Sin3b interacts with Myc and decreases Myc levels

Myc expression is deregulated in many human cancers. A yeast two-hybrid screen has revealed that the transcriptional repressor Sin3b interacts with Myc protein. Endogenous Myc and Sin3b co-localize and interact in the nuclei of human and rat cells, as assessed by co-immunoprecipitation, immunofluorescence, and proximity ligation assay. The interaction is Max-independent. A conserved Myc region (amino acids 186-203) is required for the interaction with Sin3 proteins. Histone deacetylase 1 is recruited to Myc-Sin3b complexes, and its deacetylase activity is required for the effects of Sin3b on Myc. Myc and Sin3a/b co-occupied many sites on the chromatin of human leukemia cells, although the presence of Sin3 was not associated with gene down-regulation. In leukemia cells and fibroblasts, Sin3b silencing led to Myc up-regulation, whereas Sin3b overexpression induced Myc deacetylation and degradation. An analysis of Sin3b expression in breast tumors revealed an association between low Sin3b expression and disease progression. The data suggest that Sin3b decreases Myc protein levels upon Myc deacetylation. As Sin3b is also required for transcriptional repression by Mxd-Max complexes, our results suggest that, at least in some cell types, Sin3b limits Myc activity through two complementary activities: Mxd-dependent gene repression and reduction of Myc levels

p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes

Regulation of Nucleolar Activity by MYC

The nucleolus harbors the machinery necessary to produce new ribosomes which are critical for protein synthesis. Nucleolar size, shape, and density are highly dynamic and can be adjusted to accommodate ribosome biogenesis according to the needs for protein synthesis. In cancer, cells undergo continuous proliferation; therefore, nucleolar activity is elevated due to their high demand for protein synthesis. The transcription factor and universal oncogene MYC promotes nucleolar activity by enhancing the transcription of ribosomal DNA (rDNA) and ribosomal proteins. This review summarizes the importance of nucleolar activity in mammalian cells, MYC’s role in nucleolar regulation in cancer, and discusses how a better understanding (and the potential inhibition) of aberrant nucleolar activity in cancer cells could lead to novel therapeutics

MNT knockdown effect on transcriptome and cell functions

Trabajo presentado en el 18th ASEICA International Congress, celebrado en Santiago de Compostela (España) del 16 al 18 de noviembre de 2022

CCDC6, a novel partner of the transcription factor MNT and its cellular effects

Trabajo presentado en el European Association for Cancer Research (EACR) Congress, celebrado en Sevilla (España) del 20 al 23 de junio de 2022

CCDC6, a novel partner of the transcription factor MNT

Trabajo presentado en el XIX Congreso de la Sociedad Española de Biología Celular, celebrado en Boadilla del Monte (España) del 26 al 29 de octubre de 2021

Physical and functional interaction between MNT and CCDC6

Trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018

The MYC antagonist MNT as a possible new regulator of the NF-¿B signaling pathway

Trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018

The MYC antagonist MNT beyond MAX interaction

Trabajo presentado en 25th Biennial Congress of the European Association for Cancer Research (EACR25), celebrado en Amsterdam (Países Bajos) del 30 de junio al 3 de julio de 2018.[Introduction]: MNT has been described as an antagonist and modulator of MYC, one of the most prevalent oncoproteins in human cancer. Both MYC and MNT are bHLH-LZ transcription factors that heterodimerize with MAX, bind to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MNT) their transcription. [Material and methods]: The cell lines used, URMT and URMax34, derive from MAX-deficient PC12 (rat pheochromocytoma), and carry a pHeBo-MT (empty vector) and a pHeBo-MT-MAX vector (MAX-inducible with Zn+2), respectively. Knockdown of MNT and MLX were achieved with short hairpin RNA constructs (shMNT and shMLX). Proliferation was assessed by cell counting and clonogenic assays; subG0-G1 population was determined by flow cytometry. RNA-seq was performed from two experiments of MNT silencing in URMT and URMax34 cells and confirmed by RT-qPCR. Changes in protein levels were analysed by western blot. Co-immunoprecipitation and proximity ligation assays were used to study protein-protein interactions. [Results and discussions]: Knocking-down of MNT in UR61 cells resulted in an important decrease in cell proliferation, together with a decrease in both survivin and cyclin A, which are markers of pro-survival and cell proliferation, respectively. DNA content was measured by flow cytometry, revealing an increase in sub-G0 population in shMNT cells. Thus, MNT is required for optimal proliferation of these cells. This is the first evidence of a MAX-independent function of MNT. Then, we extracted RNA from two experiments of MNT silenced and carried out RNA-seq. This resulted in 158 genes whose expression was altered. Cell cycle, DNA replication and DNA repair genes were downregulated upon MNT silencing. However, there were other up-regulated genes like the cell cycle inhibitor CDKN1C (p57). As we confirmed gene regulation by MNT without MAX, we wondered whether it could be working as an heterodimer with MLX or as an homodimer. Co-immunoprecipitation and proximity-ligation assays showed MNT’s ability to form homodimers and heterodimers with MLX. Finally, we carried out MLX knockdown and determined the genes regulated by MNT-MLX or MNT-MNT complexes. [Conclusion]: In summary, we report novel MAX-independent functions of MNT. In our MAX-deficient model, MNT can be found in homodimers (MNT-MNT) or heterodimers (MNT-MLX) and it supports proliferation and regulates cell cycle and DNA repair genes. This new data about MNT can open new insights into cell biology and tumour development promoted by MYC

NUMB inactivation confers resistance to imatinib in chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase, where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and its derivatives. NUMB is an evolutionary well-conserved protein initially described as a functional antagonist of NOTCH function. NUMB is an endocytic protein associated with receptor internalization, involved in multiple cellular functions. It has been reported that MSI2 protein, a NUMB inhibitor, is upregulated in CML blast crisis, whereas NUMB itself is downregulated. This suggest that NUMB plays a role in the malignant progression of CML. Here we have generated K562 cells (derived from CML in blast crisis) constitutively expressing a dominant negative form of NUMB (dnNUMB). We show that dnNUMB expression confers a high proliferative phenotype to the cells. Importantly, dnNUMB triggers a partial resistance to imatinib in these cells, antagonizing the apoptosis mediated by the drug. Interestingly, imatinib resistance is not linked to p53 status or NOTCH signaling, as K562 lack p53 and imatinib resistance is reproduced in the presence of NOTCH inhibitors. Taken together, our data support the hypothesis that NUMB activation could be a new therapeutic target in CML.The work was supported by grants SAF2014-53526 (to JL), BFU2007-67476 and BFU2010-21634 (to MC) from Spanish Ministry of Economy and Competitiveness (MINECO), and RD12/0036/0033 (to JL), RD12/0036/0054 (to AB) and RD12/0019/0006 and PI12/01097 (to FM) from Instituto Carlos III, and grant PI-57069 from CICE, FEDER/Fondo de Cohesion Europeo (FSE) de Andalucía 2007–2013 (to FM). The funding from MINECO and Instituto Carlos III was co-sponsored by the European Union FEDER program. EGA was supported with a JAE-doc contract form CSIC, MCL-N was supported by the FPU program from MINECO and LG-G. We thank Rosa Blanco for excellent technical advice by the FPI program from MINECO.Peer Reviewe