RESUMEN
The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5'-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5'-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5' terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Isoformas de Proteínas/genética , Proteína p53 Supresora de Tumor/genética , Supervivencia Celular/efectos de los fármacos , Codón Iniciador/antagonistas & inhibidores , Células HCT116 , Humanos , Intrones/genética , Isoformas de Proteínas/antagonistas & inhibidores , ARN Mensajero/antagonistas & inhibidores , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
Modification of the cancer-associated chromatin landscape in response to therapeutic DNA damage influences gene expression and contributes to cell fate. The central histone mark H2Bub1 results from addition of a single ubiquitin on lysine 120 of histone H2B and is an important regulator of gene expression. Following treatment with a platinum-based chemotherapeutic, there is a reduction in global levels of H2Bub1 accompanied by an increase in levels of the tumor suppressor p53. Although total H2Bub1 decreases following DNA damage, H2Bub1 is enriched downstream of transcription start sites of specific genes. Gene-specific H2Bub1 enrichment was observed at a defined group of genes that clustered into cancer-related pathways and correlated with increased gene expression. H2Bub1-enriched genes encompassed fifteen p53 target genes including PPM1D, BTG2, PLK2, MDM2, CDKN1A and BBC3, genes related to ERK/MAPK signalling, those participating in nucleotide excision repair including XPC, and genes involved in the immune response and platinum drug resistance including POLH. Enrichment of H2Bub1 at key cancer-related genes may function to regulate gene expression and influence the cellular response to therapeutic DNA damage.
Asunto(s)
Cromatina/metabolismo , Daño del ADN/genética , Transducción de Señal/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Cisplatino/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Estrogen receptor alpha (ERα) is a ligand-dependent transcription regulator, containing two transactivation functional domains, AF-1 and AF-2. The selective estrogen receptor modulators (SERMs), including 4-hydroxytamoxifen (4OHT), activate AF-1 preferentially rather than AF-2. However, it is unclear whether this specific function is related to the tissue-selective functionality of SERMs. Moreover, there is no information determining AF-1-dependent estrogenic-genes existing in tissues. We sought to identify AF-1-dependent estrogenic-genes using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an AF-2 disrupted estradiol (E2)-insensitive mutant ERα, but AF-1-dependent transcription can be activated by the estrogen-antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP-Seq analysis identified Klk1b21 as an ICI-inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell-based reporter assay. The 5'-flanking 0.4k bp region of Klk1b21 gene responded as an ERα AF-1-dependent estrogen-responsive promoter. The 150 bp minimum ERα binding element (EBE) consists of three direct repeats. These three half-site sequences were essential for the ERα-dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine-kinase promoter but could be restored by fusion with the 100 bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF-1-dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF-1 dominant estrogenic-genes exist in estrogen-responsive organs. Such findings will aid in understanding the mechanism of ERα-dependent tissue-selective activity of SERMs.
Asunto(s)
Receptor alfa de Estrógeno/genética , Activación Transcripcional/genética , Animales , Línea Celular Tumoral , Estradiol/genética , Antagonistas de Estrógenos/farmacología , Estrógenos/genética , Femenino , Fulvestrant/farmacología , Células Hep G2 , Humanos , Ligandos , Ratones , Modelos Animales , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/ultraestructura , Conformación de Ácido Nucleico/efectos de los fármacos , Sitios de Unión/genética , Factor de Unión a CCCTC/genética , ADN/genética , ADN/ultraestructura , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo II/genética , Etopósido/farmacología , Genoma Humano/genética , Inestabilidad Genómica/genética , Humanos , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2-phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II-mediated transcription in cancer cells.
Asunto(s)
Cromatina/metabolismo , Epigénesis Genética/efectos de los fármacos , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , ARN Polimerasa II/metabolismo , Transcripción Genética/efectos de los fármacos , Acetilación , Cromatina/efectos de los fármacos , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica/genética , Histonas/metabolismo , Humanos , Células MCF-7 , Nucleosomas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/genética , Dominios Proteicos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Proteínas de Drosophila/genética , Endocitosis/genética , Proteínas de Homeodominio/genética , Vesículas Sinápticas/genética , Aciltransferasas/genética , Animales , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Histona Desacetilasa 1/genética , Humanos , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Factores de Transcripción/genética , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Proteínas de Transporte Vesicular de Glutamato/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
BACKGROUND: Utilization of alternative transcription start sites through alterations in epigenetic promoter regions causes reduced expression of immunogenic N-terminal peptides, which may facilitate immune evasion in early gastric cancer. We hypothesized that tumors with high alternate promoter utilization would be resistant to immune checkpoint inhibition in metastatic gastric cancer. PATIENTS AND METHODS: Two cohorts of patients with metastatic gastric cancer treated with immunotherapy were analyzed. The first cohort (N = 24) included patients treated with either nivolumab or pembrolizumab. Alternate promoter utilization was measured using the NanoString® (NanoString Technologies, Seattle, WA, USA) platform on archival tissue samples. The second cohort was a phase II clinical trial of patients uniformly treated with pembrolizumab (N = 37). Fresh tumor biopsies were obtained, and transcriptomic analysis was carried out on RNAseq data. Alternate promoter utilization was correlated to T-cell cytolytic activity, objective response rate and survival. RESULTS: In the first cohort 8 of 24 (33%) tumors were identified to have high alternate promoter utilization (APhigh), and this was used to define the APhigh tertile of the second cohort (13 APhigh of 37). APhigh tumors exhibited decreased markers of T-cell cytolytic activity and lower response rates (8% versus 42%, P = 0.03). Median progression-free survival was lower in the APhigh group (55 versus 180 days, P = 0.0076). In multivariate analysis, alternative promoter utilization was an independent predictor of immunotherapy survival [hazard ratio 0.29, 95% confidence interval 0.099-0.85, P = 0.024). Analyzing tumoral evolution through paired pre-treatment and post-treatment biopsies, we observed consistent shifts in alternative promoter utilization rate associated with clinical response. CONCLUSION: A substantial proportion of metastatic gastric cancers utilize alternate promoters as a mechanism of immune evasion, and these tumors may be resistant to anti-PD1 immune checkpoint inhibition. Alternate promoter utilization is thus a potential mechanism of resistance to immune checkpoint inhibition, and a novel predictive biomarker for immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT#02589496.
Asunto(s)
Epigenómica , Receptor de Muerte Celular Programada 1/genética , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Secuencia de Bases/efectos de los fármacos , Biopsia , Humanos , Inmunoterapia , Metástasis de la Neoplasia , Nivolumab/administración & dosificación , Supervivencia sin Progresión , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Linfocitos T/efectos de los fármacos , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.
Asunto(s)
Segregación Cromosómica/genética , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Unión al ADN/genética , Mycobacterium smegmatis/genética , Mapeo Cromosómico , Segregación Cromosómica/efectos de los fármacos , ADN Nucleotidiltransferasas/genética , Humanos , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Rifampin/farmacología , Inhibidores de Topoisomerasa I/farmacología , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.
Asunto(s)
Proliferación Celular/genética , Metilación de ADN/genética , Leiomioma/genética , Complejo Mediador/genética , Ligando RANK/genética , Receptores de Progesterona/genética , Células Madre/patología , Neoplasias Uterinas/genética , Adulto , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Leiomioma/tratamiento farmacológico , Persona de Mediana Edad , Progesterona/genética , Promegestona/farmacología , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Neoplasias Uterinas/tratamiento farmacológicoRESUMEN
The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.
Asunto(s)
Azepinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Histonas/química , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Triazoles/farmacología , Acetilación/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Modelos Moleculares , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
BACKGROUND: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. RESULTS: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. CONCLUSIONS: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.
Asunto(s)
Perfilación de la Expresión Génica , Regulón/genética , Respuesta SOS en Genética/genética , Vibrio cholerae/genética , Regiones no Traducidas 5'/genética , Mitomicina/farmacología , Fenotipo , Respuesta SOS en Genética/efectos de los fármacos , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Vibrio cholerae/efectos de los fármacosRESUMEN
Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.
Asunto(s)
Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/agonistas , Gonadotrofos/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Nucleosomas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sitio de Iniciación de la Transcripción , Acetilación/efectos de los fármacos , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Glicoproteicas de Subunidad alfa/genética , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Gonadotrofos/efectos de los fármacos , Gonadotrofos/enzimología , Histonas/metabolismo , Lisina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Nucleosomas/enzimología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores LHRH/agonistas , Receptores LHRH/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Serina/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/genética , Código de Histonas , Inhibidores de Histona Desacetilasas/farmacología , Secuencias Repetidas Terminales/genética , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Empalme Alternativo/genética , Animales , Bencimidazoles/farmacología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/fisiología , Metilación de ADN , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Represión Epigenética , Exones/genética , Femenino , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Ácidos Hidroxámicos/farmacología , Intrones/genética , Ratones , Ratones Desnudos , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , VorinostatRESUMEN
Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.
Asunto(s)
Antivirales/toxicidad , Mitocondrias/efectos de los fármacos , Ribonucleósidos/química , Ribonucleósidos/toxicidad , Adenosina/análogos & derivados , Amidas/toxicidad , Línea Celular/efectos de los fármacos , Citidina/análogos & derivados , Citidina/toxicidad , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/toxicidad , Humanos , Concentración 50 Inhibidora , Proteínas Mitocondriales/metabolismo , Nucleósidos/toxicidad , Profármacos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Pirazinas/toxicidad , ARN/metabolismo , ARN Mitocondrial , Sofosbuvir/toxicidad , Relación Estructura-Actividad , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12â¯000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.
Asunto(s)
Inflamación/metabolismo , Pulmón/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Animales , Factor-23 de Crecimiento de Fibroblastos , Inflamación/genética , Inyección Intratimpánica , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanotubos de Carbono/química , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
The Oct4 gene codes for a transcription factor that plays a critical role in the maintenance of pluripotency in embryonic and cancer stem cells. Its expression thus has to be tightly regulated. We performed biophysical characterization of the promoter region using a combination of UV absorption, CD, and NMR spectroscopies, native PAGE and chemical probing, which was followed by functional studies involving luciferase reporter assays performed in osteosarcoma and human embryonic stem cell lines. We have shown that the evolutionarily conserved G-rich region close to the Oct4 transcription start site in the non-template strand forms a parallel G-quadruplex structure. We characterized its structure and stability upon point mutations in its primary structure. Functional studies then revealed that whereas the wild type quadruplex sequence ensures high reporter gene expression, the expression of mutated variants is significantly decreased proportionally to the destabilizing effect of the mutations on the quadruplex. A ligand, N-methyl mesoporphyrin IX that increases the stability of formed quadruplex rescued the reporter expression of single-mutated variants to the level of wild-type, but it has no effect on a mutated variant that cannot form quadruplex. These data indicate that the quadruplex acts as a strong, positive regulator of Oct4 expression and as such it might serve as a potential target for therapeutic intervention.
Asunto(s)
Factor 3 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas/genética , Línea Celular Tumoral , Dicroismo Circular/métodos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , G-Cuádruplex/efectos de los fármacos , Genes Reporteros/genética , Humanos , Imagen por Resonancia Magnética/métodos , Mesoporfirinas/farmacología , Mutación/genética , Osteosarcoma/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Sitio de Iniciación de la Transcripción/fisiologíaRESUMEN
The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse ß-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP.
Asunto(s)
Hormonas/farmacología , Macropodidae/genética , Glándulas Mamarias Animales/metabolismo , Proteínas/genética , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Secuencia Conservada/genética , Femenino , Ratones , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas/metabolismo , Ratas , Elementos de Respuesta/genética , Alineación de Secuencia , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacosRESUMEN
BACKGROUND: Resident macrophages in the CNS microglia become activated and produce proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors that drive innate immune responses following interactions with PAMPs. TLR3 and TLR4 recognize viral dsRNA Poly (I:C) and bacterial endotoxin LPS, respectively. Importantly, these receptors differ in their downstream adaptor molecules. Thus far, only a few studies have investigated the effects of TLR3 and TLR4 in macrophages. However, a genome-wide search for the effects of these TLRs has not been performed in microglia using RNA-seq. Gene expression patterns were determined for the BV-2 microglial cell line when stimulated with viral dsRNA Poly (I:C) or bacterial endotoxin LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity. RESULTS: Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 stimulation. However, the IFN/viral response gene showed a stronger response to TLR3 stimulation than to TLR4 stimulation. Unexpectedly, TLR3 and TLR4 stimulation did not activate IFN-ß and IRF3 in BV-2 microglia. Most importantly, we observed that previously unidentified transcription factors (TFs) (i.e., IRF1, IRF7, and IRF9) and the epigenetic regulators KDM4A and DNMT3L were significantly up-regulated in both TLR3- and TLR4-stimulated microglia. We also identified 29 previously unidentified genes that are important in immune regulation. In addition, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in TLR3-and TLR4-stimulated primary microglial cells. Moreover, transcriptional start sites (TSSs) and isoforms, as well as differential promoter usage, revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with TLR3 and TLR4. Furthermore, TF motif analysis (-950 to +50 bp of the 5' upstream promoters) revealed that the DNA sequences for NF-κB, IRF1, and STAT1 were significantly enriched in TLR3- and TLR4-stimulated microglia. CONCLUSIONS: These unprecedented findings not only permit a comparison of TLR3-and TLR4-stimulated genes but also identify new genes that have not been previously implicated in innate immunity.
Asunto(s)
Microglía/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Transcriptoma/genética , Animales , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Mediadores de Inflamación/metabolismo , Factor 3 Regulador del Interferón/genética , Interferón beta/genética , Ligandos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Microglía/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Sitio de Iniciación de la Transcripción/fisiología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transcriptoma/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mammalian target of rapamycin (mTOR) inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of bromodomain-containing protein 4 (BRD4) inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains the antiproliferative activity of JQ1 in OS cells.
Asunto(s)
Azepinas/farmacología , Osteosarcoma/tratamiento farmacológico , Sirolimus/farmacología , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Sinergismo Farmacológico , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Genes myc/genética , Humanos , Ratones , Ratones Desnudos , Proteínas Nucleares/metabolismo , Osteosarcoma/metabolismo , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Exposure to environmental estrogens (xenoestrogens) may play a causal role in the increased breast cancer incidence which has been observed in Europe and the US over the last 50 years. The xenoestrogen bisphenol A (BPA) leaches from plastic food/beverage containers and dental materials. Fetal exposure to BPA induces preneoplastic and neoplastic lesions in the adult rat mammary gland. Previous results suggest that BPA acts through the estrogen receptors which are detected exclusively in the mesenchyme during the exposure period by directly altering gene expression, leading to alterations of the reciprocal interactions between mesenchyme and epithelium. This initiates a long sequence of altered morphogenetic events leading to neoplastic transformation. Additionally, BPA induces epigenetic changes in some tissues. To explore this mechanism in the mammary gland, Wistar-Furth rats were exposed subcutaneously via osmotic pumps to vehicle or 250 µg BPA/kg BW/day, a dose that induced ductal carcinomas in situ. Females exposed from gestational day 9 to postnatal day (PND) 1 were sacrificed at PND4, PND21 and at first estrus after PND50. Genomic DNA (gDNA) was isolated from the mammary tissue and immuno-precipitated using anti-5-methylcytosine antibodies. Detection and quantification of gDNA methylation status using the Nimblegen ChIP array revealed 7412 differentially methylated gDNA segments (out of 58207 segments), with the majority of changes occurring at PND21. Transcriptomal analysis revealed that the majority of gene expression differences between BPA- and vehicle-treated animals were observed later (PND50). BPA exposure resulted in higher levels of pro-activation histone H3K4 trimethylation at the transcriptional initiation site of the alpha-lactalbumin gene at PND4, concomitantly enhancing mRNA expression of this gene. These results show that fetal BPA exposure triggers changes in the postnatal and adult mammary gland epigenome and alters gene expression patterns. These events may contribute to the development of pre-neoplastic and neoplastic lesions that manifest during adulthood.