RESUMEN
Alternative cleavage and polyadenylation (APA) is a gene regulatory mechanism used by cells under stress to upregulate proteostasis-promoting transcripts, but how cells achieve this remains poorly understood. Previously, we elucidated a DNA methylation-regulated APA mechanism, in which gene body DNA methylation enhances distal poly(A) isoform expression by blocking CTCF binding and chromatin loop formation at APA control regions. We hypothesized that DNA methylation-regulated APA is one mechanism cells employ to induce proteostasis-promoting poly(A) isoforms. At the DNAJB6 co-chaperone gene locus, acute heat shock resulted in binding of stress response transcription factors HSF1, ATF6, and YY1 at the APA control region and an increase in the expression of the proximal poly(A) isoform known to prevent protein aggregation. Furthermore, TET1 was recruited to rapidly demethylate DNA, facilitating CTCF binding and chromatin loop formation, thereby reinforcing preferential proximal poly(A) isoform expression. As cells recovered, the transcription factors vacated the APA control region, and DNMT1 was recruited to remethylate the region. This process resolved chromatin looping and reset the poly(A) isoform expression pattern. Our findings unveil an epigenetic mechanism enabling cells to dynamically modulate poly(A) isoforms in response to stress while shedding light on the interplay between DNA methylation, transcription factors, and chromatin looping.
RESUMEN
Tissue engineering offers a promising treatment strategy for ureteral strictures, but its success requires an in-depth understanding of the architecture, cellular heterogeneity, and signaling pathways underlying tissue regeneration. Here, we define and spatially map cell populations within the human ureter using single-cell RNA sequencing, spatial gene expression, and immunofluorescence approaches. We focus on the stromal and urothelial cell populations to enumerate the distinct cell types composing the human ureter and infer potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Furthermore, we analyze and experimentally validate the importance of the sonic hedgehog (SHH) signaling pathway in adult progenitor cell maintenance. The SHH-expressing basal cells support organoid generation in vitro and accurately predict the differentiation trajectory from basal progenitor cells to terminally differentiated umbrella cells. Our results highlight the essential processes involved in adult ureter tissue homeostasis and provide a blueprint for guiding ureter tissue engineering.
Asunto(s)
Uréter , Adulto , Diferenciación Celular , Proteínas Hedgehog/metabolismo , Humanos , Transducción de Señal , Células Madre , Uréter/metabolismoRESUMEN
Characterizing the cellular heterogeneity of human ureter tissues using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics provides a detailed atlas of cell types, signaling networks, and potential cell-cell cross talk underlying developmental and regenerative pathways. We describe an optimized protocol for generating, cryopreserving, and thawing single-cell suspensions from ureter tissues isolated post-cystectomy for scRNA-seq. In addition, we describe an optimized protocol for cryopreserving human ureter tissues for 10x Genomics Visium spatial gene expression platform. For complete details on the use and execution of this protocol, please refer to Fink et al. (2022).1.
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Transcriptoma , Uréter , Humanos , Transcriptoma/genética , Uréter/cirugía , Criopreservación , Perfilación de la Expresión Génica , BioensayoRESUMEN
Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Genoma Humano , Poliadenilación , Transcriptoma , Sitios de Unión , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HCT116 , Humanos , Transcripción Genética , CohesinasRESUMEN
Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten-/- primary melanomas. It also, as expected, promoted BrafCA/Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.
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Factores de Transcripción de Tipo Kruppel/metabolismo , Melanoma/patología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/patología , Acetilcisteína/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/inducido químicamente , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones Noqueados , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/metabolismoRESUMEN
Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress.
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Factores de Transcripción de Tipo Kruppel/metabolismo , Respuesta de Proteína Desplegada/fisiología , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Estrés del Retículo Endoplásmico , Femenino , Células HCT116 , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.
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Poliaminas Biogénicas/biosíntesis , Clofazimina/farmacología , Mieloma Múltiple , Proteínas de Neoplasias , Neoplasias Experimentales , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAFV600E-dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.
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Factores de Transcripción Forkhead/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Animales , Línea Celular Tumoral , Factores de Transcripción Forkhead/genética , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Ratones , Ratones Noqueados , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Lineage-specific regulation of tumor progression by the same transcription factor is understudied. We find that levels of the FOXQ1 transcription factor, an oncogene in carcinomas, are decreased during melanoma progression. Moreover, in contrast to carcinomas, FOXQ1 suppresses epithelial-to-mesenchymal transition, invasion, and metastasis in melanoma cells. We find that these lineage-specific functions of FOXQ1 largely depend on its ability to activate (in carcinomas) or repress (in melanoma) transcription of the N-cadherin gene (CDH2). We demonstrate that FOXQ1 interacts with nuclear ß-catenin and TLE proteins, and the ß-catenin/TLE ratio, which is higher in carcinoma than melanoma cells, determines the effect of FOXQ1 on CDH2 transcription. Accordingly, other FOXQ1-dependent phenotypes can be manipulated by altering nuclear ß-catenin or TLE proteins levels. Our data identify FOXQ1 as a melanoma suppressor and establish a mechanism underlying its inverse lineage-specific transcriptional regulation of transformed phenotypes.
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Factores de Transcripción Forkhead/genética , Melanoma/genética , Melanoma/patología , Oncogenes , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones SCID , Factor de Transcripción Asociado a Microftalmía/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Fenotipo , beta Catenina/metabolismoRESUMEN
GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.
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Proteínas Bacterianas/metabolismo , Técnicas Biosensibles , Guanosina Trifosfato/metabolismo , Proteínas Luminiscentes/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular Tumoral , Guanosina Trifosfato/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , MutaciónRESUMEN
Multiple myeloma is a form of plasma cell neoplasm that accounts for approximately 10% of all hematological malignancies. Recently, several novel drugs have been discovered that almost doubled the overall survival of multiple myeloma patients. One of these drugs, the first-in-class proteasome inhibitor bortezomib (Velcade) has demonstrated remarkable response rates in multiple myeloma patients, and yet, currently this disease remains incurable. The major factor undermining the success of multiple myeloma treatment is a rapidly emerging resistance to the available therapy. Thus, the development of stand-alone or adjuvant anti-myeloma agents becomes of paramount importance. Overproduction of intracellular reactive oxygen species (ROS) often accompanies malignant transformation due to oncogene activation and/or enhanced metabolism in tumor cells. As a result, these cells possess higher levels of ROS and lower levels of antioxidant molecules compared to their normal counterparts. Unbalanced production of ROS leads to oxidative stress which, if left unchecked, could be toxic for the cell. In multiple myeloma cells where high rates of immunoglobulin synthesis is an additional factor contributing to overproduction of ROS, further induction of oxidative stress can be an effective strategy to cope with this disease. Here we will review the available data on the role of oxidative stress in the cytotoxicity of proteasome inhibitors and the use of ROS-inducing compounds as anti-myeloma agents.
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Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteasoma/uso terapéutico , Animales , Antineoplásicos/farmacología , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Inhibidores de Proteasoma/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury.
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Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Fibrosis Pulmonar/genética , Animales , Sitios de Unión , Bleomicina , Línea Celular Tumoral , Genes Reporteros , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Células 3T3 NIH , Regiones Promotoras Genéticas , Unión Proteica , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Especies Reactivas de Oxígeno , Transducción de SeñalRESUMEN
In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.
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Senescencia Celular/genética , Daño del ADN/genética , Desoxirribonucleótidos/metabolismo , Oncogenes/fisiología , Proliferación Celular , Células Cultivadas , Senescencia Celular/fisiología , Replicación del ADN/genética , Desoxirribonucleótidos/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Ribonucleótido Reductasas/biosíntesis , Ribonucleótido Reductasas/fisiología , Timidilato Sintasa/biosíntesis , Timidilato Sintasa/fisiologíaRESUMEN
Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.