RESUMEN
In addition to genetic and epigenetic inheritance, somatic variation may contribute to cardiovascular disease (CVD) risk. CVD-associated somatic mutations have been reported in human clonal hematopoiesis, but evidence in the atheroma is lacking. To probe for somatic variation in atherosclerosis, we sought single-nucleotide private variants (PVs) in whole-exome sequencing (WES) data of aorta, liver, and skeletal muscle of two C57BL/6J coisogenic male ApoE null/wild-type (WT) sibling pairs, and RNA-seq data of one of the two pairs. Relative to the C57BL/6 reference genome, we identified 9 and 11 ApoE null aorta- and liver-specific PVs that were shared by all WES and RNA-seq datasets. Corresponding PVs in WT sibling aorta and liver were 1 and 0, respectively, and not overlapping with ApoE null PVs. Pyrosequencing analysis of 4 representative PVs in 17 ApoE null aortas and livers confirmed tissue-specific shifts toward the alternative allele, in addition to significant deviations from mendelian allele ratios. Notably, all aorta and liver PVs were present in the dbSNP database and were predominantly transition mutations within atherosclerosis-related genes. The majority of PVs were in discrete clusters approximately 3 Mb and 65 to 73 Mb away from hypermutable immunoglobin loci in chromosome 6. These features were largely shared with previously reported CVD-associated somatic mutations in human clonal hematopoiesis. The observation that SNPs exhibit tissue-specific somatic DNA mosaicism in ApoE null mice is potentially relevant for genetic association study design. The proximity of PVs to hypermutable loci suggests testable mechanistic hypotheses.
Asunto(s)
Enfermedades de la Aorta/genética , Aterosclerosis/genética , Mosaicismo , Polimorfismo de Nucleótido Simple , Animales , Aorta/patología , Enfermedades de la Aorta/patología , Aterosclerosis/patología , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Fenotipo , RNA-Seq , Secuenciación del ExomaRESUMEN
BACKGROUND: The signals that determine atherosclerosis-specific DNA methylation profiles are only partially known. We previously identified a 29-bp DNA motif (differential methylation motif [DMM]) proximal to CpG islands (CGIs) that undergo demethylation in advanced human atheromas. Those data hinted that the DMM docks modifiers of DNA methylation and transcription. METHODS AND RESULTS: We sought to functionally characterize the DMM. We showed that the DMM overlaps with the RNA polymerase III-binding B box of Alu short interspersed nuclear elements and contains a DR2 nuclear receptor response element. Pointing to a possible functional role for an Alu DMM, CGIs proximal (<100 bp) to near-intact DMM-harboring Alu are significantly less methylated relative to CGIs proximal to degenerate DMM-harboring Alu or to DMM-devoid mammalian-wide interspersed repeat short interspersed nuclear elements in human arteries. As for DMM-binding factors, LXRB (liver X receptor ß) binds the DMM in a DR2-dependent fashion, and LXR (liver X receptor) agonists induce significant hypermethylation of the bulk of Alu in THP-1 cells. Furthermore, we describe 3 intergenic long noncoding RNAs that harbor a DMM, are under transcriptional control by LXR agonists, and are differentially expressed between normal and atherosclerotic human aortas. Notably, CGIs adjacent to those long noncoding RNAs tend to be hypomethylated in symptomatic relative to stable human atheromas. CONCLUSIONS: Collectively, the data suggest that a DMM is associated with 2 distinct methylation states: relatively low methylation of in cis CGIs and Alu element hypermethylation. Based on the known atheroprotective role of LXRs, we propose that LXR agonist-induced Alu hypermethylation, a landmark of atherosclerosis, is a compensatory rather than proatherogenic response.
Asunto(s)
Elementos Alu , Aterosclerosis/genética , Islas de CpG , Metilación de ADN , Epigénesis Genética , Receptores X del Hígado/metabolismo , Motivos de Nucleótidos , Aterosclerosis/metabolismo , Benzoatos/farmacología , Bencilaminas/farmacología , Sitios de Unión , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Receptores X del Hígado/agonistas , Receptores X del Hígado/genética , Unión Proteica , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células THP-1 , Técnicas del Sistema de Dos HíbridosRESUMEN
Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage.
Asunto(s)
Metilación de ADN , Ayuno/sangre , Ácidos Grasos/sangre , Histona Desacetilasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Regiones no Traducidas 5' , Adulto , Ácido Araquidónico/sangre , Dieta Occidental/efectos adversos , Ácido Eicosapentaenoico/sangre , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Recién Nacido , Lactancia , Masculino , Periodo Posprandial , Regiones Promotoras Genéticas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Análisis de Regresión , Células THP-1RESUMEN
BACKGROUND: The deleterious effects of dietary trans fatty acids (tFAs) on human health are well documented. Although significantly reduced or banned in various countries, tFAs may trigger long-term responses that would represent a valid human health concern, particularly if tFAs alter the epigenome. METHODS: Based on these considerations, we asked whether the tFA elaidic acid (EA; tC18:1) has any effects on global DNA methylation and the transcriptome in cultured human THP-1 monocytes, and whether the progeny of EA-supplemented dams during either pregnancy or lactation in mice (n = 20 per group) show any epigenetic change after exposure. RESULTS: EA induced a biphasic effect on global DNA methylation in THP-1 cells, i.e. hypermethylation in the 1-50 µM concentration range, followed by hypomethylation up to the 200 µM dose. On the other hand, the cis isomer oleic acid (OA), a fatty acid with documented beneficial effects on human health, exerted a distinct response, i.e. its effects were weaker and only partially overlapping with EA's. The maximal differential response between EA and OA was observed at the 50 µM dose. Array expression data revealed that EA induced a pro-inflammatory and adipogenic transcriptional profile compared with OA, although with modest effects on selected (n = 9) gene promoter methylation. In mice, maternal EA supplementation in utero or via the breastmilk induced global adipose tissue DNA hypermethylation in the progeny, that was detectable postnatally at the age of 3 months. CONCLUSION: We document that global DNA hypermethylation is a specific and consistent response to EA in cell culture and in mice, and that EA may exert long-term effects on the epigenome following maternal exposure.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Oléico/efectos adversos , Tejido Adiposo/efectos de los fármacos , Animales , Células Cultivadas , Epigénesis Genética/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Lactancia , Masculino , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Ácido Oléico/farmacología , Ácidos Oléicos , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
Abnormal fatty acid metabolism and availability are landmarks of metabolic diseases, which in turn are associated with aberrant DNA methylation profiles. To understand the role of fatty acids in disease epigenetics, we sought DNA methylation profiles specifically induced by arachidonic (AA) or oleic acid (OA) in cultured cells and compared those with published profiles of normal and diseased tissues. THP-1 monocytes were stimulated with AA or OA and analyzed using Infinium HumanMethylation450 BeadChip (Illumina) and Human Exon 1.0 ST array (Affymetrix). Data were corroborated in mouse embryonic fibroblasts. Comparisons with publicly available data were conducted by standard bioinformatics. AA and OA elicited a complex response marked by a general DNA hypermethylation and hypomethylation in the 1-200 µM range, respectively, with a maximal differential response at the 100 µM dose. The divergent response to AA and OA was prominent within the gene body of target genes, where it correlated positively with transcription. AA-induced DNA methylation profiles were similar to the corresponding profiles described for palmitic acid, atherosclerosis, diabetes, obesity, and autism, but relatively dissimilar from OA-induced profiles. Furthermore, human atherosclerosis grade-associated DNA methylation profiles were significantly enriched in AA-induced profiles. Biochemical evidence pointed to ß-oxidation, PPAR-α, and sirtuin 1 as important mediators of AA-induced DNA methylation changes. In conclusion, AA and OA exert distinct effects on the DNA methylome. The observation that AA may contribute to shape the epigenome of important metabolic diseases, supports and expands current diet-based therapeutic and preventive efforts.
Asunto(s)
Ácido Araquidónico/metabolismo , Aterosclerosis/genética , Metilación de ADN/efectos de los fármacos , Ácido Oléico/metabolismo , Animales , Ácido Araquidónico/administración & dosificación , Aterosclerosis/metabolismo , Aterosclerosis/patología , Epigénesis Genética/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genoma Humano , Humanos , Metabolismo de los Lípidos/genética , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Ácido Oléico/administración & dosificación , PPAR alfa/genéticaRESUMEN
BACKGROUND: We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. RESULTS: Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. CONCLUSIONS: Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.
Asunto(s)
Metilación de ADN , Silenciador del Gen , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Línea Celular , Epigénesis Genética , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Inflamación/genética , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
Current data suggest that angiogenesis, smooth muscle cell migration, differentiation and proliferation may be epigenetically regulated. Prokaryotic DNA methyltransferases have been proposed as tools to modify mammalian DNA methylation. In order to assess the impact of DNA hypermethylation on smooth muscle pathophysiology, we expressed an HpaII site-specific methyltransferase transgene in smooth muscle cells in mice. The enzyme is expected to target only a subset (CCGG) of unmethylated CpG dinucleotides, thus avoiding possible deleterious effects of widespread hypermethylation. Transgenics of two independent lines were born at expected frequencies, showed no obvious abnormalities and were fertile. Nevertheless, ~30% of > 1 year-old transgenics developed organomegaly and ~20% showed a range of tumors. Global DNA methylation was unchanged in transgenic tissue whether hyperplastic or normal, but tumor DNA showed a pronounced global hypermethylation. DNA hypermethylation was not indiscriminate, as five tested tumor suppressor genes showed promoter CpG and non-CpG hypermethylation and transcriptional down-regulation, whereas the methylation status of one intergenic CpG islands, repeated elements (n=2) and non-tumor suppressor gene promoters (n=3) was unchanged. Our work is the first report on the effects of HpaII methyltransferase on endogenous chromatin and in a whole animal. Furthermore, our data expand previous findings that imply that global DNA hypomethylation is not an obligate oncogenic pathway at least in the tumor types examined here.
Asunto(s)
ADN-Citosina Metilasas/genética , Miocitos del Músculo Liso/enzimología , Neoplasias/genética , Animales , Línea Celular Tumoral , Cromatina/metabolismo , Islas de CpG/genética , Metilación de ADN , ADN-Citosina Metilasas/metabolismo , Regulación hacia Abajo , Genes Supresores de Tumor , Ratones , Ratones Transgénicos , Miocitos del Músculo Liso/metabolismo , Neoplasias/enzimología , Tamaño de los ÓrganosRESUMEN
Different soluble NAD+-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleum- contaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD+-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (approximately 7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD+-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol- dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.
Asunto(s)
Alcohol Deshidrogenasa/biosíntesis , Alcohol Deshidrogenasa/química , Mucor/enzimología , Mucor/aislamiento & purificación , Petróleo/microbiología , Microbiología del Suelo , Contaminantes del Suelo/farmacocinética , Alcohol Deshidrogenasa/análisis , Biodegradación Ambiental , Activación Enzimática , Glucosa/metabolismo , Mucor/clasificación , Mucor/crecimiento & desarrollo , Oxígeno/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20,38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. Km values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.