Location: Genetics and Animal Breeding
Project Number: 3040-31000-104-022-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Sep 1, 2025
End Date: Aug 31, 2028
Objective:
The USDA Animal Genome Blueprint Target Areas include a focus on characterization of genome functional elements in high throughput assays across multiple individuals and breeds. The next major challenge for functional annotation of animal genomes (FAANG) is to link genomic variation in regulatory sites to differences in gene expression and phenotype. The focus of FAANG on deep characterization of a few animals implicitly assumes those animals are representative of the whole population. For many of the data types and tissues studied within FAANG, there is limited understanding of the variability within populations, subspecies, or breeds. We will address this limitation of the FAANG approach by applying CAGE-seq in many animals. Our project will (1) generate a genome-wide atlas of genomic variation in enhancer sites in the bovine subspecies, (2) quantify the effect of enhancer variation on gene expression within animals of differing levels of subspecies hybridization. Our unique study design allows us to test the test the hypothesis that individual sequence variation in enhancer sites alters enhancer usage, within and across populations (sub-species). Our research is significant because it will contribute to understanding the way in which gene regulation occurs at enhancer sites.
Approach:
Objective #1: Generate an atlas of enhancers from whole blood for the two bovine subspecies.
We hypothesize that there is significant variability in enhancer location between and within subspecies of cattle. We will test our hypothesis by using CAGE-seq to identify enhancer sites in crossbred and multibreed populations of beef cattle, capturing a significant proportion of variable enhancer sites expected to exist within the US national beef herd. Prior FAANG efforts have focused on deep sequencing a small number of animals across many tissues, our approach will broadly sample a large number of diverse cattle to capture broad regulatory variation. Whole blood will be targeted as this is the most accessible and therefore most routinely collected tissue type in both quantitative- and molecular-genetics studies, increasing the utility of our findings.
Objective #2: Quantify the effect of bovine enhancer variation on gene expression.
We have two hypotheses for this objective: 1) that enhancer nucleotide sequence variation significantly influences gene expression; 2) that variation in enhancer expression level significantly influences gene expression. To test the extent to which these associations exist, we will assess the impact of genomic variation within enhancers on gene expression and evaluate the effect of enhancer expression level variation on gene expression. Gene expression will be measured using transcription start site abundance from CAGE-sequencing in a diverse population of beef cattle, with enhancer nucleotide (i.e.; SNP) variation identified from Objective 1, and enhancer expression levels calculated from sequence tag abundance within identified enhancers. The effect of enhancer variation on gene expression will be validated in an unrelated population and by using CRISPR-Cas9 mediated gene editing to exchange enhancer elements in cultured bovine cells.