RESUMEN
BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.
Asunto(s)
Enfermedades Cardiovasculares , Músculo Liso Vascular , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Enfermedades Cardiovasculares/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Contráctiles/metabolismo , Epigénesis Genética , Histonas/metabolismo , Humanos , Hiperplasia/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.
Asunto(s)
Eritroblastos/metabolismo , Células Eritroides/metabolismo , ARN Polimerasa II/metabolismo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Eritroblastos/citología , Células Eritroides/citología , Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , ARN Polimerasa II/genética , Transcripción GenéticaRESUMEN
An international, multicenter registry was established to collect retrospective and prospective clinical data on patients with pyruvate kinase (PK) deficiency, the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Medical history and laboratory and radiologic data were retrospectively collected at enrollment for 254 patients with molecularly confirmed PK deficiency. Perinatal complications were common, including anemia that required transfusions, hyperbilirubinemia, hydrops, and prematurity. Nearly all newborns were treated with phototherapy (93%), and many were treated with exchange transfusions (46%). Children age 5 years and younger were often transfused until splenectomy. Splenectomy (150 [59%] of 254 patients) was associated with a median increase in hemoglobin of 1.6 g/dL and a decreased transfusion burden in 90% of patients. Predictors of a response to splenectomy included higher presplenectomy hemoglobin (P = .007), lower indirect bilirubin (P = .005), and missense PKLR mutations (P = .0017). Postsplenectomy thrombosis was reported in 11% of patients. The most frequent complications included iron overload (48%) and gallstones (45%), but other complications such as aplastic crises, osteopenia/bone fragility, extramedullary hematopoiesis, postsplenectomy sepsis, pulmonary hypertension, and leg ulcers were not uncommon. Overall, 87 (34%) of 254 patients had both a splenectomy and cholecystectomy. In those who had a splenectomy without simultaneous cholecystectomy, 48% later required a cholecystectomy. Although the risk of complications increases with severity of anemia and a genotype-phenotype relationship was observed, complications were common in all patients with PK deficiency. Diagnostic testing for PK deficiency should be considered in patients with apparent congenital hemolytic anemia and close monitoring for iron overload, gallstones, and other complications is needed regardless of baseline hemoglobin. This trial was registered at www.clinicaltrials.gov as #NCT02053480.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/diagnóstico , Estudios de Asociación Genética , Piruvato Quinasa/deficiencia , Errores Innatos del Metabolismo del Piruvato/diagnóstico , Adolescente , Adulto , Anemia Hemolítica Congénita no Esferocítica/etiología , Anemia Hemolítica Congénita no Esferocítica/metabolismo , Anemia Hemolítica Congénita no Esferocítica/terapia , Transfusión Sanguínea , Niño , Preescolar , Colecistectomía/efectos adversos , Colecistectomía/métodos , Terapia Combinada , Activación Enzimática , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Piruvato Quinasa/metabolismo , Errores Innatos del Metabolismo del Piruvato/etiología , Errores Innatos del Metabolismo del Piruvato/metabolismo , Errores Innatos del Metabolismo del Piruvato/terapia , Esplenectomía/efectos adversos , Esplenectomía/métodos , Evaluación de Síntomas , Resultado del Tratamiento , Adulto JovenRESUMEN
Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Estudios de Asociación Genética/métodos , Piruvato Quinasa/deficiencia , Errores Innatos del Metabolismo del Piruvato/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Piruvato Quinasa/genética , Adulto JovenRESUMEN
Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Adulto , Anemia Hemolítica Congénita/metabolismo , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Deshidratación/genética , Deshidratación/metabolismo , Eritrocitos/metabolismo , Familia , Femenino , Células HEK293 , Humanos , Hidropesía Fetal/metabolismo , Mutación INDEL , Recién Nacido , Canales Iónicos/metabolismo , Cinética , Masculino , Mutación Missense , Presión Osmótica/fisiologíaRESUMEN
Hereditary xerocytosis (HX; MIM 194380) is an autosomal-dominant hemolytic anemia characterized by primary erythrocyte dehydration. In many patients, heterozygous mutations associated with delayed channel inactivation have been identified in PIEZO1. This report describes patients from 2 well-phenotyped HX kindreds, including from one of the first HX kindreds described, who lack predicted heterozygous PIEZO1-linked variants. Whole-exome sequencing identified novel, heterozygous mutations affecting the Gardos channel, encoded by the KCNN4 gene, in both kindreds. Segregation analyses confirmed transmission of the Gardos channel mutations with disease phenotype in affected individuals. The KCNN4 variants were different mutations in the same residue, which is highly conserved across species and within members of the small-intermediate family of calcium-activated potassium channel proteins. Both mutations were predicted to be deleterious by mutation effect algorithms. In sickle erythrocytes, the Gardos channel is activated under deoxy conditions, leading to cellular dehydration due to salt and water loss. The identification of KCNN4 mutations in HX patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mutación Missense , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Exoma/genética , Femenino , Genes Dominantes , Estudios de Asociación Genética , Heterocigoto , Humanos , Canales Iónicos/genética , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
A 17-year-old male presented with acute hemolysis with stomatocytosis, elevated mean corpuscular hemoglobin concentration (MCHC), and osmotic gradient ektacytometry consistent with marked erythrocyte dehydration. Erythrocytes from both parents also demonstrated evidence of dehydration with elevated MCHC and abnormal ektacytometry, but neither to the degree of the patient. Genetic studies revealed the patient had hereditary xerocytosis (HX) due to a novel PIEZO1 mutation inherited from his mother and hemoglobin C (HbC) trait inherited from his father. HbC trait accentuated the erythrocyte dehydration of HX. Coinheritance of interrelated disorders and/or modifier alleles should be considered whenever severe erythrocyte dehydration is observed.
Asunto(s)
Anemia Hemolítica Congénita/complicaciones , Anemia Hemolítica Congénita/genética , Eritrocitos/patología , Enfermedad de la Hemoglobina C/complicaciones , Enfermedad de la Hemoglobina C/genética , Hidropesía Fetal/genética , Adolescente , Anemia Hemolítica Congénita/sangre , Índices de Eritrocitos , Enfermedad de la Hemoglobina C/sangre , Humanos , Hidropesía Fetal/sangre , Canales Iónicos/genética , Masculino , MutaciónRESUMEN
To best define biomarkers of response, and to shed insight on mechanism of action of certain clinically important agents for early breast cancer, we used a brief-exposure paradigm in the preoperative setting to study transcriptional changes in patient tumors that occur with one dose of therapy prior to combination chemotherapy. Tumor biopsies from breast cancer patients enrolled in two preoperative clinical trials were obtained at baseline and after one dose of bevacizumab (HER2-negative), trastuzumab (HER2-positive) or nab-paclitaxel, followed by treatment with combination chemo-biologic therapy. RNA-Sequencing based PAM50 subtyping at baseline of 46 HER2-negative patients revealed a strong association between the basal-like subtype and pathologic complete response (pCR) to chemotherapy plus bevacizumab (p ≤ 0.0027), but did not provide sufficient specificity to predict response. However, a single dose of bevacizumab resulted in down-regulation of a well-characterized TGF-ß activity signature in every single breast tumor that achieved pCR (p ≤ 0.004). The TGF-ß signature was confirmed to be a tumor-specific read-out of the canonical TGF-ß pathway using pSMAD2 (p ≤ 0.04), with predictive power unique to brief-exposure to bevacizumab (p ≤ 0.016), but not trastuzumab or nab-paclitaxel. Down-regulation of TGF-ß activity was associated with reduction in tumor hypoxia by transcription and protein levels, suggesting therapy-induced disruption of an autocrine-loop between tumor stroma and malignant cells. Modulation of the TGF-ß pathway upon brief-exposure to bevacizumab may provide an early functional readout of pCR to preoperative anti-angiogenic therapy in HER2-negative breast cancer, thus providing additional avenues for exploration in both preclinical and clinical settings with these agents.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Bevacizumab/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Receptor ErbB-2/análisis , Factor de Crecimiento Transformador beta/fisiología , Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Hipoxia de la Célula , Femenino , Humanos , Análisis de Secuencia de ARN , Transducción de Señal/fisiologíaRESUMEN
T follicular helper (Tfh) cells are a subset of CD4(+) T helper cells that migrate into germinal centers and promote B-cell maturation into memory B and plasma cells. Tfh cells are necessary for promotion of protective humoral immunity following pathogen challenge, but when aberrantly regulated, drive pathogenic antibody formation in autoimmunity and undergo neoplastic transformation in angioimmunoblastic T-cell lymphoma and other primary cutaneous T-cell lymphomas. Limited information is available on the expression and regulation of genes in human Tfh cells. Using a fluorescence-activated cell sorting-based strategy, we obtained primary Tfh and non-Tfh T effector cells from tonsils and prepared genome-wide maps of active, intermediate, and poised enhancers determined by chromatin immunoprecipitation-sequencing, with parallel transcriptome analyses determined by RNA sequencing. Tfh cell enhancers were enriched near genes highly expressed in lymphoid cells or involved in lymphoid cell function, with many mapping to sites previously associated with autoimmune disease in genome-wide association studies. A group of active enhancers unique to Tfh cells associated with differentially expressed genes was identified. Fragments from these regions directed expression in reporter gene assays. These data provide a significant resource for studies of T lymphocyte development and differentiation and normal and perturbed Tfh cell function.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Transcriptoma/genética , Células Cultivadas , Citometría de Flujo , Genoma Humano/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tonsila Palatina/citología , Cultivo Primario de Células , Análisis de Secuencia de ARN , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
BACKGROUND: The optimal weekly oral dose of sirolimus and intravenous nanoparticle albumin-bound paclitaxel (nab-paclitaxel) were evaluated. METHODS: A phase 1b study was performed to evaluate escalating doses of oral sirolimus (5-60 mg) on days 2, 9, and 16 with intravenous nab-paclitaxel (100 mg/m(2) ) on days 1, 8, and 15 in a 28-day cycle. A run-in treatment of nab-paclitaxel (day -14) and sirolimus (day -7) was administered for pharmacokinetic and pharmacodynamic assessments. Clinical trial endpoints included dose-limiting toxicities (DLTs), maximum tolerated doses, and response rates. Pharmacodynamics included immunohistochemistry for phosphatase and tensin homolog, mammalian target of rapamycin (mTOR), AKT, phosphorylated AKT, S6K1, and phosphorylated S6K1; exploratory gene expression analysis; and [(18) F]fludeoxyglucose (FDG) positron emission tomography. RESULTS: Twenty-three patients with advanced solid tumors were treated. Fifteen patients had prior taxane therapy. Twenty-two patients were evaluable for responses. One patient had a complete response, and 5 patients had a partial response (3 confirmed). DLTs were seen in 1 patient each at 10 (grade 3 dyspnea/hypoxia) and 40 mg (grade 4 leukopenia/neutropenia) and in 2 patients at 60 mg (grade 3 fatigue and grade 4 pericardial effusion). Patients with higher expression of posttreatment AKT and a greater decline in FDG activity were more likely to have a treatment response or stable disease. CONCLUSIONS: Sirolimus showed an acceptable safety profile at a weekly dose of 40 mg with weekly intravenous nab-paclitaxel at 100 mg/m(2) on days 1, 8, and 15 every 28 days. The posttreatment AKT score and changes in FDG activity may have roles as early predictors of responses to mTOR inhibitors.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adulto , Anciano , Paclitaxel Unido a Albúmina , Albúminas/administración & dosificación , Albúminas/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Estudios de Cohortes , Femenino , Fluorodesoxiglucosa F18 , Humanos , Masculino , Persona de Mediana Edad , Nanopartículas/administración & dosificación , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Paclitaxel/administración & dosificación , Paclitaxel/farmacocinética , Tomografía de Emisión de Positrones/métodos , Sirolimus/administración & dosificación , Sirolimus/farmacocinética , Resultado del TratamientoRESUMEN
Constitutive activation of the PI3K/Akt/mTOR pathway has been suggested as a mechanism of resistance to trastuzumab therapy. This phase II trial was designed to evaluate the safety and clinical activity of daily oral sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in combination with trastuzumab in HER2-positive metastatic breast cancer following disease progression on prior trastuzumab therapy. Sirolimus 6 mg oral daily dose was administered with a standard dose and schedule of trastuzumab weekly or every 3 weeks. Pharmacodynamic studies included Western blot analysis of S6K1, phosphoS6K1, and mTOR in peripheral mononuclear cells, circulating tumor cells (CTC), and endothelial cells (CEC). Eleven patients were evaluable for safety; and nine were evaluable for response assessment. Subsequent enrollment was stopped due to slow accrual. Study treatment-related grade 3 toxicity included pneumonitis, myelosuppression (leukopenia/anemia), and dermatologic reactions (mucositis, nail changes and rash), with no grade 4 events. One patient received eight cycles (58 weeks) and achieved a partial response. Five patients treated for a total of 101 weeks (median 12 weeks, range 8-47 weeks) achieved stable disease as best response. Overall response rate was 1/9 (11 %) and clinical benefit rate was 4/9 (44 %). There was no statistically significant correlation between response and post-treatment change in levels of the mTOR pathway biomarkers, CTCs, HER2 CTCs, or CECs. Sirolimus 6 mg administered daily with trastuzumab appears to be well tolerated in patients with metastatic HER2-positive breast cancer following disease progression on prior trastuzumab therapy, with evidence of disease activity. mTOR inhibition may overcome resistance to trastuzumab in some HER2-positive tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Leucocitos Mononucleares/metabolismo , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Retratamiento , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/metabolismo , Trastuzumab/administración & dosificación , Resultado del TratamientoAsunto(s)
Acidosis Tubular Renal/genética , Anemia/genética , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Enfermedades del Recién Nacido/genética , Mutación Missense , Acidosis Tubular Renal/metabolismo , Acidosis Tubular Renal/patología , Sustitución de Aminoácidos , Anemia/metabolismo , Anemia/patología , Transporte Biológico Activo/genética , Humanos , Recién Nacido , Enfermedades del Recién Nacido/metabolismo , Enfermedades del Recién Nacido/patología , MasculinoRESUMEN
The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Exones , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/metabolismo , Operón Lac , Leucemia/genética , Leucocitosis/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Trombocitopenia/genéticaRESUMEN
There is an increasing interest in using single nucleotide polymorphism (SNP) genotyping arrays for profiling chromosomal rearrangements in tumors, as they allow simultaneous detection of copy number and loss of heterozygosity with high resolution. Critical issues such as signal baseline shift due to aneuploidy, normal cell contamination, and the presence of GC content bias have been reported to dramatically alter SNP array signals and complicate accurate identification of aberrations in cancer genomes. To address these issues, we propose a novel Global Parameter Hidden Markov Model (GPHMM) to unravel tangled genotyping data generated from tumor samples. In contrast to other HMM methods, a distinct feature of GPHMM is that the issues mentioned above are quantitatively modeled by global parameters and integrated within the statistical framework. We developed an efficient EM algorithm for parameter estimation. We evaluated performance on three data sets and show that GPHMM can correctly identify chromosomal aberrations in tumor samples containing as few as 10% cancer cells. Furthermore, we demonstrated that the estimation of global parameters in GPHMM provides information about the biological characteristics of tumor samples and the quality of genotyping signal from SNP array experiments, which is helpful for data quality control and outlier detection in cohort studies.
Asunto(s)
Variaciones en el Número de Copia de ADN , Pérdida de Heterocigocidad , Cadenas de Markov , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Algoritmos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Dosificación de Gen , Genes erbB-2 , Genoma Humano , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Inhibidores de Crecimiento/farmacología , Imidazoles/farmacología , Nylons/farmacología , Pirroles/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Sistemas de Liberación de Medicamentos/métodos , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/metabolismo , Humanos , Imidazoles/química , Imidazoles/metabolismo , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Nylons/química , Nylons/metabolismo , Unión Proteica/genética , Proto-Oncogenes/genética , Pirroles/química , Pirroles/metabolismo , Ratas , Retroviridae/genética , Factores de Transcripción/genéticaRESUMEN
Mammalian erythropoiesis yields a highly specialized cell type, the mature erythrocyte, evolved to meet the organismal needs of increased oxygen-carrying capacity. To better understand the regulation of erythropoiesis, we performed genome-wide studies of chromatin accessibility, DNA methylation, and transcriptomics using a recently developed strategy to obtain highly purified populations of primary human erythroid cells. The integration of gene expression, DNA methylation, and chromatin state dynamics reveals that stage-specific gene regulation during erythropoiesis is a stepwise and hierarchical process involving many cis-regulatory elements. Erythroid-specific, nonpromoter sites of chromatin accessibility are linked to erythroid cell phenotypic variation and inherited disease. Comparative analyses of stage-specific chromatin accessibility indicate that there is limited early chromatin priming of erythroid genes during hematopoiesis. The epigenome of terminally differentiating erythroid cells defines a distinct subset of highly specialized cells that are vastly dissimilar from other hematopoietic and nonhematopoietic cell types. These epigenomic and transcriptome data are powerful tools to study human erythropoiesis.
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Ensamble y Desensamble de Cromatina/genética , Cromatina/metabolismo , Células Eritroides/metabolismo , Eritropoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cromatina/genética , Metilación de ADN/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Enfermedades Hematológicas/genética , Humanos , Familia de Multigenes , Polimorfismo de Nucleótido Simple , TranscriptomaRESUMEN
The etiology of severe hemolytic anemia in most patients with recessive hereditary spherocytosis (rHS) and the related disorder hereditary pyropoikilocytosis (HPP) is unknown. Whole exome sequencing of DNA from probands of 24 rHS or HPP kindreds identified numerous mutations in erythrocyte membrane α-spectrin (SPTA1). Twenty-eight mutations were novel, with null alleles frequently found in trans to missense mutations. No mutations were identified in a third of SPTA1 alleles (17/48). Whole genome sequencing revealed linkage disequilibrium between the common rHS-linked α-spectrinBug Hill polymorphism and a rare intron 30 variant in all 17 mutation-negative alleles. In vitro minigene studies and in vivo splicing analyses revealed the intron 30 variant changes a weak alternate branch point (BP) to a strong BP. This change leads to increased utilization of an alternate 3' splice acceptor site, perturbing normal α-spectrin mRNA splicing and creating an elongated mRNA transcript. In vivo mRNA stability studies revealed the newly created termination codon in the elongated transcript activates nonsense mediated decay leading to spectrin deficiency. These results demonstrate a unique mechanism of human genetic disease contributes to the etiology of a third of cases of rHS, facilitating diagnosis and treatment of severe anemia, and identifying a new target for therapeutic manipulation.
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Anemia Hemolítica Congénita , Membrana Eritrocítica , Mutación Missense , Sitios de Empalme de ARN , Empalme del ARN/genética , Espectrina , Anemia Hemolítica Congénita/genética , Anemia Hemolítica Congénita/metabolismo , Anemia Hemolítica Congénita/patología , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patología , Femenino , Humanos , Masculino , Espectrina/biosíntesis , Espectrina/genéticaRESUMEN
BACKGROUND: CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. RESULTS: Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. CONCLUSION: These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.
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Eritropoyesis , Perfilación de la Expresión Génica/métodos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Células Eritroides/citología , Células Eritroides/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células K562 , Proteínas Nucleares/genética , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Represoras/genética , Análisis de Secuencia de ARN/métodosRESUMEN
OBJECTIVE: In polycythemia vera (PV) there is no specific clonal marker because the molecular lesion responsible for PV is unknown. The recent demonstration that the PRV-1 gene is overexpressed in granulocytes from patients with PV provided the rationale for the current study to investigate whether PRV-1 is structurally rearranged, thus explaining its aberrant expression. MATERIALS AND METHODS: Fluorescence in situ hybridization was used to determine chromosomal localization of PRV-1 and to study whether the PRV1 gene is rearranged in 26 patients with PV. RESULTS: PRV-1 was localized to chromosome 19, band region q13.12-2. Structural rearrangements of PRV-1 were evaluated in bone marrow cells from 26 patients with PV: 14 with a normal karyotype and 12 with an abnormal karyotype. None of 150 metaphase cells or more than 10,000 interphase cells demonstrated PRV-1 gene deletion, amplification, or separation of the probe signal, which would indicate a PRV-1 rearrangement. CONCLUSION: These findings are consistent with a lack of structural rearrangement of PRV-1 in patients with PV. Thus, overexpression of PRV-1 in granulocytes from patients with PV is related to mechanisms that do not involve structural genetic changes.
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Policitemia Vera/genética , Receptores de Superficie Celular/genética , Biomarcadores/análisis , Médula Ósea/patología , Aberraciones Cromosómicas , Mapeo Cromosómico , Cromosomas Humanos Par 19 , Femenino , Proteínas Ligadas a GPI , Expresión Génica , Reordenamiento Génico , Granulocitos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Isoantígenos , Masculino , Glicoproteínas de Membrana , Policitemia Vera/diagnóstico , Policitemia Vera/etiología , Receptores de Superficie Celular/análisisRESUMEN
Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.