Altered DNA methylation associated with an abnormal liver phenotype in a cattle model with a high incidence of perinatal pathologies

Abstract

Cloning enables the generation of both clinically normal and pathological individuals from the same donor cells, and may therefore be a DNA sequence-independent driver of phenotypic variability. We took advantage of cattle clones with identical genotypes but different developmental abilities to investigate the role of epigenetic factors in perinatal mortality, a complex trait with increasing prevalence in dairy cattle. We studied livers from pathological clones dying during the perinatal period, clinically normal adult clones with the same genotypes as perinatal clones and conventional age-matched controls. The livers from deceased perinatal clones displayed histological lesions, modifications to quantitative histomorphometric and metabolic parameters such as glycogen storage and fatty acid composition, and an absence of birth-induced maturation. In a genome-wide epigenetic analysis, we identified DNA methylation patterns underlying these phenotypic alterations and targeting genes relevant to liver metabolism, including the type 2 diabetes gene TCF7L2. The adult clones were devoid of major phenotypic and epigenetic abnormalities in the liver, ruling out the effects of genotype on the phenotype observed. These results thus provide the first demonstration of a genome-wide association between DNA methylation and perinatal mortality in cattle, and highlight epigenetics as a driving force for phenotypic variability in farmed animals.

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features

“…Because high-throughput analyses were necessary to identify regions that were hypomethylated in bovine sperm, we decided to assess two cost-effective approaches which are widely used to study DNA methylation: MeDIP-chip [ 44 ] and RRBS [ 37 ]. MeDIP-chip enables precise targeting of specific regions in the genome through custom design of the microarray (in our case, 3360 bp spanning the promoter and upstream region of each of the 21,296 bovine genes [ 35 ]), while RRBS offers a base-resolution analysis of CpG-rich regions through the combined use of enzymatic digestion (MspI) and the size selection of restriction fragments. Because no data were available regarding the optimal size window for RRBS in cattle, we conducted an in silico prediction of the genome coverage by RRBS and compared the results with those of MeDIP-chip (Additional file 1 : Supplementary methods, Tables S2-S3).…”

“…The microarray targeted the promoter region (− 2000 to + 1360 bp relative to the gene start) of 21,296 bovine genes, according to an annotation file downloaded from the Johns Hopkins University Center for Computational Biology FTP website ( ftp://ftp.ccb.jhu.edu/pub/data/assembly/Bos_taurus/Bos_taurus_UMD_3.0/annotation/ ; accessed Aug. 2010). The microarray design and hybridization protocol, as well as more details on the data analysis, can be found in [ 35 ]. The identification of regions of interest containing clusters of probes enriched in at least one tissue, the identification of differentially methylated regions (DMRs) among these regions of interest, as well as the calculation of mean percentages of enriched probes (Pr) in each tissue for each region r (DMR or region of interest), are detailed in the Additional file 1 : Supplementary methods.…”

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development

“…Twenty microliters of the PCR reaction were used as a template for pyrosequencing with 0.3 μM pyrosequencing primer, using the Pyromark Q24 device and Pyromark Gold Q96 reagents (Qiagen, Hilden, Germany). Each analyzed region was assayed in duplicate and the methylation value for each CpG was also obtained in duplicate and inconsistent duplicates (more than 5% difference) were repeated (Kiefer et al, ). The methylation percentage per CpG was then obtained by calculating the mean of all replicates that passed the control quality of the Pyromark Q24 software.…”

Epigenetic Reprogramming During Somatic Cell Nuclear Transfer: Recent Progress and Future Directions

“…Tissue-specific and pluripotency-related genes in cloned embryos show low and high DNA methylation levels, respectively (Figure 2, DNA methylation) (Ng and Gurdon, 2005;Kremenskoy et al, 2006;Yamazaki et al, 2006;Huan et al, 2014Huan et al, , 2015a. Following zygotic genome activation (ZGA), the erroneous reconstitution of DNA methylation pattern caused by aberrant expression of genes related to DNA methylation reprogramming and, consequently, of key genes required for the normal development of cloned embryos results in low cloning efficiency and abnormalities and death in cloned animals (Bourc'his et al, 2001;Bortvin et al, 2003;Chung et al, 2003;Kiefer et al, 2016;Gao et al, 2018). Thus, a DNA methylation pattern similar to that in normal fertilized embryos is necessary for the successful development of SCNT embryos.…”