Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50–179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4–15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.
The paternally expressed imprinted Retrotransposon-like 1 (Rtl1/Peg11) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127 and Rtl1 deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1 mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.
Epigenetic mechanisms modulate genome function by writing, reading and erasing chromatin structural features. These have an impact on gene expression, contributing to the establishment, maintenance and dynamic changes in cellular properties in normal and abnormal situations. Great effort has recently been undertaken to catalogue the genome-wide patterns of epigenetic marks—creating reference epigenomes—which will deepen our understanding of their contributions to genome regulation and function with the promise of revealing further insights into disease etiology. The foundation for these global studies is the smaller scale experimentally-derived observations and questions that have arisen through the study of epigenetic mechanisms in model systems. One such system is genomic imprinting, a process causing the mono-allelic expression of genes in a parental-origin specific manner controlled by a hierarchy of epigenetic events that have taught us much about the dynamic interplay between key regulators of epigenetic control. Here, we summarize some of the most noteworthy lessons that studies on imprinting have revealed about epigenetic control on a wider scale. Specifically, we will consider what these studies have revealed about: the variety of relationships between DNA methylation and transcriptional control; the regulation of important protein-DNA interactions by DNA methylation; the interplay between DNA methylation and histone modifications; and the regulation and functions of long non-coding RNAs.
Pw1/Peg3 is an imprinted gene expressed from the paternally inherited allele. Several imprinted genes, including Pw1/Peg3, have been shown to regulate overall body size and play a role in adult stem cells. Pw1/Peg3 is expressed in muscle stem cells (satellite cells) as well as a progenitor subset of muscle interstitial cells (PICs) in adult skeletal muscle. We therefore examined the impact of loss-of-function of Pw1/Peg3 during skeletal muscle growth and in muscle stem cell behavior. We found that constitutive loss of Pw1/Peg3 function leads to a reduced muscle mass and myofiber number. In newborn mice, the reduction in fiber number is increased in homozygous mutants as compared to the deletion of only the paternal Pw1/Peg3 allele, indicating that the maternal allele is developmentally functional. Constitutive and a satellite cell-specific deletion of Pw1/Peg3, revealed impaired muscle regeneration and a reduced capacity of satellite cells for self-renewal. RNA sequencing analyses revealed a deregulation of genes that control mitochondrial function. Consistent with these observations, Pw1/Peg3 mutant satellite cells displayed increased mitochondrial activity coupled with accelerated proliferation and differentiation. Our data show that Pw1/Peg3 regulates muscle fiber number determination during fetal development in a gene-dosage manner and regulates satellite cell metabolism in the adult.
Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5′-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.
The Virus-X—Viral Metagenomics for Innovation Value—project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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