Sin Meets NuRD and Other Tails of Repression

Fuente, Carlos;Pericas, Enri

Myc stimulates B lymphocyte differentiation and amplifies calcium signaling

Deregulated expression of the Myc family of transcription factors (c-, N-, and L-myc) contributes to the development of many cancers by a mechanism believed to involve the stimulation of cell proliferation and inhibition of differentiation. However, using B cell–specific c-/N-myc double-knockout mice and Eμ-myc transgenic mice bred onto genetic backgrounds (recombinase-activating gene 2−/− and Btk−/− Tec−/−) whereby B cell development is arrested, we show that Myc is necessary to stimulate both proliferation and differentiation in primary B cells. Moreover, Myc expression results in sustained increases in intracellular Ca2+ ([Ca2+]i), which is required for Myc to stimulate B cell proliferation and differentiation. The increase in [Ca2+]i correlates with constitutive nuclear factor of activated T cells (NFAT) nuclear translocation, reduced Ca2+ efflux, and decreased expression of the plasma membrane Ca2+–adenosine triphosphatase (PMCA) efflux pump. Our findings demonstrate a revised model whereby Myc promotes both proliferation and differentiation, in part by a remarkable mechanism whereby Myc amplifies Ca2+ signals, thereby enabling the concurrent expression of Myc- and Ca2+-regulated target genes

Metabolomics method to comprehensively analyze amino acids in different domains

Amino acids play essential roles in both metabolism and the proteome. Many studies have profiled free amino acids (FAAs) or proteins; however, few have connected the measurement of FAA with individual amino acids in the proteome. In this study, we developed a metabolomics method to comprehensively analyze amino acids in different domains, using two examples of different sample types and disease models. We first examined the responses of FAAs and insoluble-proteome amino acids (IPAAs) to the Myc oncogene in Tet21N human neuroblastoma cells. The metabolic and proteomic amino acid profiles were quite different, even under the same Myc condition, and their combination provided a better understanding of the biological status. In addition, amino acids were measured in 3 domains (FAAs, free and soluble-proteome amino acids (FSPAAs), and IPAAs) to study changes in serum amino acid profiles related to colon cancer. A penalized logistic regression model based on the amino acids from the three domains had better sensitivity and specificity than that from each individual domain. To the best of our knowledge, this is the first study to perform a combined analysis of amino acids in different domains, and indicates the useful biological information available from a metabolomics analysis of the protein pellet. This study lays the foundation for further quantitative tracking of the distribution of amino acids in different domains, with opportunities for better diagnosis and mechanistic studies of various diseases

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

Essential Functions of the Histone Demethylase Lid

Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C5HC2 zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development

Supplementary Data Only: Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN