RESUMEN
Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the ß-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one ß-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected ß-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.
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Cardiomyocytes of newborn mice proliferate after injury or exposure to growth factors. However, these responses are diminished after postnatal day-6 (P6), representing a barrier to building new cardiac muscle in adults. We have previously shown that exogenous thyroid hormone (T3) stimulates cardiomyocyte proliferation in P2 cardiomyocytes, by activating insulin-like growth factor-1 receptor (IGF-1R)-mediated ERK1/2 signaling. But whether exogenous T3 functions as a mitogen in post-P6 murine hearts is not known. Here, we show that exogenous T3 increases the cardiomyocyte endowment of P8 hearts, but the proliferative response is confined to cardiomyocytes of the left ventricular (LV) apex. Exogenous T3 stimulates proliferative ERK1/2 signaling in apical cardiomyocytes, but not in those of the LV base, which is inhibited by expression of the nuclear phospho-ERK1/2-specific dual-specificity phosphatase, DUSP5. Developmentally, between P7 and P14, DUSP5 expression increases in the myocardium from the LV base to its apex; after this period, it is uniformly expressed throughout the LV. In young adult hearts, exogenous T3 increases cardiomyocyte numbers after DUSP5 depletion, which might be useful for eliciting cardiac regeneration.
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Fosfatasas de Especificidad Dual/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/enzimología , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Triyodotironina/farmacología , Animales , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
Sequences encoding Olduvai protein domains (formerly DUF1220) show the greatest human lineage-specific increase in copy number of any coding region in the genome and have been associated, in a dosage-dependent manner, with brain size, cognitive aptitude, autism, and schizophrenia. Tandem intragenic duplications of a three-domain block, termed the Olduvai triplet, in four NBPF genes in the chromosomal 1q21.1-0.2 region, are primarily responsible for the striking human-specific copy number increase. Interestingly, most of the Olduvai triplets are adjacent to, and transcriptionally coregulated with, three human-specific NOTCH2NL genes that have been shown to promote cortical neurogenesis. Until now, the underlying genomic events that drove the Olduvai hyperamplification in humans have remained unexplained. Here, we show that the presence or absence of an alternative first exon of the Olduvai triplet perfectly discriminates between amplified (58/58) and unamplified (0/12) triplets. We provide sequence and breakpoint analyses that suggest the alternative exon was produced by an nonallelic homologous recombination-based mechanism involving the duplicative transposition of an existing Olduvai exon found in the CON3 domain, which typically occurs at the C-terminal end of NBPF genes. We also provide suggestive in vitro evidence that the alternative exon may promote instability through a putative G-quadraplex (pG4)-based mechanism. Lastly, we use single-molecule optical mapping to characterize the intragenic structural variation observed in NBPF genes in 154 unrelated individuals and 52 related individuals from 16 families and show that the presence of pG4-containing Olduvai triplets is strongly correlated with high levels of Olduvai copy number variation. These results suggest that the same driver of genomic instability that allowed the evolutionarily recent, rapid, and extreme human-specific Olduvai expansion remains highly active in the human genome.
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Proteínas Portadoras/genética , Genoma Humano , Expansión de Repetición de Trinucleótido , Animales , Secuencia de Bases , Variaciones en el Número de Copia de ADN , Evolución Molecular , G-Cuádruplex , Amplificación de Genes , Dosificación de Gen , Inestabilidad Genómica , Recombinación Homóloga , Humanos , Primates , Dominios Proteicos , Homología de SecuenciaRESUMEN
Mitochondria-generated reactive oxygen species (mROS) are frequently associated with DNA damage and cell cycle arrest, but physiological increases in mROS serve to regulate specific cell functions. T3 is a major regulator of mROS, including hydrogen peroxide (H2O2). Here we show that exogenous thyroid hormone (T3) administration increases cardiomyocyte numbers in neonatal murine hearts. The mechanism involves signaling by mitochondria-generated H2O2 (mH2O2) acting via the redox sensor, peroxiredoxin-1, a thiol peroxidase with high reactivity towards H2O2 that activates c-Jun N-terminal kinase-2α2 (JNK2α2). JNK2α2, a relatively rare member of the JNK family of mitogen-activated protein kinases (MAPK), phosphorylates c-Jun, a component of the activator protein 1 (AP-1) early response transcription factor, resulting in enhanced insulin-like growth factor 1 (IGF-1) expression and activation of proliferative ERK1/2 signaling. This non-canonical mechanism of MAPK activation couples T3 actions on mitochondria to cell cycle activation. Although T3 is regarded as a maturation factor for cardiomyocytes, these studies identify a novel redox pathway that is permissive for T3-mediated cardiomyocyte proliferation-this because of the expression of a pro-proliferative JNK isoform that results in growth factor elaboration and ERK1/2 cell cycle activation.
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Proliferación Celular , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Hormonas Tiroideas/farmacología , Animales , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Oxidación-Reducción , Peroxirredoxinas/metabolismoRESUMEN
Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as ð¾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential ð¾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated ð¾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in ð¾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of ð¾-globin. These data suggest that the 1.7 kb region is not an autonomous ð¾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , ADN Intergénico/genética , Edición Génica , Silenciador del Gen , Genotipo , Células Madre Hematopoyéticas/metabolismo , Humanos , Fenotipo , Eliminación de Secuencia/genética , Regulación hacia Arriba/genética , gamma-Globinas/genéticaRESUMEN
Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.
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Células Madre Adultas/metabolismo , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Hemoglobina Falciforme/genética , Adulto , Animales , Sistemas CRISPR-Cas , Línea Celular , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mutación , Polimorfismo de Nucleótido Simple , Investigación Biomédica TraslacionalRESUMEN
OBJECTIVE: Parental obesity can induce metabolic phenotypes in offspring independent of the inherited DNA sequence. Here we asked whether such non-genetic acquired metabolic traits can be passed on to a second generation that has never been exposed to obesity, even as germ cells. METHODS: We examined the F1, F2, and F3 a/a offspring derived from F0 matings of obese prediabetic A (vy) /a sires and lean a/a dams. After F0, only lean a/a mice were used for breeding. RESULTS: We found that F1 sons of obese founder males exhibited defects in glucose and lipid metabolism, but only upon a post-weaning dietary challenge. F1 males transmitted these defects to their own male progeny (F2) in the absence of the dietary challenge, but the phenotype was largely attenuated by F3. The sperm of F1 males exhibited changes in the abundance of several small RNA species, including the recently reported diet-responsive tRNA-derived fragments. CONCLUSIONS: These data indicate that induced metabolic phenotypes may be propagated for a generation beyond any direct exposure to an inducing factor. This non-genetic inheritance likely occurs via the actions of sperm noncoding RNA.
RESUMEN
BACKGROUND: Piwi-interacting RNAs (piRNAs) are a class of small RNAs; distinct types of piRNAs are expressed in the mammalian testis at different stages of development. The function of piRNAs expressed in the adult testis is not well established. We conducted a detailed characterization of piRNAs aligning at or near the 3' UTRs of protein-coding genes in a deep dataset of small RNAs from adult mouse testis. RESULTS: We identified 2710 piRNA clusters associated with 3' UTRs, including 1600 that overlapped genes not previously associated with piRNAs. 35% of the clusters extend beyond the annotated transcript; we find that these clusters correspond to, and are likely derived from, novel polyadenylated mRNA isoforms that contain previously unannotated extended 3'UTRs. Extended 3' UTRs, and small RNAs derived from them, are also present in somatic tissues; a subset of these somatic 3'UTR small RNA clusters are absent in mice lacking MIWI2, indicating a role for MIWI2 in the metabolism of somatic small RNAs. CONCLUSIONS: The finding that piRNAs are processed from extended 3' UTRs suggests a role for piRNAs in the remodeling of 3' UTRs. The presence of both clusters and extended 3'UTRs in somatic cells, with evidence for involvement of MIWI2, indicates that this pathway is more broadly distributed than currently appreciated.
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Regiones no Traducidas 3'/genética , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/genética , Masculino , Ratones , ARN Mensajero/genética , Testículo/metabolismoRESUMEN
Alcohol-use disorder (AUD) is prevalent and associated with substantial socioeconomic costs. While heritability estimates of AUD are â¼50%, identifying specific gene variants associated with risk for AUD has proven challenging despite considerable investment. Emerging research into heritability of complex diseases has implicated transmission of epigenetic variants in the development of behavioral phenotypes, including drug preference and drug-induced behavior. Several recent rodent studies have specifically focused on paternal transmission of epigenetic variants, which is especially relevant because sires are not present for offspring rearing and changes to offspring phenotype are assumed to result from modifications to the sperm epigenome. While considerable interest in paternal transmission of epigenetic variants has emerged recently, paternal alcohol exposures have been studied for 30+ years with interesting behavioral and physiologic effects noted on offspring. However, only recently, with improvements in technology to identify epigenetic modifications in germ cells, has it been possible to identify mechanisms by which paternal ethanol exposure alters offspring behavior. This review presents an overview of epigenetic inheritance in the context of paternal ethanol exposure and suggests future studies to identify specific effects of paternal ethanol exposure on offspring behavior and response to ethanol.
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Consumo de Bebidas Alcohólicas/genética , Alcoholismo/genética , Hijo de Padres Discapacitados/psicología , Epigénesis Genética/efectos de los fármacos , Padre , Animales , Humanos , Masculino , FenotipoRESUMEN
Drosophila melanogaster is often considered to lack genomic 5-methylcytosine (m(5) C), an opinion reinforced by two whole genome bisulfite-sequencing studies that failed to find m(5) C. New evidence, however, indicates that genomic methylation is indeed present in the fly, albeit in small quantities and in unusual patterns. At embryonic stage 5, m(5) C occurs in short strand-specific regions that cover â¼1% of the genome, at tissue levels suggesting a distribution restricted to a subset of nuclei. Its function is not obvious, but methylation in subsets of nuclei would obscure functional associations since transcript levels and epigenetic modifications are assayed in whole embryos. Surprisingly, Mt2, the fly's only candidate DNA methyltransferase, is not necessary for the observed methylation. Full evaluation of the functions of genome methylation in Drosophila must await discovery and experimental inactivation of the DNA methyltransferase, as well as a better understanding of the pattern and developmental regulation of genomic m(5) C.
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5-Metilcitosina/metabolismo , Núcleo Celular/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigénesis Genética , Genoma , Animales , Núcleo Celular/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Motivos de NucleótidosRESUMEN
The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.
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Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , ARN de Transferencia/metabolismo , Línea Celular Tumoral , Metilación de ADN , Genoma Humano , Humanos , Procesamiento Postranscripcional del ARN , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARNRESUMEN
It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here, we show that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day 15 is intermediate between that observed at postnatal days 2 and 21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.
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Diferenciación Celular , Proliferación Celular , Corazón/crecimiento & desarrollo , Miocitos Cardíacos/citología , Animales , Separación Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/fisiología , Triyodotironina/metabolismoRESUMEN
Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering â¼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is the DNMT2 homolog MT2, but lines deficient for MT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with a lower expression of specific developmental genes at stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genoma de los Insectos , Motivos de Nucleótidos , Animales , Composición de Base , Islas de CpG , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Piwi proteins and their small non-coding RNA partners are involved in the maintenance of stem cell character and genome integrity in the male germ cells of mammals. MIWI2, one of the mouse Piwi-like proteins, is expressed in the prepachytene phase of spermatogenesis during the period of de novo methylation. Absence of this protein leads to meiotic defects and a progressive loss of germ cells. There is an accumulation of evidence that Piwi proteins may be active in hematopoietic tissues. Thus, MIWI2 may have a role in hematopoietic stem and/or progenitor cell self-renewal and differentiation, and defects in MIWI2 may lead to abnormal hematopoiesis. MIWI2 mRNA can be detected in a mouse erythroblast cell line by RNA-seq, and shRNA-mediated knockdown of this mRNA causes the cells to take on characteristics of differentiated erythroid precursors. However, there are no detectable hematopoietic abnormalities in a MIWI2-deficient mouse model. While subtle, non-statistically significant changes were noted in the hematopoietic function of mice without a functional MIWI2 gene when compared to wild type mice, our results show that MIWI2 is not solely necessary for hematopoiesis within the normal life span of a mouse.
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Proteínas Argonautas/deficiencia , Diferenciación Celular , Hematopoyesis , Leucemia Eritroblástica Aguda/patología , Envejecimiento/patología , Animales , Proteínas Argonautas/metabolismo , Células Sanguíneas/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Hemoglobinas/metabolismo , Leucemia Eritroblástica Aguda/genética , Ratones Endogámicos C57BL , Especificidad de Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ARN , Bazo/metabolismo , Irradiación Corporal TotalRESUMEN
Small noncoding RNAs carry out a variety of functions in eukaryotic cells, and in multiple species they can travel between cells, thus serving as signaling molecules. In mammals multiple small RNAs have been found to circulate in the blood, although in most cases the targets of these RNAs, and even their functions, are not well understood. YRNAs are small (84-112 nt) RNAs with poorly characterized functions, best known because they make up part of the Ro ribonucleoprotein autoantigens in connective tissue diseases. In surveying small RNAs present in the serum of healthy adult humans, we have found YRNA fragments of lengths 27 nt and 30-33 nt, derived from the 5'-ends of specific YRNAs and generated by cleavage within a predicted internal loop. Many of the YRNAs from which these fragments are derived were previously annotated only as pseudogenes, or predicted informatically. These 5'-YRNA fragments make up a large proportion of all small RNAs (including miRNAs) present in human serum. They are also present in plasma, are not present in exosomes or microvesicles, and circulate as part of a complex with a mass between 100 and 300 kDa. Mouse serum contains far fewer 5'-YRNA fragments, possibly reflecting the much greater copy number of YRNA genes and pseudogenes in humans. The function of the 5'-YRNA fragments is at present unknown, but the processing and secretion of specific YRNAs to produce 5'-end fragments that circulate in stable complexes are consistent with a signaling function.
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Seudogenes/genética , Procesamiento Postranscripcional del ARN , ARN Pequeño no Traducido/genética , ARN/genética , Adulto , Animales , Secuencia de Bases , Northern Blotting , ADN Complementario/química , ADN Complementario/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Pequeño no Traducido/sangre , ARN Pequeño no Traducido/química , Ribonucleoproteínas/genética , Análisis de Secuencia de ADNRESUMEN
Activation-induced cytidine deaminase (AID), which functions in antibody diversification, is also expressed in a variety of germ and somatic cells. Evidence that AID promotes DNA demethylation in epigenetic reprogramming phenomena, and that it is induced by inflammatory signals, led us to investigate its role in the epithelial-mesenchymal transition (EMT), a critical process in normal morphogenesis and tumor metastasis. We find that expression of AID is induced by inflammatory signals that induce the EMT in nontransformed mammary epithelial cells and in ZR75.1 breast cancer cells. shRNA-mediated knockdown of AID blocks induction of the EMT and prevents cells from acquiring invasive properties. Knockdown of AID suppresses expression of several key EMT transcriptional regulators and is associated with increased methylation of CpG islands proximal to the promoters of these genes; furthermore, the DNA demethylating agent 5 aza-2'deoxycytidine (5-Aza-dC) antagonizes the effects of AID knockdown on the expression of EMT factors. We conclude that AID is necessary for the EMT in this breast cancer cell model and in nontransformed mammary epithelial cells. Our results suggest that AID may act near the apex of a hierarchy of regulatory steps that drive the EMT, and are consistent with this effect being mediated by cytosine demethylation. This evidence links our findings to other reports of a role for AID in epigenetic reprogramming and control of gene expression.
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Citidina Desaminasa/genética , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación de la Expresión Génica , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Islas de CpG/genética , Citidina Desaminasa/metabolismo , Metilación de ADN , Decitabina , Células Epiteliales/efectos de los fármacos , Prueba de Complementación Genética , Células HEK293 , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Metaloproteinasas de la Matriz/genética , Ratones , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Intrauterine nutrition can program metabolism, creating stable changes in physiology that may have significant health consequences. The mechanism underlying these changes is widely assumed to involve epigenetic changes to the expression of metabolic genes, but evidence supporting this idea is limited. Here we have performed the first study of the epigenomic consequences of exposure to maternal obesity and diabetes. We used a mouse model of natural-onset obesity that allows comparison of genetically identical mice whose mothers were either obese and diabetic or lean with a normal metabolism. We find that the offspring of obese mothers have a latent metabolic phenotype that is unmasked by exposure to a Western-style diet, resulting in glucose intolerance, insulin resistance and hepatic steatosis. The offspring show changes in hepatic gene expression and widespread but subtle alterations in cytosine methylation. Contrary to expectation, these molecular changes do not point to metabolic pathways but instead reside in broadly developmental ontologies. We propose that, rather than being adaptive, these changes may simply produce an inappropriate response to suboptimal environments; maladaptive phenotypes may be avoidable if postnatal nutrition is carefully controlled.
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Diabetes Mellitus Tipo 2/metabolismo , Epigénesis Genética , Expresión Génica , Hígado/metabolismo , Obesidad/metabolismo , Complicaciones del Embarazo/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Dieta , Femenino , Desarrollo Fetal , Hígado/patología , Masculino , Ratones , Mitocondrias Hepáticas/genética , Mitocondrias Hepáticas/metabolismo , Embarazo , Embarazo en Diabéticas/metabolismoRESUMEN
BACKGROUND: Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30-33 nt RNAs that are processed derivatives of the 5' ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5' tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. RESULTS: In sequencing small RNAs extracted from mouse serum, we identified abundant 5' tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5' tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100-300 kDa. The size of these particles suggest that the 5' tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5' tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5' tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5' tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. CONCLUSIONS: We demonstrate that 5' tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.