RESUMEN
Targeted protein degradation (TPD) has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell's endogenous protein degradation machinery. However, the susceptibility of proteins for targeting by TPD approaches, termed "degradability", is largely unknown. Here, we developed a machine learning model, model-free analysis of protein degradability (MAPD), to predict degradability from features intrinsic to protein targets. MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds [with an area under the precision-recall curve (AUPRC) of 0.759 and an area under the receiver operating characteristic curve (AUROC) of 0.775] and is likely generalizable to independent non-kinase proteins. We found five features with statistical significance to achieve optimal prediction, with ubiquitination potential being the most predictive. By structural modeling, we found that E2-accessible ubiquitination sites, but not lysine residues in general, are particularly associated with kinase degradability. Finally, we extended MAPD predictions to the entire proteome to find 964 disease-causing proteins (including proteins encoded by 278 cancer genes) that may be tractable to TPD drug development.
Asunto(s)
Lisina , Aprendizaje Automático , Proteolisis , Ubiquitinación , ProteomaRESUMEN
c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Analyses of clinical specimens reveal that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq uncover an increase in RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Genes myc , Humanos , Masculino , Próstata/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Chromatin immunoprecipitation sequencing (ChIP-seq) and the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) have become essential technologies to effectively measure protein-DNA interactions and chromatin accessibility. However, there is a need for a scalable and reproducible pipeline that incorporates proper normalization between samples, correction of copy number variations, and integration of new downstream analysis tools. Here we present Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA), a modularized computational workflow which quantifies ChIP-seq and ATAC-seq peak regions and performs unsupervised and supervised analyses. CoBRA provides a comprehensive state-of-the-art ChIP-seq and ATAC-seq analysis pipeline that can be used by scientists with limited computational experience. This enables researchers to gain rapid insight into protein-DNA interactions and chromatin accessibility through sample clustering, differential peak calling, motif enrichment, comparison of sites to a reference database, and pathway analysis. CoBRA is publicly available online at https://bitbucket.org/cfce/cobra.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional , Cromatina/genética , Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Flujo de TrabajoRESUMEN
BRAF is a serine/threonine kinase that harbors activating mutations in â¼7% of human malignancies and â¼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma, we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6, which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.
Asunto(s)
Resistencia a Antineoplásicos , Indoles/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/genética , Melanoma/patología , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and have been linked to the Notch responsiveness of a few genes. To assess the overall contribution of SPSs to Notch-dependent gene regulation, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay and applied insights from these in vitro studies to Notch-"addicted" T cell acute lymphoblastic leukemia (T-ALL) cells. We found that SPSs contributed to the regulation of about a third of direct Notch target genes. Although originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5 expression. Our work provides a general method for identifying SPSs in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación Leucémica de la Expresión Génica , Genoma Humano , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores Notch/metabolismo , Factores de Transcripción/metabolismo , Animales , Perfilación de la Expresión Génica , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Receptores Notch/genética , Transducción de Señal , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer-related death worldwide. Although epigenetic deregulation is known to be important for tumor progression, the molecular mechanisms in NSCLC remain unclear. Here, we found that G9A (known as EHMT2), a histone methyltransferase responsible for mono- or di-methylation of histone 3 (H3) lysine 9 (K9), is significantly upregulated in NSCLC. Knocking down G9A or pharmacological inhibition of its activity suppressed tumor cell growth, colony formation, invasion and migration. Furthermore, G9A exerts these functions by repressing CASP1 expression. Knocking down CASP1 in G9A-deficient cell restored capacities of tumor cell invasion and migration. Mechanistically, G9A silences the CASP1 promoter activity by increasing H3K9me2 around its promoter. Finally, high expression of G9A or low expression of CASP1 is correlated with poor overall survival in lung adenocarcinoma. Overall, our study uncovers a novel mechanism of G9A promoting tumor cell growth and invasion by silencing CASP1, and implies that G9A may serve as a therapeutic target in treating NSCLC.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasa 1/metabolismo , Movimiento Celular , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 1/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Proteínas de Neoplasias/genéticaRESUMEN
MOTIVATION: RNA-seq has been widely used in transcriptome analysis to effectively measure gene expression levels. Although sequencing costs are rapidly decreasing, almost 70% of all the human RNA-seq samples in the gene expression omnibus do not have biological replicates and more unreplicated RNA-seq data were published than replicated RNA-seq data in 2011. Despite the large amount of single replicate studies, there is currently no satisfactory method for detecting differentially expressed genes when only a single biological replicate is available. RESULTS: We present the GFOLD (generalized fold change) algorithm to produce biologically meaningful rankings of differentially expressed genes from RNA-seq data. GFOLD assigns reliable statistics for expression changes based on the posterior distribution of log fold change. In this way, GFOLD overcomes the shortcomings of P-value and fold change calculated by existing RNA-seq analysis methods and gives more stable and biological meaningful gene rankings when only a single biological replicate is available. AVAILABILITY: The open source C/C++ program is available at http://www.tongji.edu.cn/â¼zhanglab/GFOLD/index.html
Asunto(s)
Algoritmos , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Expresión Génica , Humanos , Modelos Moleculares , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/genética , Análisis de Secuencia de ARN/economíaRESUMEN
Transcriptional profiling of two isogenic models of transformation identifies a gene signature linking cancer with inflammatory and metabolic diseases. In accord with this common transcriptional program, many drugs used for treatment of diabetes and cardiovascular diseases inhibit transformation and tumor growth. Unexpectedly, lipid metabolism genes are important for transformation and are upregulated in cancer tissues. As in atherosclerosis, oxidized LDL and its receptor OLR1 activate the inflammatory pathway through NF-kappaB, leading to transformation. OLR1 is important for maintaining the transformed state in developmentally diverse cancer cell lines and for tumor growth, suggesting a molecular connection between cancer and atherosclerosis. We suggest that the interplay between this common transcriptional program and cell-type-specific factors gives rise to phenotypically disparate human diseases.
Asunto(s)
Aterosclerosis/genética , Perfilación de la Expresión Génica , Enfermedades Genéticas Congénitas/genética , Neoplasias/genética , Transcripción Genética , Transformación Celular Neoplásica/genética , Diabetes Mellitus/genética , Ligamiento Genético , Variación Genética , Humanos , Inflamación/genética , Síndrome Metabólico/genética , Obesidad/genética , Receptores Depuradores de Clase E/genéticaRESUMEN
MOTIVATION: New generation Affymetrix oligonucleotide microarrays often have blob-like image defects that will require investigators to either repeat their hybridization assays or analyze their data with the defects left in place. We investigated the effect of analyzing a spike-in experiment on Affymetrix ENCODE tiling arrays in the presence of simulated blobs covering between 1 and 9% of the array area. Using two different ChIP-chip tiling array analysis programs (Affymetrix tiling array software, TAS, and model-based analysis of tiling arrays, MAT), we found that even the smallest blob defects significantly decreased the sensitivity and increased the false discovery rate (FDR) of the spike-in target prediction. RESULTS: We introduced a new software tool, the microarray blob remover (MBR), which allows rapid visualization, detection and removal of various blob defects from the .CEL files of different types of Affymetrix microarrays. It is shown that using MBR significantly improves the sensitivity and FDR of a tiling array analysis compared to leaving the affected probes in the analysis. AVAILABILITY: The MBR software and the sample array .CEL files used in this article are available at: http://liulab.dfci.harvard.edu/Software/MBR/MBR.htm