Progress 03/01/18 to 02/28/22
Outputs
Target Audience:Our work addresses a fundamental question: how does a host respond to foreign sequence invasion? Among the adaptive responses, piRNA pathways are essential in protecting germ-line genomes by targeting foreign sequences through base-pair complementarity. We know little about how a host acquires new piRNA sequences when parasites invade. The only known mechanism underlying how new piRNA sequences evolve comes from a study in Drosophila. However, we found the first such example in vertebrates (Yu et al., 2017). In chickens, new piRNAs originate through a different mechanism from that in Drosophila. We found that the host hijacks a pre-existing provirus as a new piRNA-producing locus, reminiscent of the CRISPR system in bacteria. Interestingly, the provirus only generates piRNAs in domestic chickens but does not do so in undomesticated wild chickens. This genomic locus represents just the tip of the iceberg as we identified hundreds of loci that produce piRNAs exclusively in domestic chickens or in wild chickens, indicating extensive piRNA polymorphism in chicken populations. Our work raises new questions: how does a host activate an existing genomic element to become a piRNA-producing locus? What is the driving force that alters small RNA compositions within species? The ongoing comparative studies among chicken breeds will be one path to answer these questions (Li, 2019) (Li, 2021) (Li, 2022). Our work also addresses an agricultural question: the genetic basis of diverse commercial traits. Modern chickens have undergone intense phenotypic selection for two primary purposes: meat production and egg laying. While the poultry industry has successfully developed specialized lines with much higher productivity than their wild ancestors, incomplete understanding of the genetic basis of phenotypic variations among chicken breeds has stymied efforts to further improve their production. Among all the genetic differences among chicken breeds, structural variations (SVs, large DNA alterations ≥50 bp) are the least characterized. Our lack of understanding of SVs is due to the limitations of conventional short-read sequencing techniques in detecting SVs comprehensively and accurately across the genome. The recent application of single-molecule long-read sequencing methods in humans has helped overcome such challenges, revealing the prevalence of SVs in human populations and their significance in reproduction. Here, we performed nanopore long-read sequencing on a broiler (meat-type) line, a layer (egg-type) line, three indigenous village lines, and an undomesticated wild chicken line. We developed a new bioinformatics pipeline that allowed us to detect ~17,000 SVs in each domestic line, including insertions, deletions, duplications, and inversions, which impact 2.5% of the chicken genome. Although the chicken genome is generally believed to be devoid of transposable element (TE) activities, we identified 29 active endogenous retrovirus families and one active LINE family in chickens. These active TEs insert into protein coding genes enriched for neurological functions, likely contributing to the attenuation of behavioral responses to the domesticated environments. Taken together, our results indicate that extensive SVs exist in chickens and are generated via diverse mutational mechanisms. SVs impact a comparable number of base pairs as single nucleotide polymorphisms in domestic chicken breeds, suggesting their biological significance in phenotypic variations. These two questions are interrelated as TEs and piRNAs are a major force of genome evolution. Our studies open the possibility to mechanistically improve or alter the traits of chickens for quicker growth, more efficient reproduction, and high resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project allowed me to attend conferences bridging the poultry science community and RNA biologists together. The project also provided financial support for graduate and undergraduate students who were initially interested in RNA biology but were unaware of poultry sciences. Because of the training provided by this grant, these students have been rewarded with better career opportunities in both industry and academia, and their training in poultry science and reproductive biology will be useful expertise to further shape their careers. I envision these students will make great contributions to agriculture in the future. 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?
We have completed all these goals as listed in the publications and ongoing publications. The unpublished results spinning off from this proposal, including the mechanistic study of structural variations associated with the improved reproduction, will be further investigated in the next grant.
Publications
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Progress 03/01/21 to 02/28/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have both undergrad and graduate students working on this project, and they received great offers from industry and academia. 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?
We have completed all the proposed goal. The research has been either published or submitted to high-impact journals. The graduate student and undergrad student who arefirst authors for these paper both get good offers from famous biotech company or university. We also attend the meeting toCanadian Poultry Research Forum, Virtual at 2021, giving a talk"Nanopore sequencing reveals diverse structural variations in domestic chickens" to bridge the poultry scientists and RNA biologists. I continue to talk about avian genome biology during my courses to undergrads and graduate students at University of Rochester.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Gu, H., Yu, Y.H., Li, X.Z. (2021) Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species. Poultry Science. 100:101321
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
5. Yu, Y.H., Wang, Y.R., Du, K., Zheng, J., Xie, L.H., Pereira A., Zhang, C., Ricci, E.P., and Li, X.Z. (2021) Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs. Nature Communications. 12, 5970
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
2. Sun, Y.H., Cui, H., Song, C., Wang, Y.R., Yu, X., Zhuo, X., Ndamba, R., Mu Q., Gu, H., Wang, D., Murthy, G.G., Li, Pi., Liang, F., Liu, L., Tao, Q., Wang, Y., Orlowski, S., Zhou, H., Jagne, J., Gokcumen, O., Anthony, N. Zhao, X, and Li, X.Z. (2022) Structural variation hotspots protect germ-line genome integrity across amniotes.
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Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Since the last progress report, we have made seminal progresses in the following areas: 1 performing nanopore sequencing on diverse chicken breeds, we have identified 30 active transposable element (TE) families (29 endogenous retrovirus families and one LINE family) in the chicken genome. We detected 523 novel TE insertions in domestic chickens in genes enriched for neurological functions, including those whose human homologs are linked to autism. 2 We detected around 17,000 SVs per domestic chicken in comparison to wild chickens. Paradoxically, SV hotspots are not enriched for novel TE insertions, but instead correspond to genomic loci that produce PIWI-interacting RNAs (piRNAs), small RNAs that protect germ-line genome integrity from TE activation. Genomic instability within these loci serves to expand the diversity and abundance of the piRNA pool, allowing piRNAs to rapidly adapt to changes in TE sequences and copy numbers in chickens. 3 Although piRNA pathways are believed to be restricted to gonads in vertebrates, we identified somatic piRNAs in chicken. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1 My graduate student, Yu Sun, is about to graduate and has received multiple offers for senior scientist positions specializing in bioinformatics. Yu is practicing his leadership by mentoring undergraduates. Given Yu's dedication to his undergraduate students and exceptional mentoring capability, Yu has been listed as the co-corresponding author for works that come from his students, one of which has recently been accepted for publication in Poultry Science. 2 One of my undergraduate students, Hanwen Gu, has developed a novel method, which is an ideal method for RNA-seq and is also currently the only existing method, for constructing ribo-seq deep sequencing libraries from avian species. I believe this method developed by Hanwen will contribute significantly to poultry industry and conservation biology. Together with Yu and myself, Hanwen completed a first author manuscript for his project, which has been published in Poultry Science. 3 I was invited to present our research on chicken piRNAs at the Canadian Poultry Research Forum, the Laboratory of Cellular and Developmental Biology (LCDB), NIDDK, the College of Life Sciences at Brigham Young University (Provo), and St. John Fisher College (Rochester). 4 I have continued to modify my lectures for the Genomics Module of the Foundations course taken by all graduate students at URMC to incorporate the genomic features of agricultural animals. In particular, my lecture on comparative genomics has been revised to highlight the differences between mammals and birds. How have the results been disseminated to communities of interest?Publications: We have published the following paper: 1. Gu, H., Yu, Y.H., Li, X.Z. (2021) Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species. Poultry Science. In press. Protocols and antibodies: We have developed protocols to perform squash slides and chromosome spread slides for immunofluorescence studies on chicken testis. We validated ~20 commercial antibodies developed for human proteins to test whether they work for their chicken homologs. Bioinformatics pipeline: We have developed a pipeline to define novel TE insertions using data from nanopore sequencing. What do you plan to do during the next reporting period to accomplish the goals?1 We will complete our manuscript "Structural variation hotspots on genomes encode pachytene piRNAs", which is currently in preparation. 2 We will complete our story on somatic piRNAs in chickens and submit the manuscript. 3 We will write a manuscript on translational control throughout spermatogenesis in mice and chickens.
Impacts
What was accomplished under these goals?
Since the last progress report, we have made seminal progresses in the following areas: 1 performing nanopore sequencing on diverse chicken breeds, we have identified 30 active transposable element (TE) families (29 endogenous retrovirus families and one LINE family) in the chicken genome. We detected 523 novel TE insertions in domestic chickens in genes enriched for neurological functions, including those whose human homologs are linked to autism. 2 We detected around 17,000 SVs per domestic chicken in comparison to wild chickens. Paradoxically, SV hotspots are not enriched for novel TE insertions, but instead correspond to genomic loci that produce PIWI-interacting RNAs (piRNAs), small RNAs that protect germ-line genome integrity from TE activation. Genomic instability within these loci serves to expand the diversity and abundance of the piRNA pool, allowing piRNAs to rapidly adapt to changes in TE sequences and copy numbers in chickens. 3 Although piRNA pathways are believed to be restricted to gonads in vertebrates, we identified somatic piRNAs in chicken.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
1. Gu, H., Yu, Y.H., Li, X.Z. (2021) Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species. Poultry Science. In press.
- Type:
Other
Status:
Awaiting Publication
Year Published:
2021
Citation:
We have developed a pipeline to define novel TE insertions using data from nanopore sequencing.
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:I am able to establish the collaborations to perform piRNA sequencing on 6 additional domestic strains, the undomesticated Red Jungle Fowl, andboth male and female chicken primodiral germ cells. This allows us to define the intra-species piRNA variations by expanding the aim that only focuses on comparing two strains, one egg layer, and one broiler breeder. What opportunities for training and professional development has the project provided?1 My graduate student, Yu Sun, who is majoring in Biology, has continued to grow as a budding bioinformatician. Yu was recently invited to present at the Workshop on Long Read Sequencing at JAX Genomic Medicine in Farmington, CT. 2 I have trained an undergraduate student, Xiangyi Liu, who is majoring in Data Science, to analyze the chicken small RNA data and ribosome profiling data, initially for course credits in independent research and then as a paid student aid. 3 Another undergraduate student, Hanwen Gu, has designed the probes and protocols for depleting rRNAs in chickens and is currently working on a manuscript entitled "Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species". He will be the first author, while his mentor Yu Sun will be the co-corresponding author. 4 I have also opened a lecture on "comparative genomics", in which as well as other lectures in the Genomic module I directed for the foundation course taken by all graduate students at our medical center, we covered the genomic features of agriculture animals. How have the results been disseminated to communities of interest?Publications: We have published the following two papers: Li, X.Z. (2019) What can piRNA research learn from chickens, and vice versa? Canadian Journal of Animal Science. 99: 641-648 It represents, to the best of our knowledge, the first review on chicken piRNAs. Yu, Y.H., Zhu, J., Xie, L.H., Li, Z., Zhu, X., Song, C. Chen, C., Ricci, E.P., Weng, Z., and Li, X.Z. (2020) Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors. Nature Cell Biology. 22, 200-212 Highlighted in News and Views, Nature Cell Biology, 22,141-142, 2020 This work reveals the role of ribosomes in mediating piRNA biogenesis, which is essential for fertility. This new mechanism is evolutionarily conserved in chickens. Antibodies: We have produced custom antibodies towards CILI and CIWI, the two PIWI proteins expressed in chickens. We have validated the CIWI antibodies, whereas the validation of CILI antibodies has been delayed due to the COVID-19 outbreak. These antibodies will be available upon request. Bioinformatic pipeline: We have developed a Ribo-seq pipeline for ribosome profiling analysis. Our pipeline is available in the following link (https://github.com/LiLabZhaohua/RiboSeqPipeline). We will also organize the work in our upcoming manuscript. What do you plan to do during the next reporting period to accomplish the goals?1 Once the CILI and CIWI antibodies arrive, we will continue of our efforts to determine the intracellular localization of CILI and CIWI and perform immunoprecipitation assays to test whether there is preferential loading of CILI and CIWI. 2 We will define the intra-species piRNA variations by comparing piRNAs across different strains including the wild, undomesticated Red Jungle Fowl, as well as piRNAs from male and female primordial germ cells. 3 We will integrate our piRNA analyses with our transposon analyses, and write a manuscript focused on the arms race between piRNAs and TEs. 4 Since we have the ribosome profiling data of the chickens and mice at different time points throughout spermatogenesis, we will write a manuscript on translational control throughout spermatogenesis in mice and chickens.
Impacts
What was accomplished under these goals?
In the last progress report, we have collected testes samples from three broiler breeder strains that are randomly bred controls from the years 1950, 1995 and 2015. The 1950 line is the Athens Canadian Random Bred (ACRB) that has been widely used in the community. We collected testes at the key developmental stages of day 1, week 6, week 8, week 10, week 12, week 14, week 16, week 18, and week 30. We have fixed them for histology, as well as snap-froze for molecular analysis. We have performed small RNA sequencing of the testes at different developmental stages of the ACRB strain and defined the temporal changes in piRNA profiles, in which we identified a group of piRNAs reminiscence of the mouse pachytene piRNAs that expressed during pachytene stage of meiosis. This year, I am mainly focusing on the following: 1) continue to characterize the piRNAs in chickens beyond the ACRB strains; and 2) define the active TEs in chickens using ribosome profiling (Ribo-seq). 1 We have extended our collaboration with Dr. Xin Zhao from McGill University in Canada to sequence piRNAs from an additional 6 strains of domesticated chickens with 3 biological replicates, each of which have a total small RNA library and an oxidized small RNA library (enriched for piRNAs) 2 We have extended our collaboration with Dr. Carlos Guerrero Bosagna from Linköping University in Sweden to send us undomesticated Red Jungle Fowl tissues with 3 biological replicates for library construction of both total and oxidized small RNA libraries. 3 We have extended our collaboration with Dr. Jeffery Barrow from Brigham Young University in Provo, UT to gain knowledge in establishing chicken primordial germ cell cultures, and collect enough samples for both male and female PGCs with 3 biological replicates for library construction of both total and oxidized small RNA libraries. 4 We have performed ribosome profiling sequencing for testis at the key developmental stages of day 1, week 6, week 8, week 10, week 12, week 14, week 16, week 18, and week 30 in the 1950 strain to define the actively translated TEs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
What can piRNA research learn from chickens, and vice versa? Canadian Journal of Animal Science. 99: 641648
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors. Nature Cell Biology. 22, 200-212
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Progress 03/01/18 to 02/28/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1 I have trained a graduate student, Yu Sun, on bioinformatics and molecular biology techniques for analysis of chicken samples. Yu recently presented his research at the PAG (plant and animal genomic) conference in San Diego. 2 I also recruited an undergraduate student, Hanwen Gu, to perform molecular biology experiments on chicken samples. Hanwen has done independent research for credit in my lab for two semesters. He has designed the probes and protocols for depleting rRNAs in chickens. These will be a valuable resource for the community. I elaborate on this in the next section. How have the results been disseminated to communities of interest?1 We have been invited to write a review on chicken piRNAs for the Canadian Journal of Animal Science. It represents, to the best of our knowledge, the first review on chicken piRNAs. 2 We have developed a valuable resource for constructing RNA-seq libraries, intended for use by the poultry community. Currently, the community lacks an effective methodology to perform total RNA sequencing. As gene expression patterns from RNA-seq are based off of mRNAs and rRNAs constitute over 90% of the total RNAs, mRNAs must be enriched during library preparation. This can be done through either polyA selection or rRNA depletion. PolyA selection, however, misses some transcripts. Moreover, the majority of RNA-seq data, from mouse and humans, that has been generated are from rRNA-depleted total RNAs. Recognizing this need in the poultry community, we have developed a set of probes and a methodology for depleting rRNAs in chickens. Considering the homology of rRNAs, it should work for other birds as well. The cost is less than 10 dollars per sample. We have informed the community that we will share the protocol with the community and send the probes for free. What do you plan to do during the next reporting period to accomplish the goals?1 Going forward, we will determine the intracellular localization of CILI and CIWI and monitor for possible changes in localization as spermatogenesis progresses. Using immunohistochemistry, we will determine whether PIWI proteins localize to specific RNA granules or are diffusely distributed throughout the cytosol. In our preliminary results, we have tested the three commercial antibodies on squashed preparation from testes for immunochemistry {Page et al., 1998, #89147}, and have observed stage specific staining of CILI and CIWI that is consistent with their expression dynamics. 2 We plan to define the originating causes of SVs and elucidate their relationship with TEs. First, we will define all transposition events and determine the TE family associated with each transposition. To distinguish transposition events from genomic duplication events, we will determine the existence of target site duplication (short repeat sequences flanking the insertion site), which is the signature for transposition. We will also align the transposon sequences in a phylogeny tree to identify the master TE sequences that initiated the out-burst. We will define the transposition events that are activated only in ACRB and white Leghorn. Understanding the genetic cause for its activation will help us to avoid the activation of these selfish elements. Second, we will determine whether translocations, duplications, or large deletions are enriched at existing TE regions compared to non-TE regions, which would indicate an elevated non-allelic homologous recombination between TEs. If so, we would test whether this is associated with a specific class of TE families.
Impacts
What was accomplished under these goals?
1 We have collected testes samples from three broiler breeder strains. These strains were set up as randombred controls from the years 1950, 1995 and 2015. The 1950 line is the Athens Canadian Random Bred (ACRB) that has been widely used in the community. We collected the testes at the key developmental stages of day 1, week 6, week 8, week 10, week 12, week 14, week 16, week 18, and week 30. We have fixed the testis for histology and snap-freezed the testis for molecular analysis. 2 We have performed small RNA sequencing of the testes at different developmental stages. 3 We were able to detect piRNAs in each developmental stage and normalized their abundance to the sum total of ubiquitously expressed miRNAs. In adult testis, most piRNAs are expressed during the transition from 8 weeks to 18 weeks as the first wave of spermatogenesis undergoes meiosis{Swift, 1916, #27402}. This expression pattern is reminiscent of the piRNA dynamics in mice, where the majority of the piRNAs in adult testis are expressed at the pachytene stage of meiosis{Aravin et al., 2007, #21937}{Li et al., 2013, #14672}. Thus, both the intergenic distribution and expression pattern of piRNAs appear to be conserved between mammals and birds. We observed that a set of piRNAs that target a subgroup of TEs are also turned on at the 8 - 18 week transition, suggesting that regulation of TEs in chickens is multilayered and dynamic. 4 To define TE activities, the DNA from adult ACRB testes and adult white leghorn testes were purified to maintain genome integrity, avoiding fragmentation. We obtained a good yield of genomic DNA from 0.5 g samples of snap-frozen fresh testis. Without any amplification, we sequenced the nascent genome with 1 flow cell, which yielded 18.5 Gb of high-quality sequencing data. Considering the chicken genome is ~1.2 Gb, our sequencing dataset has a 31 X genomic coverage with N50 of 34,645 bp. This is greater than the 10X coverage required to call TE variations{Disdero and Filée, 2017, #75568}. The raw sequencing output as FAST5 files was converted to Fastq format using MINKNOW local basecaller. Long reads were aligned to the chicken genome using NGMLR with a mappability of 96.4% and an error rate of 16.4%. SV call sets were performed by Sniffles. We detected 113,623 events of SVs with lengths ranging from 50bp-1Mbp in ACRB.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
15. Li, X.Z. (2019) What can piRNA research learn from chickens, and vice versa? Canadian Journal of Animal Science. In press
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