RESUMEN
Sustaining energy homeostasis is of pivotal importance for all living organisms. In Arabidopsis thaliana, evolutionarily conserved SnRK1 kinases (Snf1-RELATED KINASE1) control metabolic adaptation during low energy stress. To unravel starvation-induced transcriptional mechanisms, we performed transcriptome studies of inducible knockdown lines and found that S1-basic leucine zipper transcription factors (S1-bZIPs) control a defined subset of genes downstream of SnRK1. For example, S1-bZIPs coordinate the expression of genes involved in branched-chain amino acid catabolism, which constitutes an alternative mitochondrial respiratory pathway that is crucial for plant survival during starvation. Molecular analyses defined S1-bZIPs as SnRK1-dependent regulators that directly control transcription via binding to G-box promoter elements. Moreover, SnRK1 triggers phosphorylation of group C-bZIPs and the formation of C/S1-heterodimers and, thus, the recruitment of SnRK1 directly to target promoters. Subsequently, the C/S1-bZIP-SnRK1 complex interacts with the histone acetylation machinery to remodel chromatin and facilitate transcription. Taken together, this work reveals molecular mechanisms underlying how energy deprivation is transduced to reprogram gene expression, leading to metabolic adaptation upon stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Redes y Vías Metabólicas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Adaptación Fisiológica , Arabidopsis/enzimología , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Oscuridad , Metabolismo Energético , Perfilación de la Expresión Génica , Homeostasis , Mitocondrias/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
Asunto(s)
Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes myc/genética , Neoplasias/genética , Transcriptoma , Regulación hacia Arriba/genética , Animales , Sitios de Unión , Línea Celular Tumoral , Elementos E-Box/genética , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
c-Myc is one of the major human proto-oncogenes and is often associated with tumor aggression and poor clinical outcome. Paradoxically, Myc was also reported as a suppressor of cell motility, invasiveness, and metastasis. Among the direct targets of Myc are many components of the protein synthesis machinery whose induction results in an overall increase in protein synthesis that empowers tumor cell growth. At present, it is largely unknown whether beyond the global enhancement of protein synthesis, Myc activation results in translation modulation of specific genes. Here, we measured Myc-induced global changes in gene expression at the transcription, translation, and protein levels and uncovered extensive transcript-specific regulation of protein translation. Particularly, we detected a broad coordination between regulation of transcription and translation upon modulation of Myc activity and showed the connection of these responses to mTOR signaling to enhance oncogenic transformation and to the TGFß pathway to modulate cell migration and invasiveness. Our results elucidate novel facets of Myc-induced cellular responses and provide a more comprehensive view of the consequences of its activation in cancer cells.
Asunto(s)
Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica , Metástasis de la Neoplasia/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Apoptosis , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Perfilación de la Expresión Génica , Genes myc , Humanos , Biosíntesis de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The ETS family member ERG is a transcription factor with physiological roles during development and in the vascular and hematopoietic systems. ERG oncogenic activity characterizes several malignancies, including Ewing's sarcoma, leukemia and prostate cancer (PCa). In PCa, ERG rearrangements with androgen-regulated genes-mostly TMPRSS2-characterize a large subset of patients across disease progression and result in androgen receptor (AR)-mediated overexpression of ERG in the prostate cells. Importantly, PCa cells overexpressing ERG are dependent on ERG activity for survival, further highlighting its therapeutic potential. Here, we review the current understanding of the role of ERG and its partners in PCa. We discuss the strategies developed in recent years to inhibit ERG activity, the current therapeutic utility of ERG fusion detection in PCa patients, and the possible future approaches to target ERG fusion-positive tumors.
RESUMEN
Pan-cancer studies sketched the genomic landscape of the tumor types spectrum. We delineated the purity- and ploidy-adjusted allele-specific profiles of 4,950 patients across 27 tumor types from the Cancer Genome Atlas (TCGA). Leveraging allele-specific data, we reclassified as loss of heterozygosity (LOH) 9% and 7% of apparent copy-number wild-type and gain calls, respectively, and overall observed more than 18 million allelic imbalance somatic events at the gene level. Reclassification of copy-number events revealed associations between driver mutations and LOH, pointing out the timings between the occurrence of point mutations and copy-number events. Integrating allele-specific genomics and matched transcriptomics, we observed that allele-specific gene status is relevant in the regulation of TP53 and its targets. Further, we disclosed the role of copy-neutral LOH in the impairment of tumor suppressor genes and in disease progression. Our results highlight the role of LOH in cancer and contribute to the understanding of tumor progression.
Asunto(s)
Pérdida de Heterocigocidad , Neoplasias , Alelos , Genómica , Humanos , Pérdida de Heterocigocidad/genética , Neoplasias/genéticaRESUMEN
21q22.2-3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease. Interestingly, ERG expression characterizes 5-30% of tumor precursor lesions - High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear. Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3ß-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions. These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta , Proteínas de Homeodominio , Próstata , Neoplasias de la Próstata , Factores de Transcripción , Regulador Transcripcional ERG , Animales , Inestabilidad Genómica , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas de Homeodominio/genética , Masculino , Ratones , Proteínas Oncogénicas , Próstata/patología , Neoplasias de la Próstata/patología , Transactivadores/metabolismo , Factores de Transcripción/genética , Regulador Transcripcional ERG/genéticaRESUMEN
Mutual Exclusivity analysis of genomic aberrations contributes to the exploration of potential synthetic lethal (SL) relationships thus guiding the nomination of specific cancer cells vulnerabilities. When multiple classes of genomic aberrations and large cohorts of patients are interrogated, exhaustive genome-wide analyses are not computationally feasible with commonly used approaches. Here we present Fast Mutual Exclusivity (FaME), an algorithm based on matrix multiplication that employs a logarithm-based implementation of the Fisher's exact test to achieve fast computation of genome-wide mutual exclusivity tests; we show that brute force testing for mutual exclusivity of hundreds of millions of aberrations combinations can be performed in few minutes. We applied FaME to allele-specific data from whole exome experiments of 27 TCGA studies cohorts, detecting both mutual exclusivity of point mutations, as well as allele-specific copy number signals that span sets of contiguous cytobands. We next focused on a case study involving the loss of tumor suppressors and druggable genes while exploiting an integrated analysis of both public cell lines loss of function screens data and patients' transcriptomic profiles. FaME algorithm implementation as well as allele-specific analysis output are publicly available at https://github.com/demichelislab/FaME.
RESUMEN
Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Edición Génica , ARN Guía de Kinetoplastida/genética , Mutación , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Especificidad por SustratoRESUMEN
In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.
RESUMEN
Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.