN

CD21/CR2 as a possible Myc target gene in hematopoietic cell lines

c-Myc is a transcription factor which belongs to the family of basic-helix/loop/helix-leucine zipper proteins. It is found deregulated in more than 50% of human tumors, including Burkitt lymphoma, a human B-cell tumor, where Myc is chromosomally translocated. There is an African epidemic version of Burkitt lymphoma associated with the infection of Epstein-Barr virus (EBV). CD21/CR2 is the co-receptor of B-cell receptor (BCR) that serves as receptor for this virus. We explored whether CR2 could be a potential Myc target gene, since previous results in our laboratory showed that CR2 and Myc were correlated. We have used several hematopoietic cell lines (Raji and K562) in which we have measured CR2 mRNA levels by qPCR upon different conditions of Myc expression. Our results indicate that Myc overexpression in K562 cells results in CR2 overexpression and this effect is reproduced in the absence of protein synthesis. Moreover, in Burkitt cells, inhibition of Myc through the drug JQ1 (which impairs Myc transcription) resulted in CR2 mRNA decrease. The bioinformatic analysis of the ChIP-seq data of the ENCODE project revealed that Myc is bound to the CR2 promoter. Altogether the data strongly suggest that CR2 is a novel Myc target gene and this may explain the association of Burkitt lymphoma in EBV infection.Peer Reviewe