RESUMEN
EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.
Asunto(s)
ARN Helicasas DEAD-box , Elementos de Facilitación Genéticos , Factores de Transcripción de Tipo Kruppel , Eritropoyesis , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismoRESUMEN
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
Asunto(s)
Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores del ARN/metabolismo , Sarcoma de Ewing/metabolismo , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Fenotipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Piperazinas/farmacología , Unión Proteica , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Sarcoma de Ewing/tratamiento farmacológicoRESUMEN
Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.
Asunto(s)
Arginina/metabolismo , Cromatina/enzimología , Secuencia Rica en GC , Histonas/metabolismo , Proteína Metiltransferasas/metabolismo , Alelos , Animales , Células Cultivadas , Metilación de ADN , Fibroblastos/enzimología , Genoma , Impresión Genómica , Histonas/química , Metilación , Ratones , Regiones Promotoras Genéticas , Proteína-Arginina N-MetiltransferasasRESUMEN
The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Proteínas Señalizadoras YAP , Animales , Humanos , Ratones , Proliferación Celular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP/agonistas , Proteínas Señalizadoras YAP/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1-null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome-wide defect. Here we show that conditional deletion of the Setdb1 gene in ES cells results in the upregulation of lineage differentiation markers, especially trophectoderm-specific factors, similar to effects observed upon loss of Oct3/4 expression in ES cells. We demonstrate that KMT1E deficiency in ES cells leads to a decrease in histone H3K9 methylation at and derepression of trophoblast-associated genes such as Cdx2. Furthermore, we find genes that are derepressed upon Setdb1 deletion to overlap with known targets of polycomb mediated repression, suggesting that KMT1E mediated H3K9 methylation acts in concert with polycomb controlled H3K27 methylation. Our studies thus demonstrate an essential role for KMT1E in the control of developmentally regulated gene expression programs in ES cells.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Metiltransferasas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Immunoblotting , Metilación , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Metiltransferasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tamoxifeno/farmacologíaRESUMEN
AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/ß-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/ß-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of ß-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/ß-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/ß-Catenin activity, balancing metabolic function and hepatocyte proliferation.
Asunto(s)
Hígado , Ubiquitina-Proteína Ligasas/genética , Animales , Proliferación Celular , Hepatocitos/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Ratones , Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
Asunto(s)
Hepatocitos , Hígado , Animales , Proteína Axina , Homeostasis , Regeneración Hepática , Ratones , Células MadreRESUMEN
Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.
Asunto(s)
Carcinoma de Células Renales/genética , Ceruloplasmina/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Renales/genética , Factor de Transcripción PAX8/genética , Acetilación , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proliferación Celular/genética , Ceruloplasmina/metabolismo , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.
Asunto(s)
Factor 2 Regulador del Interferón/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Células Madre/citología , Células Madre/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Humanos , Factor 2 Regulador del Interferón/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Activación Transcripcional/fisiologíaRESUMEN
Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Células Eritroides/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Oncogénicas/metabolismo , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/análisis , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/análisis , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas/análisis , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
The switch from proliferation to differentiation during the terminal stages of erythropoiesis is a tightly controlled process that relies in part on transcription factor-mediated activation of cell cycle components. EKLF is a key transcription factor that is necessary for the initial establishment of the red cell phenotype. Here, we find that EKLF also plays a role during the subsequent differentiation process, as it induces p21(WAF1/CIP1) expression independent of p53 to regulate the changes in the cell cycle underlying erythroid maturation. EKLF activates p21 not only by directly binding to an EKLF site within a previously characterized GC-rich region in the p21 proximal promoter but also by occupancy at a novel, phylogenetically conserved region that contains consensus CACCC core motifs located downstream from the p21 TATA box. Our findings demonstrate that EKLF, likely in coordination with other transcription factors, directly contributes to the complex set of events that occur at the final erythroid cell divisions and accentuates terminal differentiation directly by activation of CDK inhibitors such as p21.
Asunto(s)
Diferenciación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Eritroides/metabolismo , Intrones , Factores de Transcripción de Tipo Kruppel/metabolismo , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Células Eritroides/citología , Regulación del Desarrollo de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However, the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis, as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver, we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies, we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population, followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis.
Asunto(s)
Células Precursoras Eritroides/fisiología , Factor de Transcripción GATA2/fisiología , Hematopoyesis , Factores de Transcripción de Tipo Kruppel/fisiología , Proteína Smad5/fisiología , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Factor de Transcripción GATA2/genética , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Ácido Nucleico , Proteína Smad5/genéticaRESUMEN
Megakaryocytes and erythroid cells are thought to derive from a common progenitor during hematopoietic differentiation. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We now show by gain- and loss-of-function studies that erythroid Krüppel-like factor (EKLF), a transcription factor whose role in erythroid gene regulation is well established, plays an unexpected directive role in the megakaryocyte lineage. EKLF inhibits the formation of megakaryocytes while at the same time stimulating erythroid differentiation. Quantitative examination of expression during hematopoiesis shows that, unlike genes whose presence is required for establishment of both lineages, EKLF is uniquely down-regulated in megakaryocytes after formation of the megakaryocyte-erythroid progenitor. Expression profiling and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels.