Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
GENOME WIDE IDENTIFICATION AND ANNOTATION OF FUNCTIONAL REGULATORY REGIONS IN LIVESTOCK SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1005339
Grant No.
2015-67015-22940
Project No.
CA-D-ASC-2283-CG
Proposal No.
2014-05920
Multistate No.
(N/A)
Program Code
A1201
Project Start Date
Jan 1, 2015
Project End Date
Dec 31, 2017
Grant Year
2015
Project Director
Zhou, H.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Animal Science
Non Technical Summary
Genomics is playing an increasingly significant role in improving animal production, health, and well-being. The utility of a genome depends critically on how well it is annotated. The goal of this project is to improve the annotation of regulatory regions for three of the most important farm animal genomes: chicken, cow, and pig. These genomes have been assembled, but there is very limited information on the functional roles of regulatory regions of their genes. Recent work by the human and mouse ENCODE projects provide a blueprint for identifying the functional roles of regulatory elements in the human and mouse genomes, that can be implemented for similar efforts on animal genomes. Our specific goals are to 1) identify regulatory elements of chicken, cow and pig genomes, 2) determine functional roles of regulatory regions, and 3) freely distribute all data using the UCSC and Ensembl genome browsers. The proposed research addresses the program area priority: Application of genome-wide methods for identification of gene regulatory regions. This study will set a cornerstone for initiating animal ENCODE project by providing a valuable resource for exploring the funtional landscapes of the chicken, bovine, and swine genomes, and provide a valuable tool for a deeper and more meaningful understanding of complex biological systems.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
(N/A)
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043210108034%
3043310108033%
3043510108033%
Goals / Objectives
The overall goal of this project is to generate a comprehensive resource of functional regulatory elements for the chicken, cattle, and pig genomes. Our specific goals are:Annotate chromatin states corresponding to DNase I hypersensitivity, four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H3K4me1), and one insulator element (insulator-binding protein CCCTC-binding factor).Identify and annotate promoters, enhancers, and silencers by integrating information from RNA-seq, DNase I hypersensitivity, and ChIP-seq for four histone modifications and one insulator element.Freely distribute all raw and annotated data via UCSC Genome Browser and Ensembl.
Project Methods
DNase-seq, RNA-seq, and ChIP-seq from 8 tissues will be generated. For DNase-seq, nuclei from different tissues will be isolated and libraries will be prepared and sequenced by Illumina HiSeq 2000. For RNA-seq, total RNA will be isolated and libraries will be prepared and sequenced by Illumina HiSeq 2000. For ChIP-seq, chromatin will be isolated from each tissue and solated chromatin will be fragmented by sonication to an average size of 200-300 bp using a Bioruptor apparatus. Sonicated chromatin will be then used for immunoprecipitation. The antibodies will be used for each histonemodification marks. Chromatin precipitation will be made using protein A/G coated magnetic beads. Individual antibodies will be first bound to the beads. Then, beads with bound antibodies and targets will be purified using a magnet technology. DNA will be precipitated from each of the antibodies and from the input control sample and used for preparation of sequencing libraries indexed with TruSeq adaptors. Libraries will be sequenced using HiSeq2000 with up to 8 samples per lane in a 100 bp single read run. For RNA-seq, total RNA will be isolated and will be used for libraries preparation and sequenced using HiSeq2000.The promoter, enhancer and insulator elements based on mapped features will be identified and the hidden Markov models will be used to determine functional roles of regulatory regions. Annotated genomes will be distributed via UCSC browser and Ensembl browser.

Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Animal genome scientific community, livestock industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students, undergraduate students, and postdoctoral scholar have training in genomics assays and bioinformatics analysis. How have the results been disseminated to communities of interest?Oral and poster presentations were given in scientific meetings, and institutes in the US , New Zealand and China. Participated in workshop with diverse participants including scientists from institutes, funding agencies, NGO. What do you plan to do during the next reporting period to accomplish the goals?Continue to work other three ChIP-seq assays in all three species. For fat and muscle, additional optimization is needed for ChIP-seq. ATAC-seq assay will be further optimized. Integrative analysis of DNase-seq, ChIP-seq and RNA-seq will be conducted.

Impacts
What was accomplished under these goals? In order to identify regulatory elements in the genomes by integrating RNA-seq, DNase-seq and ChIP-seq data from each tissue. We have generated and analyzed data from these three important species, including analysis of forty-eight RNA-seq libraries (sixteen per species) collected from two biological replicates across eight tissues: adipose, cerebellum, cortex, hypothalamus, liver, lung, muscle and spleen. For chicken, an analysis of 15 DNase-seq data from all issues with two replicates, except hypothalamus (one replicate) show that identified tissue-specific DNase hypersensitivity (DHS) sites are associated with genes that relate to unique biological functions of the organs or tissues. Using the two replicates to construct a set of DHS sites that are present in each replicate, we found 29190 sites in cerebellum, 43672 in cortex, 52337 in liver, 64149 in lung, 27433 in muscle, and 63605 in spleen. In addition, 24 ChIP-seq data from all tissues (two replicates) except adipose and muscle in chickens and 12 ChIP-seq from liver, spleen and lung in pigs (H3K4me3 and H3K27me3 histone modification marks) were generated. Integrative analysis of DHS sites, ChIP-seq, and RNA-seq allows the identification of genome-wide active and inactive promoter regions in chickens, which enables an in-depth comparison of the regulatory landscapes of multiple tissues within these species. ATAC-seq in chicken tissues was optimized and some promising results have been achieved.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Tuggle, C., E. Giuffra, S. N. White, L. Clarke, H. Zhou, P. J. Ross, H. Acloque, J. M. Reecy, A. Archibald, R. R. Bellone, M. Boichard, A. Chamberlain, H. Cheng, R. P.M.A. Crooijmans, M. E. Delany, C. J. Finno, M. A. M. Groenen, B. Hayes, J. K. Lunney, J. L. Petersen, G. S. Plastow, C. J. Schmidt, J. Song, M. Watson. 2016. GO-FAANG meeting: a Gathering On Functional Annotation of ANimal Genomes Animal Genetics DOI: 10.1111/age.12466.
  • Type: Other Status: Published Year Published: 2016 Citation: Zhou, H. P. J. Ross, Kern, C. P. Saelao, Y. Wang, J. L. Chitwood, I. Korf, M. Delany, H. Cheng,. 2016. Genome-wide Functional Annotation of Regulatory Elements in chickens. Pp:48-52. The Proceedings of XXV World's Poultry Congress, Beijing, China.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kern, C. P. Saelao, Y. Wang, M. Halstead, J. L. Chitwood, T. Kim, P. J. Ross, I. Korf, M. Delany, H. Cheng, Zhou, H. 2016. Identification of tissue-specific promoters in chickens. 35th Conference for the International Society of Animal Genetics. Salt Lake City, UT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: M. Halstead, Kern, C. P. Saelao, Y. Wang, Zhou, H. P. J. Ross. 2016. Profiling of open chromatin in chicken tissues using ATAC-seq. 35th Conference for the International Society of Animal Genetics. Salt Lake City, UT.


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Animal genome scientists, livestock industry 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?Research findings have been presented in both oral and poster formats. What do you plan to do during the next reporting period to accomplish the goals?Continue to work on other three ChIP-seq assays for chicken, and will work on DNase-seq and all ChIP-seq for pig and cattle. Integrative analysis of 3 assays will be conducted.

Impacts
What was accomplished under these goals? The identification of regulatory elements is a key step in understanding how an organism's genotype determines the phenotype. The technologies and assays developed in human and mouse ENCODE projects provide a solid foundation to functionally annotate chicken, pig and cattle genomes. These three livestock species, which are core to the world's food production, lack robust functional annotations that could be leveraged to improve the production efficiency of these industries. We will present the current progress in generating and analyzing data from these three important species, including analysis of forty-eight RNA-seq libraries (sixteen per species) collected from two biological replicates across eight tissues: adipose, cerebellum, cortex, hypothalamus, liver, lung, muscle and spleen. Transcripts detected in these tissues show good coverage of the Ensembl gene sets for the three species, and an initial analysis has identified putative long non-coding RNAs, both tissue-specific and expressed across all tissues. For chicken, an analysis of 12 DNase-seq libraries from liver, lung, cerebellum and spleen (two replicates) and hypothalamus, cortex, muscle and adipose (one replicate) show that identified tissue-specific DNase hypersensitivity (DHS) sites are associated with genes that relate to unique biological functions of the organs or tissues. Twelve dataset from H3K4me3 and H3K27me3 histone modification in chickens were analyzed. Integrative analysis of DHS sites, ChIP-seq (H3K4me3 and H3K27me3 histone modification marks), and RNA-seq allows the identification of genome-wide active and inactive promoter regions in chickens, which enables an in-depth comparison of the regulatory landscapes of multiple tissues within these species.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: H. Zhou, P. Ross, C. Kern, P. Saelao, Y. Wang, M. Halstead, J. Chitwood, T. Kim, I. Korf, M. E. Delany, H. H. Cheng, J. F. Medrano, A. L. Van Eenennaam, C. K. Tuggle, C. Ernst. 2015. Genome wide identification and annotation of functional regulatory regions in livestock species. GO-FAANG Workshop, Washington D.C. October