Progress 05/01/22 to 04/30/23
Outputs
Target Audience:poultry scientific community, livestock and poultry 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 in animal genetics, bioinformatics and genomic analysis. How have the results been disseminated to communities of interest?The research findings have been presented in a variety of national and international conferences with audiences from both academia and livestock industry. What do you plan to do during the next reporting period to accomplish the goals?We will focus on performing MPRAs in chicken DF-1 cells. We will optimize the conditions of the critical steps for MPRAs (barcode addition, electroporation, plasmid maxi prep, transfection, maxi scale RNA extraction, GFP capture, etc.) and test them on a smaller scale (~1000 oligos) first, then we will apply it on a larger scale (~14,000 oligos). For each synthesized oligo, we will add multiple random barcodes by PCR and will then clone them into vectors. We will then perform illumina sequencing to determine the association of candidate enhancer and barcode. We will generate MPRA plasmids pool by assembling GFP amplicon into the vectors containing a candidate enhancer and barcode. We will then transfect the MPRA plasmids pool into DF-1 cells and then harvest RNA and DNA from the transfected cells, amplify barcodes, and perform illumina sequencing for barcodes. We will then determine the enhancer activity of each candidate sequence by calculating the RNA/DNA ratio of its barcodes.
Impacts
What was accomplished under these goals?
A comprehensive characterization of regulatory elements in the chicken genome across tissues 41 will have substantial impacts on both fundamental and applied research. Here we systematically 42 identified and characterized regulatory elements in the chicken genome by integrating 377 43 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million 44 regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million 45 enhancer-gene pairs and 7,662 super-enhancers. This functional annotation of the chicken genome 46 should have wide utility on identifying regulatory elements accounting for gene regulation 47 underlying domestication, selection, and complex trait regulation, which we explored. In short, 48 this comprehensive atlas of regulatory elements provides the scientific community with a valuable 49 resource for chicken genetics and genomics. For the enhancers we have predicted, it is essential to functionally validate these predicted enhancers. We selected a total of 3501 predicted enhancers whose target genes exhibit relatively high expression in DF-1 cells. This includes 1180 enhancers active among at least 5 tissue categories, 793 enhancers specifically active in spleen (149), bone marrow (159), bursa (457) or thymus (28), and 1528 enhancers active in immune-related tissues but not tissue-specific. We chose a 269-bp region from each candidate enhancer and add 15 nt adaptors at both ends. These resulting 299 nt oligos will be synthesized along with 100 positive control sequences and 100 negative control sequences. Each oligo will be coupled to multiple barcodes through PCR to facilitate reproducible and quantitative measurements of regulatory activity. The vectors containing a minimal promoter, synthesized sequence, enhanced green fluorescent protein (eGFP), and associated barcode will be transfected into DF-1 cells. We will harvest RNA and DNA from the transfected cells, amplify barcodes, and perform sequencing for barcodes (Illumina NextSeq). We will then determine the enhancer activity of each candidate sequence by calculating the RNA/DNA ratio of its barcodes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
D. Guan, M. L. Fang, and H. Zhou. 2022. The Chicken Genotype-Tissue Expression (ChickenGTEx): a comprehensive atlas of genetic regulatory variants in chicken transcriptome. FAANG Workshop.
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:poultry scientific community, livestock and poultry 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 in animal genetics, bioinformatics and genomic analysis. 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?
Functional annotation of the chicken genome, especially the identification of regulatory elements, will play a significant role in identifying causative genetic variants associated with economically important traits. Using a bioinformatics pipeline that incorporates and analyzes (Chromatin Immunoprecipitation Sequencing (ChIP-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and RNA-seq datasets, we extend our annotation efforts to seven gut-associated tissues from two male chickens used in the FAANG pilot project. ChromHMM was used to predict a total of 15 chromatin states and 1,197,186 regulatory elements (excluding Quiescent) spanning 15 tissues (include 8 core tissues done previously) with 398,050 new regulatory elements were identified. These regulatory elements included 29,823 strong promoters, 104,560 active strong enhancers, and 73,582 repressors. This result provides a great resource for the farm animal genome community to further explore causative variants of complex traits. Active enhancer loci can be identified on the basis of certain epigenetic signatures, chromatin accessibility, and transcription factor and cofactor binding. Thousands of enhancers have been identified through the functional annotation of regulatory elements in the chicken genome by the Functional Annotation of Animal Genomes (FAANG) consortium. While an important first step, the FAANG annotated enhancers may have low concordance with their bona fide functional activities, thus, experimental validation of enhancer activity is required. In this study, we aim to achieve high-throughput functional validation of enhancers in the chicken genome. We first validated two splenic-specific enhancers (Proly 4-Hydroxylase Subunit Beta and Migration and Invasion Inhibitory Protein) using the standard luciferase reporter assay in the DF-1 chicken cell line. We are currently developing massively parallel reporter assays (MPRAs) in DF1 cells to overcome the throughput limit of reporter gene assays. Our MPRA library involves the synthesis of ~2,000 putative enhancers, each 250-nt in length, and each coupled to 15 unique 15-nt molecular barcodes separately. Our candidate enhancers include two sets: (1) predicted strong enhancers associated with high expression genes in DF-1 cells, and (2) variants of predicted strong enhancers with allele specific expression genes related to genetic resistance to Marek's disease. A positive control and a negative control sets are also included. The development of this high throughput enhancer validation system will enable the genome-wide discovery and functional characterization of enhancers in the chicken genome, which could provide a growing knowledge base for the systematic exploration of their role in chicken biology and disease susceptibility.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
An L, Y. Wang, Z. Pan, D. Guan, H.H. Cheng, H. Zhou. 2022. Functionally Validate Enhancers in the Chicken Genome using MPRAs. FAANG Workshop.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Z. Pan, Y. Wang, An L, D. Guan, H.H. Cheng, H. Zhou. 2022 A multi-tissue atlas of regulatory elements in the chicken genome. Annual poultry science meeting, San Antonio, TX
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:poultry scientific community, livestock and poultry 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?Graduate students, undergraduate students, and postdoctoral scholar have training in in animal genetics, bioinformatics and genomic analysis. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
For the chicken regulatory elements annotation, we have generated 135 epigenomic data from 15 tissues including thymus, trachea, bone marrow, kidney, heart, follicle, shell gland, testis, gizzard, jejunum, colon, ileum, duodenum, cecum, pventriculus (2 biological replicates per tissue except follicle) from F1 cross of line 6 by line 7 on H3K4me3, H3K27ac, H3K4me1, K3k27me3 and ATAC-seq. RNA-seq libraries for all above tissues have been done and are currently sequencing the libraries. In addition, in order to validate spleen-specific enhancers identified from FAANG pilot project, a Renilla luciferase assay was developed to validate 2 enhancers using DF1 cell line.
Publications
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