FROM THE GENOME TO THE EPIGENOME: UNRAVELING OF PARENT-SPECIFIC REGULATORY ELEMENTS AND IDENTIFICATION OF ENRICHED FERTILITY-RELATED QUANTITATIVE TRAIT LOCI IN THE DEVELOPING BOVINE PLACENTA

Morgan Wagle

In both the beef and dairy cattle industries, pregnancy loss is a major concern, contributing to several billion dollars of loss per year in the United States. Proper placental development and function is integral to pregnancy maintenance. Important drivers of placental development are trophoblast cells, enriched in cotyledons in the placenta. The cotyledon is the portion of the placenta that forms the placentome together with maternal caruncle, where nutrient and gas exchange occur during pregnancy. The objective of the first study was to investigate the enrichment of fertility-specific quantitative trait loci (QTL) within gene regulatory regions of two types of trophoblast cells, uninucleate (UNC) and binucleate (BNC) cells. This was investigated using single-nuclei assay for transposase-accessible chromatin sequencing (snATAC-seq) in the developing (d40) and mature (d170) bovine placenta, obtained from a previous study. Fertility QTL, acquired from AnimalQTLdb, that resided within open chromatin regions were identified, then transcription factor binding motifs within QTL regions were determined using Hypergeometric Optimization of Motif EnRichment (HOMER). Transcription factor expression was validated with single-nuclei RNA sequencing (snRNA-seq) data in UNC and BNC cells at both time points. Several QTL, including but not limited to conception rate, calving interval, and heifer pregnancy, were identified. Additionally, several transcription factor binding sites were enriched in these QTL locations, including GATA3, TFAP2A, TFAP2C, JUN, JUND, and TEAD4, and were previously identified as part of a regulatory network critical for UNC-to-BNC cell differentiation. The objective of the second study was to determine the parent-of-origin specific DNA methylation patterns in bovine placenta at day 50 of gestation. Cotyledon DNA was extracted and sequenced to 30X coverage using PacBio Hifi sequencing. Parent DNA was sequenced to 30X coverage using an Illumina NovaSeq 6000 platform. Variants were called using GATK HaplotypeCaller and DeepVariant, and pedigree phasing and haplotagging was completed for each trio using WhatsHap, to identify sire and dam haplotypes. Pb-CpG-tools was used to call methylation, then differential methylation was determined with MethBat. Annotation of differentially methylated regions (DMRs) to the reference genome was completed using HOMER. A total of 333 significant (minimum Z-score ± 5) DMRs were discovered, within or in cis-regulatory regions relative to 252 candidate genes. In total, 224 hypomethylated sire DMRs and 109 hypomethylated dam DMRs were identified. Pregnancy related genes hypomethylated in sire haplotypes included PEG10, PEG3, KBTBD6, MEST, NAP1L5, PLAGL1, and VEGFA, while hypomethylated dam DMRs corresponded to TWIST2, PGF, and PAPPA. Understanding both the regulatory elements within UNC and BNC trophoblast populations, and the parent-specific mechanisms of placental development in the cotyledon will enhance understanding of biological mechanisms driving placental development. Dysregulation of placental development could be impacted by genetic variation in transcription factor binding sites near critical fertility genes, in addition to sire and dam specific DNA methylation patterns. These studies will improve our understanding of genetic and epigenetic mechanisms important for cattle cotyledon development and pregnancy maintenance. This knowledge is an important step in improving selection strategies and reproductive in the beef and dairy industries.

FROM THE GENOME TO THE EPIGENOME: UNRAVELING OF PARENT-SPECIFIC REGULATORY ELEMENTS AND IDENTIFICATION OF ENRICHED FERTILITY-RELATED QUANTITATIVE TRAIT LOCI IN THE DEVELOPING BOVINE PLACENTA

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