ABSTRACT
The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
Subject(s)
Chromatin , Retinoblastoma Protein , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin/genetics , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Promoter Regions, Genetic , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Transcription Factor AP-1/geneticsABSTRACT
Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.
Subject(s)
Breast Neoplasms/metabolism , Cell Proliferation , Immediate-Early Proteins/metabolism , Mitosis , Neoplastic Cells, Circulating/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , 3' Untranslated Regions , Animals , Antineoplastic Agents/pharmacology , Binding Sites , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Chromatin Assembly and Disassembly , Female , Gene Expression Regulation, Neoplastic , Genomic Instability , HEK293 Cells , Humans , Immediate-Early Proteins/genetics , Indoles/pharmacology , MCF-7 Cells , Mice, Inbred NOD , Mice, SCID , Mitosis/drug effects , Neoplastic Cells, Circulating/drug effects , Neoplastic Cells, Circulating/pathology , Nuclear Receptor Subfamily 4, Group A, Member 1/antagonists & inhibitors , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Phenylacetates/pharmacology , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , R-Loop Structures , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Signal Transduction , Transcription Elongation, Genetic , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.
ABSTRACT
Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.
Subject(s)
Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Pluripotent Stem Cells , Humans , Microglia/metabolism , Transcription Factors/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolismABSTRACT
Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients.
Subject(s)
Cell Cycle Proteins , Head and Neck Neoplasms , Nuclear Proteins , Transcription Factors , YAP-Signaling Proteins , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chromatin , Head and Neck Neoplasms/genetics , Humans , Nuclear Proteins/genetics , Proteomics , Squamous Cell Carcinoma of Head and Neck , Transcription Factors/genetics , Transcription Factors/metabolism , YAP-Signaling Proteins/genetics , YAP-Signaling Proteins/metabolismABSTRACT
The subfamily Phlebotominae is principally composed of the Lutzomyia and Phlebotomus genera: the main vectors of several protozoan, bacterial and viral pathogens. Since the 1990s molecular markers have enabled us to effectively address many issues concerning this taxon by, for example, solving systematic conflicts, increasing our understanding of speciation and host-parasite co-evolution, and determining the genetic structure of populations. In this paper we review the research undertaken using molecular markers in this taxonomic group. We hope that this will make it easier for scientists to identify markers and data analyses appropriate to their particular research interests. The principal trends we found are a move towards the use of mitochondrial DNA as molecular markers, DNA sequencing as the characterization method of choice, and phylogenetic analysis for analyzing the data. Most of the studies reviewed center on Lutzomyia longipalpis, the main vector for visceral leishmaniasis in the American tropics and Phlebotomus papatasi, the main vector for cutaneous leishmaniasis in Europe, Asia and Africa. Taxonomic problems and the description of genetic structure are the issues most addressed by researchers, followed by resolving systematic conflicts. Future research using molecular markers in the study of sandflies should be aimed towards: a) the development of genetic barcoding as a complementary tool for morphological identification and b) genome sequencing to increase our understanding of host-parasite interactions.
La subfamilia Phlebotominae está compuesta principalmente por los géneros Lutzomyia y Phlebotomus, vectores principales de patógenos virales, bacterianos y protozoarios. Desde los años 90 marcadores moleculares han ayudado a abordar problemas dentro del taxón, como por ejemplo: determinar la estructura genética, resolver conflictos sistemáticos, especiación, co-evolución de parasito y vector. Esta revisión pretende crear un compendio de la investigación realizada con marcadores moleculares en este grupo taxonómico, para así facilitar el trabajo de investigadores que pretenda identificar marcadores y el análisis de datos apropiados para responder sus preguntas. La tendencia principal encontrada fue el uso de ADN mitocondrial como marcador molecular, la secuenciación de ADN como técnica de caracterización y el análisis filogenético como método de análisis predilecto. La mayoría de los estudios revisados se centran en la especie Lutzomyia longipalpis, vector principal de leishmaniosis visceral en las regiones tropicales de América, y Phlebotomus papatasi vector principal de leishmaniosis cutánea en Europa, Asia y América. Los problemas taxonómicos y las descripciones de estructura genética fueron los problemas más abordados por los investigadores, seguidos por la resolución de conflictos sistemáticos. La investigación a futuro empleando marcadores moleculares en flebotomíneos deben apuntar hacia: el desarrollo de barcoding genético como técnica complementaria a la identificación morfológica y la secuenciación de genomas para así avanzar en el área de relaciones parasito-vector.