Involvement of aberrant chromosome architecture and locus-specific vulnerability to DNA methylation epimutations in bovine large offspring syndrome

Abstract

Large/abnormal offspring syndrome (LOS/AOS) and Beckwith-Wiedemann syndrome (BWS) are similar congenital overgrowth syndromes which occur naturally in ruminants and humans, respectively. The incidence of these syndromes increases when offspring are conceived with the use of assisted reproductive technologies (ART; i.e. in vitro oocyte maturation, in vitro fertilization, and embryo culture). Molecular defects reported in both syndromes include global gene misregulation, DNA methylome epimutations, and disruption of genomic imprinting (parental-allele-specific gene expression). Although we have reported that bovine LOS occurs spontaneously (SLOS) based on phenotypic similarities to ART-LOS, to date no study has been conducted to determine if SLOS has the same methylome epimutations as ART-LOS. One goal of my dissertation research is to characterize DNA methylation profiles in bovine SLOS and ART-LOS to determine whether there are conserved genomic loci with DNA methylation defects between these overgrowth conditions. In addition, while it is known that LOS is characterized by global alterations in DNA methylation, it is largely unknown how altered DNA methylation drives the development of LOS, as the methylation errors (i.e., differentially methylated regions; DMRs) observed in the syndrome only explain [less than] 4 percent of the gene misregulation in short range (the flanking 20,000 DNA bases from the DMR). Therefore, another goal of my dissertation research is to determine whether long-range regulatory mechanisms of gene expression, such as chromosome architecture, is altered in LOS as a result of aberrant DNA methylation. In this dissertation, Chapter 1 is the literature review and will introduce epigenetic regulation of gene expression including chromosome architecture and clinical features and molecular findings of LOS and BWS. Chapter 2 and 3 are the research chapters. In Chapter 2, I characterize allele-specific chromosome architecture of IGF2R imprinted domain in fibroblast cells derived from control bovine fetuses and identified disrupted chromosome architecture in LOS. I also observed genomic location-based clustering tendency of misregulated genes in LOS. This study has been published in the Journal iScience (https://doi.org/10.1016/j.isci.2022.104269) (Li et al., 2022). In Chapter 3, I determined that bovine SLOS has DNA methylation defects with some similarities and differences when compared to ART-LOS. I also identified vulnerable genomic loci for DNA methylation defects in LOS, which could serve as molecular markers for the diagnosis of the syndrome during early pregnancy. This study has been published in the journal Epigenetics (https://doi.org/10.1080/15592294.2022.2067938). Chapter 4 is the general discussion in which my research findings are incorporated into the general knowledge of the field and implications and directions of future studies are discussed. In Appendix 1, I briefly introduce our ongoing Hi-C (global chromosome architecture), methylome and transcriptome project in which samples from LOS and BWS will be analyzed together to further shed light into the etiology of these syndromes, knowledge that will equally help Agriculture and Biomedicine. I anticipate submitting this manuscript for peer review and publication in July of 2022, thus becoming the third primary literature manuscript from my dissertation research. Appendix 2 is a review paper in which I am main contributor author published in Veterinary Clinics of North America: Food Animal Practice in 2019 (PMID: 31103180, https://doi.org/10.1016/j.cvfa.2019.02.007). This review summarized clinical and molecular findings in LOS and for the first time reported the existence of SLOS. Lastly, Appendix 3 summarizes my contributions of five other publications in which I collaborated with groups in the Division, at Mizzou, and other Academic institutions in the USA during my tenure as a PhD student.