RESUMEN
Progesterone prevents development of endometrial cancers through its receptor (PR) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that expressed PR endogenously or exogenously. We found progesterone strongly inhibits multiple components of the platelet derived growth factor receptor (PDGFR), Janus kinase (JAK), signal transducer and activator of transcription (STAT) pathway through PR. The PDGFR/JAK/STAT pathway signals to control numerous downstream targets including AP-1 transcription factors Fos and Jun. Treatment with inhibitors of the PDGFR/JAK/STAT pathway significantly blocked proliferation in multiple novel patient-derived organoid models of endometrial cancer, and activation of this pathway was found to be a poor prognostic signal for the survival of patients with endometrial cancer from The Cancer Genome Atlas. Our study identifies this pathway as central to the growth-limiting effects of progesterone in endometrial cancer and suggests that inhibitors of PDGFR/JAK/STAT should be considered for future therapeutic interventions.
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Neoplasias Endometriales , Quinasas Janus , Femenino , Humanos , Progesterona/farmacología , Transducción de Señal , Factores de Transcripción STAT/genética , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genéticaRESUMEN
Multiple Myeloma (MM) is a plasma cell neoplasm originating in the bone marrow and is the second most common blood cancer in the United States. One challenge in understanding the pathogenesis of MM and improving treatment is a lack of immunocompetent mouse models. We previously developed the IL6Myc mouse that generates plasmacytomas at 100% penetrance that phenotypically resemble aggressive MM. Using comprehensive genomic analysis, we found that the IL6Myc tumors resemble aggressive MM by RNA and protein expression. We also found that IL6Myc tumors accumulated fusions and missense mutations in genes that overlap significantly with human myeloma, indicating that the mouse is good model for studying disease etiology. Lastly, we derived cell lines from IL6Myc tumors that express cell surface markers typical of MM and readily engraft into mice, home to the bone marrow, and induce osteolytic disease. The cell lines may be useful in developing immunotherapies directed against BAFF-R and TACI, though not BCMA, and may also be a good model for studying dexamethasone resistance. These data indicate that the IL6Myc model is useful for studying development of aggressive MM and for developing new treatments against such forms of the disease.
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Mieloma Múltiple , Ratones , Humanos , Animales , Mieloma Múltiple/patología , Médula Ósea/patologíaRESUMEN
Transcription factors (TF) are proteins that bind DNA in a sequence-specific manner to regulate gene transcription. Despite their unique intrinsic sequence preferences, in vivo genomic occupancy profiles of TFs differ across cellular contexts. Hence, deciphering the sequence determinants of TF binding, both intrinsic and context-specific, is essential to understand gene regulation and the impact of regulatory, non-coding genetic variation. Biophysical models trained on in vitro TF binding assays can estimate intrinsic affinity landscapes and predict occupancy based on TF concentration and affinity. However, these models cannot adequately explain context-specific, in vivo binding profiles. Conversely, deep learning models, trained on in vivo TF binding assays, effectively predict and explain genomic occupancy profiles as a function of complex regulatory sequence syntax, albeit without a clear biophysical interpretation. To reconcile these complementary models of in vitro and in vivo TF binding, we developed Affinity Distillation (AD), a method that extracts thermodynamic affinities de-novo from deep learning models of TF chromatin immunoprecipitation (ChIP) experiments by marginalizing away the influence of genomic sequence context. Applied to neural networks modeling diverse classes of yeast and mammalian TFs, AD predicts energetic impacts of sequence variation within and surrounding motifs on TF binding as measured by diverse in vitro assays with superior dynamic range and accuracy compared to motif-based methods. Furthermore, AD can accurately discern affinities of TF paralogs. Our results highlight thermodynamic affinity as a key determinant of in vivo binding, suggest that deep learning models of in vivo binding implicitly learn high-resolution affinity landscapes, and show that these affinities can be successfully distilled using AD. This new biophysical interpretation of deep learning models enables high-throughput in silico experiments to explore the influence of sequence context and variation on both intrinsic affinity and in vivo occupancy.
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The classical dogma of glucocorticoid-induced insulin resistance is that it is caused by the transcriptional activation of hepatic gluconeogenic and insulin resistance genes by the glucocorticoid receptor (GR). Here, we find that glucocorticoids also stimulate the expression of insulin-sensitizing genes, such as Irs2. The transcriptional coregulator EHMT2 can serve as a transcriptional coactivator or a corepressor. Using male mice that have a defective EHMT2 coactivation function specifically, we show that glucocorticoid-induced Irs2 transcription is dependent on liver EHMT2's coactivation function and that IRS2 play a key role in mediating the limitation of glucocorticoid-induced insulin resistance by EHMT2's coactivation. Overall, we propose a model in which glucocorticoid-regulated insulin sensitivity is determined by the balance between glucocorticoid-modulated insulin resistance and insulin sensitizing genes, in which EHMT2 coactivation is specifically involved in the latter process.
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Glucocorticoides , N-Metiltransferasa de Histona-Lisina , Resistencia a la Insulina , Animales , Masculino , Ratones , Glucocorticoides/farmacología , Insulina/metabolismo , Resistencia a la Insulina/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor-Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.
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Núcleo Celular , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Núcleo Celular/metabolismo , Cromatina , Proteínas de Unión al ADN/metabolismo , FibrinógenoRESUMEN
Glucocorticoids, including dexamethasone and prednisone, are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency is a route to improving outcomes. However, systematic toxicities prevent the use of higher dose and more potent glucocorticoids. We therefore took a functional genomic approach to identify targets to enhance glucocorticoid activity specifically in B-ALL cells. Here we show that inhibition of the lymphoid-restricted PI3Kδ, signaling through the RAS/MAPK pathway, enhances both prednisone and dexamethasone activity in almost all ex vivo B-ALL specimens tested. This potentiation is most synergistic at sub-saturating doses of glucocorticoids, approaching the EC50. Potentiation correlates with global enhancement of glucocorticoid-induced gene regulation, including regulation of effector genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at MAPK1/ERK2 targets S203 and S226, and ablation of these phospho-acceptor sites enhances glucocorticoid potency. We further show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro, which impairs DNA binding. We therefore propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. The overall enhancement of GR function suggests that idelalisib will provide benefit to most patients with B-ALL by improving outcomes for patients whose disease is less responsive to glucocorticoid-based therapy, including high-risk disease, and allowing less toxic glucocorticoid-sparing strategies for patients with standard-risk disease.
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Glucocorticoids are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency should improve treatment. We previously showed that inhibition of the lymphoid-restricted PI3Kδ with idelalisib enhances glucocorticoid activity in B-ALL cells. Here, we show that idelalisib enhances glucocorticoid potency in 90% of primary B-ALL specimens and is most pronounced at sub-saturating doses of glucocorticoids near the EC50. Potentiation is associated with enhanced regulation of all glucocorticoid-regulated genes, including genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at PI3Kδ/MAPK1 (ERK2) targets S203 and S226. Ablation of these phospho-acceptor sites enhances sensitivity to glucocorticoids with ablation of S226 in particular reducing synergy. We also show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro. We propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. This mechanism and the response of patient specimens suggest that idelalisib will benefit most patients with B-ALL, but particularly patients with less responsive, including high-risk, disease. This combination is also promising for the development of less toxic glucocorticoid-sparing therapies.
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Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.
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Carcinoma Endometrioide , Neoplasias Endometriales , Femenino , Humanos , Progesterona/farmacología , Progesterona/uso terapéutico , Estradiol/farmacología , Células Tumorales Cultivadas , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Estrógenos/farmacología , Estrógenos/uso terapéutico , Carcinoma Endometrioide/tratamiento farmacológico , Carcinoma Endometrioide/genética , Línea CelularRESUMEN
The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.
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Proteínas de Drosophila , Lámina Nuclear , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Expresión Génica , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Glucocorticoids (GCs) are widely used, potent anti-inflammatory and chemotherapeutic drugs. They work by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor, inducing translocation to the nucleus and regulation of genes that influence a variety of cellular activities. Despite being effective for a broad number of conditions, GC use is limited by severe side effects. To identify ligands that are more selective, we synthesized pairs of regioisomers in the pyrazole ring that probe the expanded binding pocket of GR opened by deacylcortivazol (DAC). Using an Ullmann-type reaction, a deacylcortivazol-like (DAC-like) backbone was modified with five pendant groups at the 1'- and 2'-positions of the pyrazole ring, yielding 9 ligands. Most of the compounds were cytotoxic to leukemia cells, and all required GR expression. Both aliphatic and other aromatic groups substituted at the 2'-position produced ligands with GC activity, with phenyl and 4-fluorophenyl substitutions exhibiting high cellular affinity for the receptor and >5× greater potency than dexamethasone, a commonly used strong GC. Surprisingly, phenyl substitution at the 1'-position produced a high-affinity ligand with â¼10× greater potency than dexamethasone, despite little apparent room in the expanded binding pocket to accommodate 1'-modifications. Other 1'-modifications, however, were markedly less potent. The potency of the 2'-substituted and 1'-substituted DAC-like compounds tracked linearly with cellular affinity but had different slopes, suggesting a different mode of interaction with GR. These data provide evidence that the expanded binding pocket opened by deacylcortivazol is more accommodating that expected, allowing development of new, and possibly selective, GCs by substitution within the pyrazole ring.
RESUMEN
Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.
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Proteínas de Unión al ADN/química , Conformación Molecular , Ácidos Nucleicos Heterodúplex/química , Proteínas de Arabidopsis/química , Emparejamiento Base , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Termodinámica , Factores de Transcripción/químicaRESUMEN
The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to investigate the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.
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ADN Helicasas/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Dominios Proteicos , Factores de Transcripción/metabolismo , Emparejamiento Base , Cromatina , Ensamble y Desensamble de Cromatina , ADN Helicasas/química , ADN Helicasas/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Simulación de Dinámica Molecular , Mutación , Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformación Proteica , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.
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Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , Acidaminococcus/enzimología , Disparidad de Par Base , Emparejamiento Base , Proteínas Asociadas a CRISPR/genética , ADN/química , ADN/metabolismo , División del ADN , Deltaproteobacteria/enzimología , Endodesoxirribonucleasas/genética , Mutación , Proteína Homeótica Nanog/genética , Unión Proteica , ARN/química , Técnica SELEX de Producción de Aptámeros , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Providing students with training in advanced laboratory skills is an essential part of scientific education. At the same time, engaging students in research is becoming equally important. Classroom-based undergraduate research experiences (CUREs) have emerged to fill this need, and can take many forms. In this article we describe reengineering an advanced organic synthesis laboratory at a primarily undergraduate institution into a CURE. This objective of this CURE is to provide small molecules relevant to an ongoing research program at a research-intensive institution. This new model cross trains students and provides a new structure for a CURE that could be adapted to other partnerships and institutions.
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Bioquímica/educación , Educación Interprofesional/métodos , Biología Molecular/educación , Investigación/educación , Curriculum , Humanos , Indiana , Laboratorios , Aprendizaje , Modelos Educacionales , Estudiantes , UniversidadesRESUMEN
The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.
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Dexametasona/farmacología , Inflamación/metabolismo , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cromatina/metabolismo , Dexametasona/metabolismo , Elementos de Facilitación Genéticos , Células HEK293 , Humanos , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death. This complex selectively supports GC-induced expression of genes contributing to cell death. A metaanalysis of gene expression data from B-ALL patient specimens revealed that Aurora kinase B (AURKB), which restrains GC signaling by phosphorylating EHMT1-2, is overexpressed in relapsed B-ALL, suggesting it as a potential contributor to relapse. Inhibition of AURKB enhanced GC-induced expression of cell death genes, resulting in potentiation of GC cytotoxicity in cell lines and relapsed B-ALL patient samples. This function for AURKB is distinct from its canonical role in the cell cycle. These results show the utility of functional genomics in understanding mechanisms of resistance and rapidly identifying combination chemotherapeutics.
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Aurora Quinasa B/genética , Muerte Celular/genética , Resistencia a Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Regulación Leucémica de la Expresión Génica/genética , Glucocorticoides/genética , Glucocorticoides/farmacología , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , ARN Interferente Pequeño/genética , RecurrenciaRESUMEN
Synthetic glucocorticoids (GCs) are used to treat lymphoid cancers, but many patients develop resistance to treatment, especially to GC. By identifying genes that influence sensitivity to GC-induced cell death, we found that histone methyltransferases G9a and G9a-like protein (GLP), two glucocorticoid receptor (GR) coactivators, are required for GC-induced cell death in acute lymphoblastic leukemia (B-ALL) cell line Nalm6. We previously established in a few selected genes that automethylated G9a and GLP recruit heterochromatin protein 1γ (HP1γ) as another required coactivator. Here, we used a genome-wide analysis to show that HP1γ is selectively required for GC-regulated expression of the great majority of GR target genes that require G9a and GLP. To further address the importance of G9a and GLP methylation in this process and in cell physiology, we found that JIB-04, a selective JmjC family lysine demethylase inhibitor, increased G9a methylation and thereby increased G9a binding to HP1γ. This led to increased expression of GR target genes regulated by G9a, GLP and HP1γ and enhanced Nalm6 cell death. Finally, the KDM4 lysine demethylase subfamily demethylates G9a in vitro, in contrast to other KDM enzymes tested. Thus, inhibiting G9a/GLP demethylation potentially represents a novel method to restore sensitivity of treatment-resistant B-ALL tumors to GC-induced cell death.
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Muerte Celular/genética , Glucocorticoides/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Precursoras de Linfocitos B/patología , Células A549 , Línea Celular , Línea Celular Tumoral , Células HEK293 , Histona Metiltransferasas/genética , Humanos , Metilación , Procesamiento Proteico-Postraduccional/genética , Receptores de Glucocorticoides/genéticaRESUMEN
In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.
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The DNA-binding interfaces of the androgen (AR) and glucocorticoid (GR) receptors are virtually identical, yet these transcription factors share only about a third of their genomic binding sites and regulate similarly distinct sets of target genes. To address this paradox, we determined the intrinsic specificities of the AR and GR DNA-binding domains using a refined version of SELEX-seq. We developed an algorithm, SelexGLM, that quantifies binding specificity over a large (31-bp) binding site by iteratively fitting a feature-based generalized linear model to SELEX probe counts. This analysis revealed that the DNA-binding preferences of AR and GR homodimers differ significantly, both within and outside the 15-bp core binding site. The relative preference between the two factors can be tuned over a wide range by changing the DNA sequence, with AR more sensitive to sequence changes than GR. The specificity of AR extends to the regions flanking the core 15-bp site, where isothermal calorimetry measurements reveal that affinity is augmented by enthalpy-driven readout of poly(A) sequences associated with narrowed minor groove width. We conclude that the increased specificity of AR is correlated with more enthalpy-driven binding than GR. The binding models help explain differences in AR and GR genomic binding and provide a biophysical rationale for how promiscuous binding by GR allows functional substitution for AR in some castration-resistant prostate cancers.
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Antagonistas de Receptores Androgénicos , Proteínas de Neoplasias , Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides , Técnica SELEX de Producción de Aptámeros/métodos , Antagonistas de Receptores Androgénicos/síntesis química , Antagonistas de Receptores Androgénicos/química , Antagonistas de Receptores Androgénicos/farmacología , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Línea Celular Tumoral , Humanos , Masculino , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismoRESUMEN
The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.