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Acute kidney injury (AKI) is a common condition associated with significant morbidity, mortality, and cost. Injured kidney tissue can regenerate after many forms of AKI. However, there are no treatments in routine clinical practice to encourage recovery. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that orchestrate kidney recovery. The advent of high-throughput sequencing technologies and genetic mouse models has opened an unprecedented window into the transcriptional dynamics that accompany both successful and maladaptive repair. AKI recovery shares similar cell-state transformations with kidney development, which can suggest common mechanisms of gene regulation. Several powerful bioinformatic strategies have been developed to infer the activity of gene regulatory networks by combining multiple forms of sequencing data at single-cell resolution. These studies highlight not only shared stress responses but also key changes in gene regulatory networks controlling metabolism. Furthermore, chromatin immunoprecipitation studies in injured kidneys have revealed dynamic epigenetic modifications at enhancer elements near target genes. This review will highlight how these studies have enhanced our understanding of gene regulation in injury response and regeneration.
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PURPOSE OF REVIEW: Highlight the mechanisms through which vasopressin and hypertonic stress regulate protein metabolism. RECENT FINDINGS: Mammals have an 'aestivation-like' response in which hypertonic stress increases muscle catabolism and urea productionVasopressin can directly regulate ureagenesis in the liver and the kidneyIn humans chronic hypertonic stress is associated with premature aging, diabetes, cardiovascular disease, and premature mortality. SUMMARY: There is an evolutionarily conserved 'aestivation-like' response in humans in which hypertonic stress results in activation of the vasopressin system, muscle catabolism, and ureagenesis in order to promote water conservation.
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Vasopresinas , Humanos , Animales , Vasopresinas/metabolismo , Urea/metabolismo , Riñón/metabolismo , Proteínas/metabolismo , Hígado/metabolismoRESUMEN
We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.
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Desamino Arginina Vasopresina , Túbulos Renales Colectores , Ratones Noqueados , Animales , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Desamino Arginina Vasopresina/farmacología , Capacidad de Concentración Renal/efectos de los fármacos , Arginina Vasopresina/metabolismo , Masculino , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Ratones , Acuaporina 2/metabolismo , Acuaporina 2/genética , Fármacos Antidiuréticos/farmacología , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Ratones Endogámicos C57BL , Privación de Agua , Concentración Osmolar , Sodio/orina , Sodio/metabolismo , Vasopresinas/metabolismo , BenzazepinasRESUMEN
The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in cells. Guanine-rich sequences form structures called guanine quadruplexes (G4) that result from non-canonical base pairing between guanine residues. These stable structures are enriched in gene promoters and have been correlated with the locations of RNA polymerase II pausing (Pol II). While promoter-proximal RNA polymerase pausing regulates gene expression, the effects of guanine quadruplexes on gene transcription have been less clear. We determined the pattern of mitochondrial RNA polymerase (mtRNAP) pausing in human fibroblasts and found that it pauses over 400 times on the mitochondrial genome. We identified quadruplexes as a mediator of mtRNAP pausing and show that stabilization of quadruplexes impeded transcription by mtRNAP. Gene products encoded by the mitochondrial genome are required for oxidative phosphorylation and the decreased transcription by mtRNAP resulted in lower expression of mitochondrial genes and significantly reduced ATP generation. Energy from mitochondria is essential for transport function in renal epithelia, and impeded mitochondrial transcription inhibits transport function in renal proximal tubule cells. These results link formation of guanine quadruplex structures to regulation of mtRNAP elongation and mitochondrial function.
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Vasopressin is a highly conserved peptide hormone that has been traditionally associated with water homeostasis. There is accumulating evidence in both humans and animal models that vasopressin is implicated in the regulation of metabolism. This review focuses on the effects that vasopressin exerts on the regulation of glucose and fatty acids with a particular emphasis on the potential repercussions of metabolic dysregulation in kidney disease.
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Transducción de Señal , Vasopresinas , Humanos , Animales , Vasopresinas/metabolismo , Glucosa/metabolismo , Homeostasis/fisiologíaRESUMEN
The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (â¼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.
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Acuaporina 2 , Insulina , Animales , Masculino , Ratones , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Insulina/metabolismo , Mamíferos/metabolismo , Presión Osmótica , Vasopresinas/farmacología , Vasopresinas/metabolismo , Agua/metabolismoRESUMEN
RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.
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Enfermedad de Alzheimer , Estructuras R-Loop , Humanos , ARN/genética , Enfermedad de Alzheimer/genética , Transcripción Genética , Apolipoproteínas E/genéticaRESUMEN
Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.
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Túbulos Renales Colectores , Ratones , Humanos , Animales , Túbulos Renales Colectores/metabolismo , Cloruro de Sodio/farmacología , Cloruro de Sodio/metabolismo , Vasopresinas/metabolismo , Agua/metabolismo , ARN Mensajero/metabolismoRESUMEN
RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.
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Secuencia de Bases/genética , ARN/química , ARN/genética , Sitios de Unión , ADN/química , Daño del ADN/genética , ADN Glicosilasas/metabolismo , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Desoxirribonucleasa I/metabolismo , Humanos , Nucleótidos/genética , Estructuras R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Levaduras/genéticaRESUMEN
OBJECTIVE: To determine the clinical and molecular features in patients with amyotrophic lateral sclerosis 4 (ALS4) due to mutations in the senataxin (SETX) gene and to develop tools for evaluating SETX variants. METHODS: Our study involved 32 patients, including 31 with mutation in SETX at c.1166 T>C (p.Leu389Ser) and 1 with mutation at c.1153 G>A (p.Glu385Lys). Clinical characterization of the patients included neurological examination, blood tests, magnetic resonance imaging (MRI), and dual-energy x-ray absorptiometry (DEXA). Fibroblasts and motor neurons were obtained to model the disease and characterize the molecular alteration in senataxin function. RESULTS: We report key clinical features of ALS4. Laboratory analysis showed alteration of serum creatine kinase and creatinine in the Leu389Ser ALS4 cohort. MRI showed increased muscle fat fraction in the lower extremities, which correlates with disease duration (thigh fat fraction R2 = 0.35, p = 0.01; lower leg fat fraction R2 = 0.49, p < 0.01). DEXA measurements showed lower extremities are more affected than upper extremities (average fat z scores of 2.1 and 0.6, respectively). A cellular assay for SETX function confirmed that like the Leu389Ser mutation, the Glu385Lys variant leads to a decrease in R loops, likely from a gain of function. INTERPRETATION: We identified clinical laboratory and radiological features of ALS4, and hence they should be monitored for disease progression. The molecular characterization of R-loop levels in patient-derived cells provides insight into the disease pathology and assays to evaluate the pathogenicity of candidate mutations in the SETX gene. ANN NEUROL 2020;87:547-555.
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Esclerosis Amiotrófica Lateral/metabolismo , ADN Helicasas/metabolismo , Enzimas Multifuncionales/metabolismo , ARN Helicasas/metabolismo , Absorciometría de Fotón , Tejido Adiposo/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/diagnóstico por imagen , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Western Blotting , Creatina Quinasa/metabolismo , Creatinina/metabolismo , ADN Helicasas/genética , Electromiografía , Femenino , Fibroblastos , Humanos , Células Madre Pluripotentes Inducidas , Lactante , Extremidad Inferior/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Enzimas Multifuncionales/genética , Músculo Esquelético/diagnóstico por imagen , Mutación , Conducción Nerviosa , Estructuras R-Loop/genética , ARN Helicasas/genética , ARN Mensajero , Extremidad Superior/diagnóstico por imagen , Adulto JovenRESUMEN
Aberrant gene expression underlies many human diseases. RNA polymerase II (Pol II) pausing is a key regulatory step in transcription. Here, we mapped the locations of RNA Pol II in normal human cells and found that RNA Pol II pauses in a consistent manner across individuals and cell types. At more than 1,000 genes including MYO1E and SESN2, RNA Pol II pauses at precise nucleotide locations. Characterization of these sites shows that RNA Pol II pauses at GC-rich regions that are marked by a sequence motif. Sixty-five percent of the pause sites are cytosines. By differential allelic gene expression analysis, we showed in our samples and a population dataset from the Genotype-Tissue Expression (GTEx) consortium that genes with more paused polymerase have lower expression levels. Furthermore, mutagenesis of the pause sites led to a significant increase in promoter activities. Thus, our data uncover that RNA Pol II pauses precisely at sites with distinct sequence features that in turn regulate gene expression.
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Regulación de la Expresión Génica , ARN Polimerasa II/metabolismo , Adulto , Alelos , Células Cultivadas , Humanos , Recién NacidoRESUMEN
R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor ß (TGF-ß), is reduced; that then leads to the activation of the TGF-ß pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.
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Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas , ARN Helicasas/genética , ARN Helicasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , ADN/genética , ADN/ultraestructura , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN Helicasas , Metilación de ADN/genética , Humanos , Proteínas de la Membrana/metabolismo , Enzimas Multifuncionales , Mutación , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , ARN/genética , ARN/ultraestructura , Motivos de Unión al ARN , Activación Transcripcional/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.
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Elementos de Facilitación Genéticos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-gamma del Hepatocito/metabolismo , Nucleosomas/metabolismo , Animales , Histonas/metabolismo , Hígado/metabolismo , Ratones , Nucleosomas/genética , Especificidad de Órganos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic "shadow" enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming.
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Adenocarcinoma/genética , Esófago de Barrett/patología , Proteínas de Unión al ADN/genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Activación Transcripcional , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Esófago de Barrett/metabolismo , Secuencia de Bases , Sitios de Unión , Factor de Transcripción CDX2 , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Elementos de Facilitación Genéticos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Silenciador del Gen , Genoma , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción del Factor Regulador X , Factor Regulador X1RESUMEN
We reported previously that well-characterized enhancers but not promoters for typical tissue-specific genes, including the classic Alb1 gene, contain unmethylated CpG dinucleotides and evidence of pioneer factor interactions in embryonic stem (ES) cells. These properties, which are distinct from the bivalent histone modification domains that characterize the promoters of genes involved in developmental decisions, raise the possibility that genes expressed only in differentiated cells may need to be marked at the pluripotent stage. Here, we demonstrate that the forkhead family member FoxD3 is essential for the unmethylated mark observed at the Alb1 enhancer in ES cells, with FoxA1 replacing FoxD3 following differentiation into endoderm. Up-regulation of FoxD3 and loss of CpG methylation at the Alb1 enhancer accompanied the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Studies of two genes expressed in specific hematopoietic lineages revealed that the establishment of enhancer marks in ES cells and iPS cells can be regulated both positively and negatively. Furthermore, the absence of a pre-established mark consistently resulted in resistance to transcriptional activation in the repressive chromatin environment that characterizes differentiated cells. These results support the hypothesis that pluripotency and successful reprogramming may be critically dependent on the marking of enhancers for many or all tissue-specific genes.
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Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Factores de Transcripción Forkhead/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transcripción Genética/genética , Animales , Línea Celular , Reprogramación Celular , Islas de CpG/genética , Metilación de ADN , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state.
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Islas de CpG/genética , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Silenciador del Gen , Nucleótidos/genética , Albúminas/genética , Animales , Línea Celular , Metilación de ADN , Células Madre Embrionarias/citología , Marcadores Genéticos , Hematopoyesis , Histonas/metabolismo , Humanos , Interleucina-12/genética , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Alineación de Secuencia , Timo/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
Transcription factor GATA-1 is required for erythropoiesis, yet its full actions are unknown. We performed transcriptome analysis of G1E-ER4 cells, a GATA-1-null erythroblast line that undergoes synchronous erythroid maturation when GATA-1 activity is restored. We interrogated more than 9000 transcripts at 6 time points representing the transition from late burst forming unit-erythroid (BFU-E) to basophilic erythroblast stages. Our findings illuminate several new aspects of GATA-1 function. First, the large number of genes responding quickly to restoration of GATA-1 extends the repertoire of its potential targets. Second, many transcripts were rapidly down-regulated, highlighting the importance of GATA-1 in gene repression. Third, up-regulation of some known GATA-1 targets was delayed, suggesting that auxiliary factors are required. For example, induction of the direct GATA-1 target gene beta major globin was late and, surprisingly, required new protein synthesis. In contrast, the gene encoding Fog1, which cooperates with GATA-1 in beta globin transcription, was rapidly induced independently of protein synthesis. Guided by bioinformatic analysis, we demonstrated that selected regions of the Fog1 gene exhibit enhancer activity and in vivo occupancy by GATA-1. These findings define a regulatory loop for beta globin expression and, more generally, demonstrate how transcriptome analysis can be used to generate testable hypotheses regarding transcriptional networks.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eritroblastos/fisiología , Eritropoyesis/genética , Perfilación de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Elementos de Facilitación Genéticos/fisiología , Eritroblastos/citología , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Prueba de Complementación Genética , Humanos , Ratones , Proteínas Nucleares/genética , Reproducibilidad de los Resultados , Transcripción Genética/fisiologíaRESUMEN
The defining characteristic of recessive diseases is the absence of a phenotype in the heterozygous carriers. Nonetheless, subtle manifestations may be detectable by new methods, such as expression profiling. Ataxia telangiectasia (AT) is a typical recessive disease, and individual carriers cannot be reliably identified. As a group, however, carriers of an AT disease allele have been reported to have a phenotype that distinguishes them from normal control individuals: increased radiosensitivity and risk of cancer. We show here that the phenotype is also detectable, in lymphoblastoid cells from AT carriers, as changes in expression level of many genes. The differences are manifested both in baseline expression levels and in response to ionizing radiation. Our findings show that carriers of a recessive disease may have an "expression phenotype." In the particular case of AT, this suggests a new approach to the identification of carriers and enhances understanding of their increased cancer risk. More generally, we demonstrate that genomic technologies offer the opportunity to identify and study unaffected carriers, who are hundreds of times more common than affected patients.