Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Department of Animal Science
Non Technical Summary
Understanding of genetic, biochemical, and physiological interrelationships influencing animal growth and development is essential for improving the efficiency of production for our livestock species. The relationship between the deposition of muscle and adipose tissue are at the core of production efficiency of animal protein. Molecular variation within the LCORL gene provides a unique opportunity to potentially define key events during animal development that appear to have continued effects throughout the growth phase of production, especially in muscle and adipose tissue. Characterizing the mechanism by which this genetic variation influences the function of downstream gene networks to modify animal growth will provide insight to both genetic and environmental factors that can be used to improve production efficiency in cattle. However, the significance of understanding this phenomenon extends beyond cattle as there is significant evidence that regulation of these genetic mechanisms are likely conserved among species. Thus, the proposed research will provide fundamental knowledge that has the potential to have a much broader impact on our understanding ofanimal biology as well as improve the production efficiency of livestock species.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
(N/A)
Developmental
20%
Goals / Objectives
Over-Arching Goal.The overall objective of this proposal is to understand how LCORLis able to exert its effects on animal growth and composition. Successful completion of this work will provide insight to both genetic and environmental factors that can be used to improve production efficiency in cattle and provide fundamental knowledge about animal growth that has the potential to increase the efficiency of animal protein production in several species. Furthermore, the fundamental knowledge gained may also provide insight to human health and development.Objective 1. Assess population level variation of LCORL expression in muscle tissue and its association with phenotypic characteristics and predicted molecular signatures.Experimentally, we propose to 1) measure the population variation of LCORL expression in muscle tissue within a large population of cattle, 2) confirm the relationship between LCORL QTN genotype and LCORL expression, 3) evaluate the relationship between growth and carcass phenotypes and the level of LCORL expression, 4) further define the influence of variation in LCORL expression on the muscle transcriptome, and 5) examine the influence of allelic variation at the LCORL locus on the regulation of individual genes and gene networks.Objective 2. Define changes in the epigenome, specifically H3K4 and H3K27 modification, generated by LCORL variation and their relationship to transcriptional changes.Experimentally, we propose to 1) measure changes in H3K4 and H3K27 epigenetic marks using ChIP-seq analysis, 2) determine the relationship between changes in histone modifications and LCORL genotype and level of expression, and 3) link epigenetic modifications with the corresponding changes in the muscle transcriptome. By defining these elements we can better understand the causal relationship between changes in LCORL expression, chromatin structure, and downstream regulation of gene networks.Objective 3. Demonstrate that induction of LCORL expression is causal for the generation of molecular and physical phenotypes we have associated with the genetic and environmental variation in LCORL expression.Experimentally, we propose to 1) develop and characterize a transgenic mouse model capable of inducible expression of LCORL both globally and in a tissue-specific manner, and 2) measure both the molecular and physical phenotypes associated with induction of LCORL expression.
Project Methods
Objective 1Progeny growth and feedlot performance data will be collected on 300 Shorthorn or Simmental-Angus sired animals. Bodyweight will be taken at birth and at weaning. Individual estimates of feed intake will be obtained using the GrowSafe System Ltdfeed monitoring system. Weights being taken on 28d intervals. Ultrasound will be used to determine 12th rib backfat and marblingat successive time points on 28d intervals. Yield and quality grade will be collected on all cattle at harvest, approximately 430 d of age. A total of 200 animals, with equal distribution between sexes, born within a three-week calving window will be selected for whole blood collection and muscle biopsy at ~300d of age. In addition to the calving period, health status will also be assessed prior to biopsy and only animals with positive health status will be considered for biopsy. Whole blood will be collected by jugular venipuncture and used for the isolation of genomic DNA. A biopsy sample of the longissimus dorsi muscle will be taken from each animal for gene expression analyses. Total RNA will be extracted from muscle samples. Quantitative RT-PCR of LCORL will be performed. The calculation of relative expression between individuals will be performed using the relative standard curve method. The relationship between LCORL expression and phenotypic traits will be analyzed. Genotyping, CNV-calling, and RNA-seq analyses will be performed on 48 animals from each tail of the distribution for LCORL expression (n=96 total). Selected animals will be genotyped using the Illumina Bovine HD Beadchip™ containing approximately 777,000 markers. RNA-seq libraries will be constructed. All libraries will be indexed, quantified by qPCR, and pooled. The pool will be sequencedfor a target of >40 million raw reads per sample. Initial quality control, read mapping, normalization, and removal of unknown batch effects will be performed. Samples will be fitted to a negative-binomial general linear model and tested for differential gene expression between the high and low LCORL expression groups. Further analyses to examine the relationship between the expression of individual genes and their networks will also be performed. A phenotype by marker/CNV GWAS will be conducted. Normalized read counts for each transcript meeting a minimal count threshold will be used as molecular phenotypes to conduct an eQTL analysis. All animals will be used in the eQTL analysis as this analysis aims to map genetic variation to changes in gene expression.Objective 2Chromatin immunoprecipitation will be performed using longissimus dorsi muscle tissue collected in Objective 1. A total of 48 animals will be used, 24 animals from both high and low LCORL expression groups. Selection will be based on both LCORL genotype and the relationship between gene expression patterns within each group. Samples with the highest pairwise gene expression correlations will be selected based on cluster analysis. Antibodies directed against H3K4me3 and H3K27me3 will be obtained. Guidelines recommended by ENCODE and FAANG will be followed. ChIP samples and controls will be sequencedto a target depth of >50 million reads for H3K27me3, >25 million for H3K4me3, and >20 million for input DNA controls. Following pruning for quality, reads will be aligned to the appropriate reference genome. Identification of regions containing high read densities, or peak calling, will be performed. Reads from non-immunoprecipitation control libraries are used to assess enrichment of sequences associated with the specific target during analysis. RNA-seq and ChIP-seq data will be analyzed to investigate the relationship between changes in epigenetic marks and gene expression by comparing gene lists generated from each analysis.Objective 3Transgenic mice will be produced with a knock-in of LCORL into the endogenous safe-harbor locus, ROSA26. The general design involves generating a construct that contains the murine LCORL cDNA; isoform 1 of murine LCORL (NM_001163073) will be used. For tissue-specific activation of LCORL designs include reporter/stop cassettes that are flanked by loxP sites. Mating of transgenic mice carrying this LCORL construct with Cre-recombinase expressing transgenic mouse strains removes this upstream cassette and allows expression of the inserted gene via the ROSA26 promoter and/or the exogenous promoter used to drive reporter expression. Therefore, mating mice with muscle-specific Cre-recombinase mice would allow muscle-specific expression. Heterozygous mice for each transgene can be mated to produce pups that have no transgene, either of the transgenes or both transgenes within a single litter. As only mice with both transgenes will respond to doxycycline administration with LCORL overexpression, all other litter mates can be used as controls.Mice will be housed in breeding pairs or groups, and pups will be weaned at 3 weeks of age into individual or group cages. Pups will be genotyped by collecting ear notches or tail clips at weaning. Once validation is complete, heterozygous mice for each transgene will be mated to produce pups; 25% of these pups will carry both transgenes and respond to doxycycline administration with LCORL overexpression. Bodyweight is expected to increase due to LCORL overexpression. To detect a 10% increase in body weight (α=0.05, β=0.80) at 8 weeks of age, 13 mice are needed. Two periods of LCORL induction are planned. Ten litters of mice will be exposed to doxycycline in utero. Pregnant females will be given doxycycline for the final 14d of pregnancy. After giving birth, all dams will be given water without doxycycline, and progeny will be weighed at 5 days of age and weaned at 3 weeks of age into individual cages. Ten litters will be used resulting in 20 mice expected to carry both transgenes. Mice from litters unexposed to doxycycline in utero will be weaned into individual cages and genotyped. A total of 30 mice (15 with both transgenes and 15 without) will be administered doxycycline from 3 to 8 weeks of age. After weaning, all mice will be housed individually and sacrificed at 8 weeks of age. Bodyweight and feed intake of mice will be measured weekly from 3-8 weeks of age. Additionally, body composition will be assessed at 4, 6, and 8 weeks of age using EchoMRI. After sacrifice, weights of liver, heart, muscles (tibialis anterior, biceps femoris, gastrocnemius/soleus, longissimus dorsi, triceps brachii ), and fat pads (inguinal, retroperitoneal, gonadal) will be recorded.Data will be analyzed as two separate experiments based on induction timing. The mouse will serve as the experimental unit and litter serve as a block. This study is designed as a 2x2 factorial of LCORL induction and sex; the interaction of sex and LCORL induction will be considered but sample sizes between sexes may be unequal. The expression of LCORL will be measured in muscle, adipose tissue, heart, and liver of all mice. RNA-seq and ChIP-seq protocols will be conducted as described in Objectives 1 & 2. Provided the expected results are obtained for LCORL overexpression, we will select four mice having both transgenes and four mice having neither transgene from the postnatal induction period for RNA-seq analysis (8 mice total). To determine the effects of in utero induction of LCORL overexpression, 3 additional pregnant mice will be administered doxycycline, and progeny will be sacrificed at birth. Hindlimb and forelimb muscles from individual mice will be combined for RNA isolation and immunoprecipitation in order to generate sufficient RNA and DNA for sequencing. Four mice having both transgenes and four mice having neither transgene will be selected for RNA-seq analysis (8 mice total).