DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

<div><p>Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or <em>in vitro</em> fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.</p> </div

Proportion of DNA methylation.

<p>Proportion of methylation at the alpha satellite I sequence CpG site analyzed during embryonic, fetal and post-natal development from SCNT and control tissues. All control samples were generated using AI, except for the blastocyst stage embryos that were generated using IVF. Blastocysts were analyzed as pools of 10 embryos while all other samples were analyzed on an individual basis.</p

Genome-Wide DNA Methylation Patterns and Transcription Analysis in Sheep Muscle

<div><p>DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep <i>Longissimus dorsi</i> muscles. RRBS analysis of ∼1% of the genome from <i>Longissimus dorsi</i> muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep <i>Longissimus dorsi</i> muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.</p></div

Average DNA methylation in regions annotated as genes and intergenic regions.

<p>Histogram of average DNA methylation calculated for (a) annotated genes and (b) intergenic regions.</p

CpG content around transcription start sites in the sheep genome (OARv3.1).

<p>CpG content 6(A), and a histogram of gene counts with 0–14% CpG content in 1000 bp around the TSS (B).</p

Single nucleotide resolution analysis of DNA methylation.

<p>Histogram of average DNA methylation calculated for all single CpG sites with a minimum read depth coverage of 10.</p

Accuracy of RRBS.

<p>Comparison of DNA methylation levels at single nucleotide resolution obtained using RRBS and levels measured at the same sites using Sequenom analysis. Four neighbouring CpG sites are shown as an example of concordant results.</p

Sequenom PCR primer sequences used to determine DNA methylation on chromosome 18.

<p>Sequenom PCR primer sequences used to determine DNA methylation on chromosome 18.</p

DNA methylation around transcription start sites relative to CpG content and gene expression.

<p>Average DNA methylation of highly expressed vs. repressed genes in three sheep across 6; a) genes with high CpG content, b) low CpG content. Standard error of the difference was less than 1.3% for each data point and therefore too small to be represented in the figure.</p

Average methylation of the analyzed DNA sequences in rederived and original cell lines.

ab<p>values for the methylation within a specific region with these superscripts differ significantly; P<0.0005.</p>cd<p>values for the methylation within a specific region with these superscripts differ significantly; P<0.026.</p>ef<p>values for the methylation within a specific region with these superscripts differ significantly; P<0.009.</p>eg<p>the difference of the methylation values with these superscripts are just outside the significance level (P<0.052). The P-value is 0.007 with a t-test assuming equal variances.</p