GENETIC REGULATION OF MUSCLE GROWTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0229916
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Animal Sciences

Non Technical Summary
The growth and maintenance of skeletal muscle is fundamentally important for production of meat in domestic animals. Callipyge sheep are a naturally occurring model that shows that carcass composition can be improved with better feed efficiency and without changing net live weights. Research on the callipyge model will improve our understanding of the control postnatal muscle growth lead to methods to improve the efficiency of meat animal production.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3620	1050	10%
304	3620	1080	50%
305	3620	1030	20%
305	3620	1040	20%

Knowledge Area
305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3620 - Meat, sheep;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1080 - Genetics; 1050 - Developmental biology;

Keywords

sheep

muscle

hypertrophy

callipyge

dlk1

rtl1

Goals / Objectives
The callipyge trait in sheep provides a unique model to understand mechanisms affecting skeletal muscle growth and meat quality in domestic animals. Callipyge lambs have a 30-40% increase in muscle mass and 6-7% decrease in carcass fat without a net effect on animal growth. The trait has a novel mode of inheritance called polar overdominance because only heterozygous (+/C) animals that inherit a normal allele from the dam and the callipyge allele from the sire have the muscle hypertrophy phenotype. The callipyge mutation is a regulatory mutation in the DLK1-DIO3 imprinted gene cluster that increases the expression of Delta-like-1 homologue (DLK1) and Retrotransposon Like-1 (RTL1) in the muscles that undergo hypertrophy. The objectives of this project renewal will address two major unknowns in the callipyge trait: 1) the genetic mechanism of polar overdominant inheritance and 2) the physiological mechanism that underlie the induced muscle hypertrophy. Objective 1. Determine the effect of the callipyge mutation on epigenetic modifications and regulation of transcripts within the DLK1-DIO3 locus. Objective 2. Determine the effect of differentially expressed genes on regulatory networks that control myosin isoform expression in skeletal muscle. These objectives are expected to provide new knowledge of the coordinated regulation of a chromosomal region that has profound effects on growth and identify regulatory networks that are rate limiting for muscle growth.

Project Methods
We have made a library of restriction fragments from the two BACs in the luciferase reporter vector pGL3P that spans the DLK1 to MEG8 region of the DLK1-DIO3 domain. We will initially focus on the sequences surrounding the callipyge mutation and identification of the regulatory elements that control DLK1, MEG3 and RTL1 genes. A series of overlapping clones will be assayed for enhancer or repressor activity in both C2C12 myoblasts and NIH3T3L1 adipocytes using the Renilla luciferase reporter as a co-transfection control. The activities of the individual clones will be determined by comparison to the parental vector (pGL3P) that has minimal promoter activity using Dunnets t-test which corrects for multiple testing to a common control. In order to identify genes that are components of the fundamental mechanism of callipyge induced hypertrophy, we will extend our qPCR validation by examining 7 muscles, 4 that undergo hypertrophy and three that do not hypertrophy. We predict that primary transcriptional responders to DLK1 and/or RTL1 should be differentially expressed in all 4 hypertrophied muscles but not differentially expressed in the 3 non-hypertrophied muscles. These transcripts would appear to be co-regulated with DLK1and RTL1 expression and are considered candidate genes for components of the regulatory networks controlling muscle hypertrophy. This approach will allow generation of a shorter list of candidate genes that have a more direct role in callipyge induced hypertrophy. Gene ontology analysis and Gene Expression Omnibus data will then be used to select genes for functional analysis of their role in myogenesis and myosin isoform expression. Muscle cell culture models are available that will enable measuring myosin gene expression under defined conditions. This will include C2C12 cells or mouse primary satellite cell cultures in collaboration with Dr. Shihuan Kuang. When myoblast cells fuse into myotubes, they begin to express a number of myosin isoforms. We will utilize an effector/luciferase reporter co-transfection assay in C2C12 or satellite cells. We have two luciferase reporter plasmids that Myh4 promoter or Myh7 promoter that can be co-transfected with a cloned cDNA for a selected gene as the effector and a Renilla luciferase as a transfection control. The cells will be induced to differentiate into myotubes for 72 hours and normalized luciferase activity relative to an inert effector plasmid was analyzed by Dunnets t-test for multiple comparisons. Successful candidate genes can be confirmed for effects on the endogenous myosin loci using more refined models including IGF1 treatment as a hypertrophy model or RNAi knockdown models assayed by qPCR or antibody detection of myosin protein. Based on predicted function and cellular localization, more specific assays such as DNA interaction, (ChIP) or protein interaction (immunoprecipitation) will be used to determine the molecular mechanism of the target gene.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:Sheep breaders / geneticists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD has trainedgraduate students, post-doc's and visiting scientists to use microarrays, RNA sequencing and quantitative PCRfor their sheep, pigand mouse projects.The PD has developed skills in functional genomics and RNA sequencing analysis that has been integrated into his graduate level genetic course (ANSC514 Animal Biotechnology).? How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The PD has investigated callipyge sheep because the trait shows that much higher muscle growth rates are biologically possible without increasing feed inputs or using drugs or hormones. In 2015, an investigation by Gao and colleagues solved the genetic mechanism of polar overdominance using a series of gene knockout experiments in mice. This work is also important as it confirms in an animal model the DLK1 gene is the primary driver of callipyge muscle hypertrophy. The functional genomics analysis conducted for this project have identified genes that are part of the physiological pathways that are induced by DLK1 to cause higher levels of muscle growth. The genes that are regulated by DLK1 may be acting to limit muscle growth in normal animals. This project also showed that increased expression of PARK7 in resulted in higher muscle protein (myosin) expression and made muscle cells more responsive to the growth factor IGF-1. This indicated that elevated PARK7 expression makes callipyge lamb muscle more responsive the natural growth signals present in young rapidly growing animals.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Penick, M., H-W. Kim, D. Setyabrata, J.N. Waddell, C.A. Bidwell, Y. H. Kim. 2017. Callipyge genotypic effects on meat quality attributes and oxidation stability of ovine M. longissimus. Small Ruminant Research 146, 5-12.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kelly, A.C., C.A. Bidwell, F. McCarthy, D.J. Taska, M.J. Anderson, L.E. Comacho and S.W. Limesand. 2017. RNA sequencing exposes novel adaptive and immune responses to intrauterine growth restriction in fetal sheep islets. Endocrinology 158(4):743-755

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Research data was presented at a conference for scientists, researchers, graduate student and life sciences industry participants and federal granting agency officials. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?An abstract and presentationon the callipyge goat cloning progress was presented at theLarge Animal Genetic Engineering Summit, in Bethesda, MD on September 16-18, 2016. What do you plan to do during the next reporting period to accomplish the goals?The cloned goats will be used as sires and will produce F1 progeny that inherit a paternal copy of the callipyge allele that induces muscle hypertrophy in sheep. The F1 progeny will determine if the muscle hypertrophy trait will also occur in goats. Testing polar overdominance inheritance and maternal effects of the mutation will require generating females that carry the new callipyge allele.

Impacts
What was accomplished under these goals? It is generally thought that quantitative variation in animals is due to single base pair variation in regulatory regions of genes. The callipyge trait of muscle hypertrophy in sheep is due to a regulatory mutation in the DLK1-DIO3 imprinted gene cluster. This mutation changes the expression of several genes over a 300 Kb region. We wanted to determine if the regulatory effects of the mutation and the muscle growth would be preserved when introduced into a different species such as goats. Collaborators D. Carlson and S. Fahrenkrug, (Recombinetics Inc) used TALENS (Transcription Activator-like Effector Nucleases) gene editing technology to introduce the specific A to G substitution in a male Spanish goat cell line provided by I. Polejaeva , Utah State University. One cell line that was homozygous for the goat callipyge allele (C/C) produced three live male goats by somatic cell nuclear transfer at Utah State University in Spring 2016. The goats are healthy and have a normal Spanish goat phenotype and muscling. Biopsies of the longissimus dorsi were used to assess gene expression in the DLK1-DIO3 imprinted cluster from 3 CC clones and 6 age-matched control using RT-qPCR. Gene expression was detected for five loci from the DLK1-DIO3 region including DLK1, three maternal expressed long ncRNA and the CLPG1 transcript that spans the mutation site. All transcripts were detected in all samples but the transcript abundance for the five genes were highly variable between the three clones. Elevated levels of expression of DLK1, MEG3 and CLPG1 were all detected in individual clones. As a group, there were no significant differences between the CC goats and the controls. The high variability in gene expression among the clones is likely due to incomplete reprogramming and epigenetic modification associated with somatic cell nuclear transfer.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bi, P., F. Yue, A. Karki, S.E. Wirbisky, C. Wang, A. Durkes, O.M. Andrisani, C.A. Bidwel, J.L. Freeman, S.F. Konieczny, S. Kuang. 2016. Notch activation drives adipocyte dedifferentiation and tumorigenic transformation. Journal of Experimental Medicine 213(10) 2019-2037.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Kelly, A., C. A. Bidwell, X. Chen, M. Anderson and S.W. Limesand. 2016. Identifying the adrenergic component of intrauterine growth restriction in fetal pancreatic ?-cells. Society for Reproductive Investigation 63rd Annual Scientific Meeting, Montreal, Canada March 2016
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Kelly, A., C.A. Bidwell, L. Camacho, F. McCarthy and S.W. Limesand. 2016. Transcriptome expression profiles identify increased metabolic capacity in adipose tissue from fetal sheep with intrauterine growth restriction. Society for Reproductive Investigation 63rd Annual Scientific Meeting, Montreal, Canada March 2016
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Yang, M., D. Webster, Q. Meng, Z. Fan, D. Carlson, M. Regouski1, T. Hadfield, C. Bidwell, S. Fahrenkrug, N. Cockett and I. Polejaeva. 2016 Introduction of callipyge mutation into the goat genome using transcription activator-like effector nucleases (TALENs). Large Animal Genetic Engineering Summit, Bethesda, MD. September, 2016

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience is other researchers, post-doctoral fellows, graduate and undergraduate students investigating muscle biology or meat science Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD has been developing skills in bioinformatics and RNA-sequencing analysis that has been integrated into his graduate level genetic course (ANSC514 Animal Biotechnology) as well as training graduate students and post-doc's to do RNA sequencing analysis for their sheep, cattle and mouse projects. How have the results been disseminated to communities of interest?This work was presented to the NRSP8 Cattle/Sheep/Goat Workshop at the Plant and Animal Genome Conference in San Diego CA in January 2015. What do you plan to do during the next reporting period to accomplish the goals?The PD will pursue funding to understand the maternal non-coding RNA structure of the DLK1-DIO3 locus. Short read sequencing technology can identify exon-intron splice junctions but determine actual associations of exons will require either long-read RNA sequencing or cDNA cloning and sequencing to determine which exons are actually included or associated in the various spliced long-non-coding RNA isoforms. A further need is characterization of messenger RNA and small RNA in the same muscle samples.

Impacts
What was accomplished under these goals? The current research focus has been on objective 1 and determining the effect of the callipyge mutation on the expression of the maternal allele-specific expression of several long non-coding RNA. The PD has been using the Tuxedo suite of programs with existing RNA sequencing data to develop a more accurate model of the gene structure for the maternal long non-coding RNA. These genes are highly up-regulated due to the callipyge mutation but the annotation of these genes is very sparse. The identification of splice junctions using splice read mapping program,Tophat2, shows a highly variable region of transcript splicing in MEG8 (Maternal Expressed Gene 8) that is a host gene for both small nucleolar RNA and microRNA. An accurate gene model will be a prerequisite for quantifying the effects of the mutation on gene expression.

Publications

• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: Cramer, T., Y.H.B. Kim, M. Penick,, J.N. Waddell, C.A. Bidwell. 2015. Small heat shock protein 27 may be related to toughness in loins of callipyge lamb. 68th Reciprocal Meat Conference, June 2015 Lincoln, NE.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: Penick, M., Y.H.B. Kim, T. Cramer, J.N. Waddell, C.A. Bidwell, B. Cooper. 2015. Metabolomics approach to elucidate meat quality traits of loins from callipyge sheep. 68th Reciprocal Meat Conference, June 2015 Lincoln, NE.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Bidwell, C.A., H. Yu, J.N. Waddell, R.L. Tellam, and N.E. Cockett 2015 Muscle Hypertrophy and Polar Overdominance in Callipyge Sheep. W138 NRSP8 Cattle Sheep Goat Workshop, Plant and Animal Genome XXIII, San Diego CA January 2015.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audience is other researchers, post-doctoral fellows, graduate and undergraduate students interested in animal science, animal growth or meat science. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student completed her Ph.D and one undergraduate student completed her honors thesis. How have the results been disseminated to communities of interest? This work was presented to the NRSP8 Cattle/Sheep/Goat committee and the Epigenetics Workshop at the Plant and Animal Genome Conference in San Diego CA in January 2014 What do you plan to do during the next reporting period to accomplish the goals? We are continuing to analyze the RNA-seq data (Bidwell et al 2014) to identify key transcripts and their splice variants that are regulated by DLK1-RTL1 and can influence muscle growth.

Impacts
What was accomplished under these goals? Our current work is attempting to determine which genes have a physiological role between the two endpoints of DLK1/RTL1 induction and the increased myosin expression. We have identified PARK7 as a candidate gene that has a transcriptional response to DLK1. PARK7 expression is up-regulated in hypertrophied muscles of callipyge sheep at both mRNA and protein levels. The PARK7 protein is a component of growth factor signaling such as IGF-I. We have developed a test to determine if the changes PARK7 gene expression can alter myosin gene expression in response to growth factor stimulation (IGF-I) using mouse primary myoblast cultures from normal (+/+) and Park7 knock-out (-/-) mice. Park7 (+/+) myotubes have a significantly higher myosin expression than Park7 (-/-) myotubes at both protein and mRNA levels. Furthermore, the phosphorylation state of AKT, a critical component of IGF-I signal transduction was tested using the myotube cell culture assay. IGF-I stimulation resulted in higher levels of AKT phosphorylation in Park7 (+/+) myotubes than Park7 knockout (-/-) myotubes. The ability of PARK7 to have a direct role in IGF-I signal transduction and cause an increase in myosin expression in a myotube cell culture model suggests that PARK7 is part of the physiological pathway for callipyge induced muscle hypertrophy. Furthermore, methods to increase PARK7 gene expression in skeletal muscle should enable increased growth response to normal growth factor signaling in livestock species.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bidwell C.A., Waddell J.N., Taxis T.M., Yu H., Tellam R.L., Neary M.K. & Cockett N.E. (2014) New insights into polar overdominance in callipyge sheep. Anim Genet 45 Suppl 1, 51-61.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yu H., Waddell J.N., Kuang S. & Bidwell C.A. (2014) Park7 expression influences myotube size and myosin expression in muscle. PLoS ONE 9, e92030.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Lutz, Kimberly (2014) "Determining the Role of DNTTIP1: Piecing Together the Callipyge Sheep Muscle Hypertrophy Pathway," The Journal of Purdue Undergraduate Research: Vol. 4, Article 4 DOI: http://dx.doi.org/10.5703/jpur.04.1.03.
• Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: Yu, H. 2013. Genes with physiological roles in callipyge muscle hypertrophy. Purdue University

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience is undergraduate students, graduate students in muscle biology and Animal Sciences as well as researchers in genetics and genomics and muscle biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided training for one graduate student and one undergraduate student. How have the results been disseminated to communities of interest? This work was presented to the NRSP8 Cattle/Sheep/Goat committee and the Epigenetics Workshop at the XXI Plant and Animal Genome Conference in San Diego CA in January 2013. What do you plan to do during the next reporting period to accomplish the goals? The analysis of gene expression using RNA-sequencing will continue. The ability of specific effector genes identified by functional genomics methods will be tested for their ability to regulate myosin gene expression in order to define the components regulatory networks.

Impacts
What was accomplished under these goals? We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA-sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the down-regulated genes are putative targets of the maternal allele-specific microRNA with gene ontology indicating regulatory and cell signaling functions. These results suggest that the trans effect of the maternal non-coding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 1) Bahls, M., C. A. Bidwell, J. Hu, A. Tellez, G.L. Kaluza, J.F. Granada, C.G. Krueger, J.D. Reed, M.H. Laughlin, W.G. Van Alstine and S.C. Newcomer 2013. Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine. BMC Genomics 14:443
• Type: Journal Articles Status: Published Year Published: 2013 Citation: 2) Bahls, M., R.D. Sheldon, P. Taheripour, K.A. Clifford, K. B Foust, E.D. Breslin, J.N. Marchant-Forde, R.A. Cabot, M. H. Laughlin, C.A. Bidwell and S.C. Newcomer 2013. Mothers exercise during pregnancy programs vasomotor function in adult offspring Experimental Physiology DOI expphysiol.2013.075978.
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: 3) Bidwell, C.A., J.N. Waddell, T.M. Taxis, H. Yu, R.L. Tellam, M.K. Neary and N.E. Cockett. New insight into polar overdominance in callipyge sheep. Animal Genetics (In Press)