The Multiple Faces of MNT and Its Role as a MYC Modulator

MNT is a crucial modulator of MYC, controls several cellular functions, and is activated in most human cancers. It is the largest, most divergent, and most ubiquitously expressed protein of the MXD family. MNT was first described as a MYC antagonist and tumor suppressor. Indeed, 10% of human tumors present deletions of one MNT allele. However, some reports show that MNT functions in cooperation with MYC by maintaining cell proliferation, promoting tumor cell survival, and supporting MYC-driven tumorigenesis in cellular and animal models. Although MAX was originally considered MNT?s obligate partner, our recent findings demonstrate that MNT also works independently. MNT forms homodimers and interacts with proteins both outside and inside of the proximal MYC network. These complexes are involved in a wide array of cellular processes, from transcriptional repression via SIN3 to the modulation of metabolism through MLX as well as immunity and apoptosis via REL. In this review, we discuss the present knowledge of MNT with a special focus on its interactome, which sheds light on the complex and essential role of MNT in cell biology.J.L.-P. was supported by a postdoctoral scholarship from the Radiumhemmet Research Funds, Stockholm. M.A.-H. was supported by grants from the Swedish Cancer Society, the Swedish Childhood Cancer Fund, the Swedish Research Council, Radiumhemmet Research Funds, and Karolinska Institutet, and J.L. was supported by grant SAF2017-88026-R from Agencia Estatal de Investigación, from the Spanish Government

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

Study of the novel interaction between MNT and c-REL and its impact on cell proliferation and viability

MNT is a basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein from the MXD family. MNT and other MXD proteins play an important role as MYC antagonists, which is one of the most frequently altered oncogenes in human cancer. Both MYC and MXD proteins heterodimerize with MAX, binding to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MXD) their transcription. However, some MAX-deficient cell lines and tumours with MAX mutations have been found, pointing out the existence of MYC and MXD functions which are MAX-independent. Our preliminary results in UR61 cells (derived from rat pheochromocytoma and deficient in MAX) suggest a possible interaction between MNT and c-REL that is MAX-independent. c-REL belongs to the REL/NF-kB family which takes part in several relevant biological processes. Here, performing co-immunoprecipitation assays, we confirm this interaction in other rodent cell lines (rat C6, mouse Neuro-2a) but not in the human cell lines tested so far (HeLa, 293T, Raji, K562, SH-SY5Y, T98G). In order to identify the MNT interaction domain, a MNT deletion mutant (ΔBr) was transfected into UR61 cells and the c-REL binding was analysed by immunoprecipitation with anti-MNT antibodies. The results showed that the basic region (including the DNA binding domain) is required for the c-REL-MNT interaction. Furthermore, we showed that MNT and c-REL silencing by siRNAs (generated with short-hairpin vectors) reduced cell proliferation and viability of UR61, C6 and H1417 cell line (human lung cancer cells deficient in MAX), as assessed by clonogenic assays and/or cell counting.Máster en Biología Molecular y Biomedicin

Novel interactions of MNT protein and their effect on transcriptional regulation

RESUMEN: MNT es un factor de transcripción de la familia MXD, conocido por ser un modulador importante de la actividad de la oncoproteína MYC. Tanto MYC como las proteínas MXD son factores de transcripción de tipo hélice-bucle-hélice/cremallera de leucina (bHLHLZ) que heterodimerizan con MAX, se unen a secuencias específicas en el ADN denominadas E-boxes, generalmente activando (MYC-MAX) o reprimiendo (MXD-MAX) la transcripción de genes diana. Sin embargo, poco se sabe sobre las funciones de MYC y las MXDs al margen de su interacción con MAX. Los resultados obtenidos en este trabajo revelan que MNT interacciona con REL y que es un regulador de la vía de señalización de NF-kB. Asimismo, confirmamos la habilidad de MNT de formar homodímeros y heterodímeros con MLX, y estudiamos su función en la regulación transcripcional en ausencia de MAX. Por último, describimos nuevos genes dianas de MNT mediante un modelo de células humanas knockout para MNT. Estos genes están implicados en una gran variedad de procesos celulares, tales como desarrollo, proliferación, diferenciación o respuesta a estímulos externos. En conclusión, nuestro trabajo expande el conocimiento que se posee sobre MNT y confirma su papel clave en la red proximal de MYC.ABSTRACT: MNT is a transcription factor from the MXD family known to be an important modulator of the activity of the oncoprotein MYC. Both MYC and MXD proteins are basic helix-loop­ helix leucine zipper (bHLHLZ) transcription factors that heterodimerize with MAX, bind to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MXD) their transcription. However, little is known about the MYC and MXD functions beyond MAX interaction. Here we provide evidence of a novel MAX­ independent interaction between MNT and REL, and a new function of MNT as a regulator of the NF-KB pathway. In addition, we confirm the ability of MNT to form homodimers and heterodimers with MLX and we study their function as transcriptional regulators in the absence of MAX. Finally, we describe new MNT target genes by using a human MNTknockout model. These novel target genes are involved in different cellular processes, such as development, proliferation, differentiation or response to externa! stimulus. Taken together, our results widen our knowledge about MNT beyond MAX interaction and provide new evidences of the key role of MNT in the MYC proximal network.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), en Santander (Cantabria, España). 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). El autor de esta Tesis ha disfrutado de una ayuda para la formación de profesorado universitario (FPU) (referencia FPU14/01786), concedida por el Ministerio de Educación y Formación Profesional

A druggable conformational switch in the c-MYC transactivation domain

Abstract The c-MYC oncogene is activated in over 70% of all human cancers. The intrinsic disorder of the c-MYC transcription factor facilitates molecular interactions that regulate numerous biological pathways, but severely limits efforts to target its function for cancer therapy. Here, we use a reductionist strategy to characterize the dynamic and structural heterogeneity of the c-MYC protein. Using probe-based Molecular Dynamics (MD) simulations and machine learning, we identify a conformational switch in the c-MYC amino-terminal transactivation domain (termed coreMYC) that cycles between a closed, inactive, and an open, active conformation. Using the polyphenol epigallocatechin gallate (EGCG) to modulate the conformational landscape of coreMYC, we show through biophysical and cellular assays that the induction of a closed conformation impedes its interactions with the transformation/transcription domain-associated protein (TRRAP) and the TATA-box binding protein (TBP) which are essential for the transcriptional and oncogenic activities of c-MYC. Together, these findings provide insights into structure-activity relationships of c-MYC, which open avenues towards the development of shape-shifting compounds to target c-MYC as well as other disordered transcription factors for cancer treatment

Target Genes of c-MYC and MYCN with Prognostic Power in Neuroblastoma Exhibit Different Expressions during Sympathoadrenal Development

Deregulation of the MYC family of transcription factors c-MYC (encoded by MYC), MYCN, and MYCL is prevalent in most human cancers, with an impact on tumor initiation and progression, as well as response to therapy. In neuroblastoma (NB), amplification of the MYCN oncogene and over-expression of MYC characterize approximately 40% and 10% of all high-risk NB cases, respectively. However, the mechanism and stage of neural crest development in which MYCN and c-MYC contribute to the onset and/or progression of NB are not yet fully understood. Here, we hypothesized that subtle differences in the expression of MYCN and/or c-MYC targets could more accurately stratify NB patients in different risk groups rather than using the expression of either MYC gene alone. We employed an integrative approach using the transcriptome of 498 NB patients from the SEQC cohort and previously defined c-MYC and MYCN target genes to model a multigene transcriptional risk score. Our findings demonstrate that defined sets of c-MYC and MYCN targets with significant prognostic value, effectively stratify NB patients into different groups with varying overall survival probabilities. In particular, patients exhibiting a high-risk signature score present unfavorable clinical parameters, including increased clinical risk, higher INSS stage, MYCN amplification, and disease progression. Notably, target genes with prognostic value differ between c-MYC and MYCN, exhibiting distinct expression patterns in the developing sympathoadrenal system. Genes associated with poor outcomes are mainly found in sympathoblasts rather than in chromaffin cells during the sympathoadrenal development

Liberal education and fiction in the Swedish "folkhögskola" adult education college

The purpose of this thesis is to evaluate the present importance of liberal education in the Swedish "folkhögskola" by studying its expressions in the teaching of literature and fiction. I have interviewed teachers at four different schools about their opinions of liberal education, literature and cultural heritage, means of instruction, and influence of their students. Liberal education in this thesis is considered as a process. Starting from our own experiences we meet something strange to us and while absorbing this in our experiences we change. But its purpose also is to convey the canon of Western literature. My intention is to study the emphasis of these two poles at the various schools. My conclusion is that the concept "liberal education" or the Swedish word "bildning" is still vividly present in the practical daily teaching at the various schools.Uppsatsnivå:

A novel role of MNT as a negative regulator of REL and the NF-κB pathway

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer