RESUMEN
Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression.
Asunto(s)
Metilación de ADN , Momento de Replicación del ADN , Endotelio Vascular/citología , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Fase S/fisiología , Animales , Antígenos CD/genética , Aorta/citología , Aorta/metabolismo , Cadherinas/genética , Bovinos , Moléculas de Adhesión Celular/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Dermis/citología , Dermis/metabolismo , Endotelio Vascular/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Factor de von Willebrand/genéticaRESUMEN
BACKGROUND: The Falkland Islands and Patagonia are traditionally assigned to the Magellan Biogeographic Province. Most marine species in Falkland waters are also reported from southern Patagonia. It remains unclear if relatively immobile, marine benthic, shallow-water species maintain gene flow, and by what mechanism. Recurrent fluctuations in sea level during glacial cycles are regarded as a possible mechanism that might have allowed genetic exchange between the regions. However, the realized genetic exchange between the Falkland Islands and Patagonia has never been estimated. RESULTS: This study analyses the genetic structure of three populations of the marine shallow-water isopod Serolis paradoxa (Fabricius, 1775) from the Falkland Islands and southern Patagonia (central Strait of Magellan and the Atlantic opening) applying seven nuclear microsatellites and a fragment of the mitochondrial 16S rRNA gene. Both marker systems report highest genetic diversity for the population from the central Strait of Magellan and lowest for the Falkland Islands. The estimated effective population sizes were large for all populations studied. Significant differentiation was observed among all three populations. The magnitude of differentiation between Patagonia and the Falkland Islands (16S: uncorrected p-distance 2.1%; microsatellites: standardized F'ST > 0.86) was an order of magnitude higher than between populations from within Patagonia. This indicates that there is currently no effective gene flow for nominal S. paradoxa between these two regions and it has been absent for time exceeding the last glacial maximum. We argue that specimens from the Strait of Magellan and the Falkland Islands very likely represent two distinct species that separated in the mid-Pleistocene (about 1 MY BP). CONCLUSION: The results of this study indicate limited gene flow between distant populations of the brooding isopod Serolis paradoxa. The patterns of genetic diversity suggest that the only recently inundated Strait of Magellan was colonized by different source populations, most likely from Atlantic and Pacific coastal waters. Our results demonstrate that more systematic testing of shared faunal inventory and realized genetic exchange between Patagonia and the Falkland Islands is needed before a consensus concerning the position of the Falkland Islands relative to the Magellan zoogeographic province can be reached.
RESUMEN
This study reports the successful isolation of highly informative microsatellite marker sets for two marine serolid isopod species. For Serolis paradoxa (Fabricius, 1775), 13, and for Septemserolis septemcarinata (Miers, 1875), eight polymorphic microsatellite markers were isolated using the reporter genome enrichment protocol. The number of alleles per locus (N(A) ) and the observed heterozygosity (H(O) ) encompass a wide range of variation within S. paradoxa (N(A) 3-31, H(O) 6-89%) and S. septemcarinata (N(A) 2-18, H(O) 9-94%). The suitability of the newly isolated markers for population genetic studies is evaluated.