Jani De Vos

The genome provides the entire set of DNA instructions of an organism, while the epigenome involves modifications that do not alter the DNA sequence. Gene expression is regulated by the intricate interplay between the genome and epigenome. We can explore the epigenome and regulatory elements through functional assays such as bisulfite sequencing, ChIP-seq, and ATAC-seq. Cell lines provide a valuable model for studying the genomic architecture and the regulatory genome of species. In vivo experiments are often complex and cell lines offer ethical alternatives.

Moreover, development is a dynamic and complex process which is regulated by gene expression. One epigenetic modification is DNA methylation, which plays an essential role in regulating development. The general dynamics of DNA methylation in the growing embryo and fetus, however, are still poorly understood. The aim of the research described in this thesis was two-fold, starting with understanding the (epi)genetic makeup of a pig and chicken cell line. The second aim was to disentangle the dynamic changes occurring within the epigenome, regulating gene expression during embryonic and fetal development in pig and chicken.

To do so, we first investigated the molecular characteristics of a pig intestine epithelial cell line, and chicken fibroblast cell line, using an integrative omics approach (Chapter 2). Functional assays such as ChIP-seq of histone modifications associated with regulatory elements, ATAC-seq indicative of open chromatin, reduced representation- and whole genome bisulfite sequencing (RRBS and WGBS) for DNA methylation, RNA-seq for transcriptome profiling and whole genome sequencing, were profiled both on an individual level as well as by integrating the various epigenetic modifications. This provided insights into the complex interactions between the genome and epigenetic modifications regulating the gene expression of these cell lines. Chromosomal abnormalities, copy-number variations, and aneuploidy, typical for a cell line, were identified for several chromosomes in both cell lines. Furthermore, higher gene expression for genes located on aneuploid chromosomes compared to diploid chromosomes was observed. Although these cell lines are described as nontumorigenic and untransformed, aneuploidy occurs not only due to the number of passages, but with each passage, the probability of its occurrence within a cell increases. This can lead to the emergence of cells with a growth advantage, ultimately causing them to become the predominant cell type in the culture over time.

It is important for future studies to take note of these cell lines’ features and it is recommended that researchers proceed cautiously when interpreting findings. Understanding the characteristics and (epi)genetic composition of these cell lines, help to improve our knowledge about their limitations and potential use as an in vivo research model.

A DNA methylation pipeline, specifically focussed on processing bisulfite sequencing data is described in Chapter 3, which was developed to include methylation calling, and visualisation of various methylation statistics. To gain further insights into methylation levels across various genomic characteristics, the processed data produced by this pipeline may be easily imported into visualization tools. The aim of this pipeline was to ensure reproducibility of the results generated within this thesis, specifically for the GENE-SWitCH project. This pipeline was implemented and used to analyse both the RRBS and WGBS data described in Chapters 4 and 5 to investigate the dynamics of the DNA methylome that regulate gene expression during development.

Investigating this role of the epigenome and the functional regulatory genome was facilitated by identification of the methylome dynamics in seven tissues during embryonic and fetal development for both pig (Chapter 4) and chicken (Chapter 5). In both species we identified differentially methylated sites and regions, together with categories of methylation, which were combined for insight into the developmental methylation dynamics (Chapter 4 and Chapter 5). By integrating transcriptomic data with methylation data, we gained further insight into the dynamic nature of the methylome during fetal development. We identified tissue specific changes, and in the pig at early development (30 dpf) germ layer specific methylation characteristics are evident in lung, muscle, kidney, skin, and small intestine, which then transition to tissue specific methylation from 70 dpf until newborn stages (Chapter 4). In the chicken we found enrichment of processes indicating development of brain and skin function from an early developmental stage (E8) (Chapter 5).

Remarkable differences in the methylome patterns between chicken and pig were observed, with the most notable differences found in the liver tissue during development. Liver is fully developed at an early stage of development in pig in comparison to chicken, which is due to the hematopoietic function of the liver in mammals during development in comparison to birds. This research provides fundamental insights into the methylome dynamics that regulate development and highlights key distinctions between avian and mammalian systems.