Annotation of Functional Regulatory Elements in Livestock Species
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1 Annotation of Functional Regulatory Elements in Livestock Species Midwest ASAS March 17, 2015 Huaijun Zhou 1, Pablo Ross 1, Ian Korf 1, Mary Delany 1, Hans Cheng 2, Juan Medrano 1, Alison Van Eenennaam 1, Chris Tuggle 3, Cathy Ernst 4, Vicki Leesburg 5 1 Animal Science, University of California, Davis, CA 2 ADOL, USDA-ARS, East Lansing, MI 3 Animal Science, Iowa State University, Ames, IA 4 Animal Science, Michigan State University, East Lansing, MI 5 LRRL, USDA-ARS, Miles City, MT
2 Outline ENCODE (Encyclopedia of DNA Elements) Farm animal ENCODE pilot projects FAANG (Functional Annotation of Animal Genomes) initiative Future plan
3 Human ENCODE Project 95% of genome is junk (<3% of genome is coding) cis regulatory elements occur within a limited genome distance Objective: Catalog functional elements in the human genome
4 Human ENCODE Project Roadmap (200M $) 1990 Human genome published 2003 GWAS 90% lies outside coding 2007 ENCODE published Human genome project kicked off 2000 NHGRI solicited pilot proposal for ENCODE 2005 First publication on ENCODE 2012 RFAs were sought for full ENCODE
5 Nature 489, (06 September 2012) doi: /489049a
6 Nature 489, (06 September 2012) doi: /489052a
7 Mate Ongenaert at Galapagos
8 Plos Biology DOI: /journal.pbio
9 30 papers published in 3 journals at the same time
10 Key Findings 80% of the human genome participates in at least one functional activity Environment is critical in switch on or off for many genes 4 millions switches control ~21,000 genes switches are software and genes are hardware
11 Key Findings 1% of the genome is distally regulated (Kbs to Mbs) using 5C On an average 3.9 distal elements interacted with TSS 2.9 million DHS in 125 diverse cells and tissue types expressed isoforms per gene per cell
12 How about farm animals? Genome to Phenome Wealth genetic and phenotypic diversity Diverse evolution: Mammal, ruminant, avian species Extensive pedigree records for economically important complex traits
13 Challenges Many species Many genetic lines Different development stages Limited resource Quality of genome assembly
14 Genome-wide identification and annotation of functional regulatory regions in livestock species (New USDA grant) H. Zhou (PD), P. Ross, I. Korf (co-pds), UC Davis Collaborators: Poultry Genome Coordinators: M. Delany, H. Cheng Cattle Genome Coordinators: J. Medrano, A. Van Eenennaam Swine Genome Coordinators: C. Tuggle, C. Ernst V. Leesburg, USDA ARS Jim Kent, UCSC Laura Clarke, Paul Flicek, EBI Bin Ren, UCSD
15 Overall Goal Generate a comprehensive resource of functional regulatory elements for the chicken, cattle, and pig genomes
16 Specific Objectives Annotate chromatin states corresponding to DNase I hypersensitivity, four histone modifications, and one insulator element. Identify and annotate promoters, enhancers, and silencers by integrating information from RNA-seq, DNase I hypersensitivity, and ChIPseq. Freely distribute all raw and annotated data via UCSC Genome Browser and Ensembl.
17 Genetic lines Adult stages Chickens: F1 (Line 6 X 7) from ADOL, 2 males and 2 females at 20 weeks of age Cattle: Line 1 Hereford from Fort Keogh Livestock and Range Research Lab, 2 males and 2 females from the same sire at 14 months of age Pigs: Yorkshire from MSU, 2 littermates males at 6 months of age
18 Tissues Cow Pig chicken Liver X X X Kidney X X X Skeletal muscle X X X Adipose X X X Spleen X X X Pancreas X X X Tissues Brain cortex/whole X X X Cerebellum X X X Hypothalamus X X Heart X X X Lung X X X Trachea X X X Tissues collection Bladder X X Thyroid gland X X X Bursa X Esophagus X X X Thymus X X X Skin X X X Mammary gland X X Ovary X X X RNAlater Liquid nitrogen Fresh tissues Oviduct X X X Uterus X X Testis X X X Bone marrow X X X Blood X X X Cartilage X X X Duodenum X X X Jejunum X X X Ileum X X X Cecum X RNA work Chromatin work Nuclei work Colon X X X Abomasum/Stomach/Gizzard X X X
19 Assays Stranded RNA-seq Chromatin accessibility: DNase-seq (ATAC-seq) Histone modification marks H3K4me3 (promoters of active genes and transcription starts) H3K27me3 (silence) H3K27AC (active regulatory elements) H3K4Me1 (enhancers and other distal elements) Transcription factor CTCF (insulator)
20 Distribution of H3K4me3 and H3K27me3 across chicken genome
21 H3K4me3 across chicken genome in 3 tissues Liver Spleen Breast muscle
22 Candidate genes demonstrating tissue-specific chromatin state H3k4me3 Spleen Liver Breast muscle H3k27me3 Spleen Liver Breast muscle RNA-seq Spleen Liver Breast muscle
23 Other pilot projects WUR-pigENCODE (Groenen et al.) Pig Duroc, Large White, Pietrain RNA-seq, Small-RNA-seq, Methylation FR-AgENCODE (Giuffra et al.) Pig: Large White; Chicken; White Leghorn; Cattle: Holstein; Goat: Alpine RNA-seq, Small RNA-seq, and Hi-C Sheep ENCODE (Kijas et al) USDA MARC, Baylor College of Medicine, Utah State University and CSIRO Rambouillet ewe Iso-seq, RNA-seq, Small-RNA-seq Pig ENCODE in China (PENCODE China, Huang et al) 300 F6 mosaic pigs
24 FAANG Consortium Roadmap ($$$???) 11/2011 Hinxton, UK Animal Biotechnology Working group 1/2014 Xi an, China FAANG white paper drafted at the ISAG 1/2015 Washington DC GO-FAANG Washington DC EU-US ABWG proposed 9/2012 AgENCODE workshop at the PAG meeting 7/2014 FAANG consortium launched at the PAG meeting 10/2015? www. FAANG.org
25
26 FAANG Organization Chart Steering Committee Sample/Assay Matadata Analysis Communication
27 Future plan Sub-committee will work with animal genome community to coordinate FAANG activities Sample protocol Assay protocols Data standard Infrastructure Foster collaborations Reach out more funding agencies worldwide USDA, NSF, NIH, BBSRC, Europe Commission, Genome Canada, NSF China, Australia?, Korea?
28 Future FAANG workshops/meetings July, 2015, Piacenza, Italy, ISAFG October, 2015, Washington DC, GO-FAANG January, 2016, FAANG workshop, PAG July, 2016, Salk city, UT, ISAG July, 2017, Dublin, ISAG
29 Financial support USDA NIFA grant # Poultry, Cattle, Swine Genome Coordination Funds
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