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author:("Song, liuzhou")
1.  Genomic Signatures Reveal New Evidences for Selection of Important Traits in Domestic Cattle 
Molecular Biology and Evolution  2014;32(3):711-725.
We investigated diverse genomic selections using high-density single nucleotide polymorphism data of five distinct cattle breeds. Based on allele frequency differences, we detected hundreds of candidate regions under positive selection across Holstein, Angus, Charolais, Brahman, and N'Dama. In addition to well-known genes such as KIT, MC1R, ASIP, GHR, LCORL, NCAPG, WIF1, and ABCA12, we found evidence for a variety of novel and less-known genes under selection in cattle, such as LAP3, SAR1B, LRIG3, FGF5, and NUDCD3. Selective sweeps near LAP3 were then validated by next-generation sequencing. Genome-wide association analysis involving 26,362 Holsteins confirmed that LAP3 and SAR1B were related to milk production traits, suggesting that our candidate regions were likely functional. In addition, haplotype network analyses further revealed distinct selective pressures and evolution patterns across these five cattle breeds. Our results provided a glimpse into diverse genomic selection during cattle domestication, breed formation, and recent genetic improvement. These findings will facilitate genome-assisted breeding to improve animal production and health.
PMCID: PMC4441790  PMID: 25431480
cattle genome; population structure; positive selection; haplotype network
3.  Genome-wide identification of copy number variations between two chicken lines that differ in genetic resistance to Marek’s disease 
BMC Genomics  2015;16:843.
Copy number variation (CNV) is a major source of genome polymorphism that directly contributes to phenotypic variation such as resistance to infectious diseases. Lines 63 and 72 are two highly inbred experimental chicken lines that differ greatly in susceptibility to Marek’s disease (MD), and have been used extensively in efforts to identify the genetic and molecular basis for genetic resistance to MD. Using next generation sequencing, we present a genome-wide assessment of CNVs that are potentially associated with genetic resistance to MD.
Three chickens randomly selected from each line were sequenced to an average depth of 20×. Two popular software, CNVnator and Pindel, were used to call genomic CNVs separately. The results were combined to obtain a union set of genomic CNVs in the two chicken lines.
A total of 5,680 CNV regions (CNVRs) were identified after merging the two datasets, of which 1,546 and 1,866 were specific to the MD resistant or susceptible line, respectively. Over half of the line-specific CNVRs were shared by 2 or more chickens, reflecting the reduced diversity in both inbred lines. The CNVRs fixed in the susceptible lines were significantly enriched in genes involved in MAPK signaling pathway. We also found 67 CNVRs overlapping with 62 genes previously shown to be strong candidates of the underlying genes responsible for the susceptibility to MD.
Our findings provide new insights into the genetic architecture of the two chicken lines and additional evidence that MAPK signaling pathway may play an important role in host response to MD virus infection. The rich source of line-specific CNVs is valuable for future disease-related association studies in the two chicken lines.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-2080-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4619206  PMID: 26492869
Copy number variation; Chicken; Susceptibility; Marek’s disease; MAPK signaling pathway; Next generation sequencing
4.  The conservation and signatures of lincRNAs in Marek’s disease of chicken 
Scientific Reports  2015;5:15184.
Long intergenic non-coding RNAs (lincRNAs) associated with a number of cancers and other diseases have been identified in mammals, but they are still formidable to be comprehensively identified and characterized. Marek’s disease (MD) is a T cell lymphoma of chickens induced by Marek’s disease virus (MDV). Here, we used a MD chicken model to develop a precise pipeline for identifying lincRNAs and to determine the roles of lincRNAs in T cell tumorigenesis. More than 1,000 lincRNA loci were identified in chicken bursa. Computational analyses demonstrated that lincRNAs are conserved among different species such as human, mouse and chicken. The putative lincRNAs were found to be associated with a wide range of biological functions including immune responses. Interestingly, we observed distinct lincRNA expression signatures in bursa between MD resistant and susceptible lines of chickens. One of the candidate lincRNAs, termed linc-satb1, was found to play a crucial role in MD immune response by regulating a nearby protein-coding gene SATB1. Thus, our results manifested that lincRNAs may exert considerable influence on MDV-induced T cell tumorigenesis and provide a rich resource for hypothesis-driven functional studies to reveal genetic mechanisms underlying susceptibility to tumorigenesis.
PMCID: PMC4608010  PMID: 26471251
5.  Transcriptomic Profiling of Spleen in Grass-Fed and Grain-Fed Angus Cattle 
PLoS ONE  2015;10(9):e0135670.
The grass-fed cattle obtain nutrients directly from pastures containing limited assimilable energy but abundant amount of fiber; by contrast, grain-fed steers receive a diet that is comprised mainly of grains and serves as an efficient source of high-digestible energy. Besides energy, these two types of diet differ in a large number of nutritional components. Additionally, animals maintained on rich-energy regimen are more likely to develop metabolic disorders and infectious diseases than pasture raised individuals. Thus, we hypothesize that spleen–a relevant immune organ–may function differently under disparate regimes. The objective of this study was to find the differentially expressed genes in the spleen of grass-fed and grain-fed steers, and furtherly explore the potential involved biopathways. Through RNA sequencing (RNA-Seq), we detected 123 differentially expressed genes. Based on these genes, we performed an Ingenuity Pathway Analysis (IPA) and identified 9 significant molecular networks and 13 enriched biological pathways. Two of the pathways, Nur77 signaling in T lymphocytes and calcium-induced T lymphocyte apoptosis which are immune related, contain a pair of genes HLA-DRA and NR4A1 with dramatically altered expression level. Collectively, our results provided valuable insights into understanding the molecular mechanism of spleen under varied feeding regimens.
PMCID: PMC4569079  PMID: 26367387
6.  ∆ DNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis 
EBioMedicine  2015;2(10):1340-1350.
Aberrant DNA methylation is a hallmark of cancer but mechanisms contributing to the abnormality remain elusive. We have previously shown that ∆DNMT3B is the predominantly expressed form of DNMT3B. In this study, we found that most of the lung cancer cell lines tested predominantly expressed DNMT3B isoforms without exons 21, 22 or both 21 and 22 (a region corresponding to the enzymatic domain of DNMT3B) termed DNMT3B/∆DNMT3B-del. In normal bronchial epithelial cells, DNMT3B/ΔDNMT3B and DNMT3B/∆DNMT3B-del displayed equal levels of expression. In contrast, in patients with non-small cell lung cancer NSCLC), 111 (93%) of the 119 tumors predominantly expressed DNMT3B/ΔDNMT3B-del, including 47 (39%) tumors with no detectable DNMT3B/∆DNMT3B. Using a transgenic mouse model, we further demonstrated the biological impact of ∆DNMT3B4-del, the ∆DNMT3B-del isoform most abundantly expressed in NSCLC, in global DNA methylation patterns and lung tumorigenesis. Expression of ∆DNMT3B4-del in the mouse lungs resulted in an increased global DNA hypomethylation, focal DNA hypermethylation, epithelial hyperplastia and tumor formation when challenged with a tobacco carcinogen. Our results demonstrate ∆DNMT3B4-del as a critical factor in developing aberrant DNA methylation patterns during lung tumorigenesis and suggest that ∆DNMT3B4-del may be a target for lung cancer prevention.
•DNMT3B/∆DNMT3B-del is the predominantly expressed isoform in a large number of lung cancers.•∆ DNMT3B4-del can cause aberrant DNA methylation patterns similar to tumorigenesis.•∆ DNMT3B4-del facilitates carcinogen-induced lung tumorigenesis in a mouse model.
Lung cancer is the leading cause of cancer-related deaths in the United States. Epigenetic alterations, particularly alterations in DNA methylation patterns, play critical roles in lung tumorigenesis. We show that DNMT3B/ΔDNMT3B-del is predominantly expressed in a significant percentage of lung cancers including both cell lines and primary tumors. We demonstrate that ΔDNMT3B4-del is critical in the formation of aberrant DNA methylation patterns in mouse lungs similar to human lung cancers and contributes to neoplasia formation when exposed to carcinogens, supporting ΔDNMT3B4-del as a novel target for lung cancer prevention.
PMCID: PMC4634842  PMID: 26629529
DNMT3B; ΔDNMT3B; ΔDNMT3B-del; DNA methylation; Lung cancer; Mouse model; Tumorigenesis
7.  Correction: Ruminal Transcriptomic Analysis of Grass-Fed and Grain-Fed Angus Beef Cattle 
PLoS ONE  2015;10(7):e0134067.
PMCID: PMC4510587  PMID: 26197431
8.  Methylome Analysis in Chickens Immunized with Infectious Laryngotracheitis Vaccine 
PLoS ONE  2015;10(6):e0100476.
In this study we investigated the methylome of chickens immunized with Infectious laryngotracheitis (ILT) vaccine derived from chicken embryos. Methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) method was employed in the detection of the 1,155 differentially methylated regions (DMRs) across the entire genome. After validation, we ascertained the genomic DMRs distribution and annotated them regarding genes, transcription start sites (TSS) and CpG islands. We found that global DNA methylation decreased in vaccinated birds, presenting 704 hypomethylated and 451 hypermethylated DMRs, respectively. Additionally, we performed an enrichment analysis detecting gene networks, in which cancer and RNA post-transcriptional modification appeared in the first place, followed by humoral immune response, immunological disease and inflammatory disease. The top four identified canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, axonal guidance signaling and mTOR signaling, providing new insight regarding the mechanisms of ILT etiology. Lastly, the association between DNA methylation and differentially expressed genes was examined, and detected negative correlation in seventeen of the eighteen genes.
PMCID: PMC4481310  PMID: 26107953
9.  Ruminal Transcriptomic Analysis of Grass-Fed and Grain-Fed Angus Beef Cattle 
PLoS ONE  2015;10(6):e0116437.
Beef represents a major diet component and one of the major sources of protein in human. The beef industry in the United States is currently undergoing changes and is facing increased demands especially for natural grass-fed beef. The grass-fed beef obtained their nutrients directly from pastures, which contained limited assimilable energy but abundant amount of fiber. On the contrary, the grain-fed steers received a grain-based regime that served as an efficient source of high-digestible energy. Lately, ruminant animals have been accused to be a substantial contributor for the green house effect. Therefore, the concerns from environmentalism, animal welfare and public health have driven consumers to choose grass-fed beef. Rumen is one of the key workshops to digest forage constituting a critical step to supply enough nutrients for animals’ growth and production. We hypothesize that rumen may function differently in grass- and grain-fed regimes. The objective of this study was to find the differentially expressed genes in the ruminal wall of grass-fed and grain-fed steers, and then explore the potential biopathways. In this study, the RNA Sequencing (RNA-Seq) method was used to measure the gene expression level in the ruminal wall. The total number of reads per sample ranged from 24,697,373 to 36,714,704. The analysis detected 342 differentially expressed genes between ruminal wall samples of animals raised under different regimens. The Fisher’s exact test performed in the Ingenuity Pathway Analysis (IPA) software found 16 significant molecular networks. Additionally, 13 significantly enriched pathways were identified, most of which were related to cell development and biosynthesis. Our analysis demonstrated that most of the pathways enriched with the differentially expressed genes were related to cell development and biosynthesis. Our results provided valuable insights into the molecular mechanisms resulting in the phenotype difference between grass-fed and grain-fed cattle.
PMCID: PMC4475051  PMID: 26090810
10.  Genome-Wide H3K4me3 Analysis in Angus Cattle with Divergent Tenderness 
PLoS ONE  2015;10(6):e0115358.
Tenderness is one of the most important properties of meat quality, which is influenced by genetic and environmental factors. As an intensively studied epigenetic marker, histone methylation, occurring on arginine and lysine residues, has pivotal regulatory functions on gene expression. To examine whether histone methylation involves in beef tenderness variation, we analyzed the transcriptome and H3K4me3 enrichment profiles of muscle strips obtained from the longissimus dorsi (LD) of Angus steers previously classify to the tender or tough group. We first plotted a global bovine H3K4me3 map on chromosomes and called peak-enriched regions and genes. We found that majorities of H3K4me3 on genes were occupying the first intron and intergenic regions and its maps displayed similar patterns in tender and tough groups, with high H3K4me3 enrichment surrounding the transcription start site (TSS). We also explored the relationship of H3K4me3 and gene expression. The results showed that H3K4me3 enrichment is highly positively correlated with gene expression across the whole genome. Cluster analysis results confirmed the relationship of H3K4me3 enrichment and gene expression. By using a pathway-based approach in genes with H3K4me3 enrichment in promoter regions from the tender cluster, we revealed that those genes involved in the development of different tissues–connective tissue, skeletal and muscular system and functional tissues–; while in tough group those genes engaged in cell death, lipid metabolism and small molecule biochemistry. The results from this study provide a deep insight into understanding of the mechanisms of epigenetic regulations in meat quality and beef tenderness.
PMCID: PMC4473007  PMID: 26086782
11.  Histone modifications induced by MDV infection at early cytolytic and latency phases 
BMC Genomics  2015;16(1):311.
Marek’s disease (MD) is a highly contagious, lymphomatous disease of chickens induced by a herpesvirus, Marek’s disease virus (MDV) that is the cause of major annual losses to the poultry industry. MD pathogenesis involves multiple stages including an early cytolytic phase and latency, and transitions between these stages are governed by several host and environmental factors. The success of vaccination strategies has led to the increased virulence of MDV and selective breeding of naturally resistant chickens is seen as a viable alternative. While multiple gene expression studies have been performed in resistant and susceptible populations, little is known about the epigenetic effects of infection.
In this study, we investigated temporal chromatin signatures induced by MDV by analyzing early cytolytic and latent phases of infection in the bursa of Fabricius of MD-resistant and –susceptible birds. Major global variations in chromatin marks were observed at different stages of MD in the two lines. Differential H3K27me3 marks were associated with immune-related pathways, such as MAP kinase signaling, focal adhesion and neuroactive ligand receptor interaction, and suggested varying degrees of silencing in response to infection. Immune-related microRNAs, e.g. gga-miR-155 and gga-miR-10b, bore chromatin signatures, which suggested their contribution to MD-susceptibility. Finally, several members of the focal adhesion pathway, e.g. THBS4 and ITGA1, showed marked concordance between gene expression and chromatin marks indicating putative epigenetic regulation in response to MDV infection.
Our comprehensive analysis of chromatin signatures, therefore, revealed further clues about the epigenetic effects of MDV infection although further studies are necessary to elucidate the functional implications of the observed variations in histone modifications.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1492-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4404578  PMID: 25896894
12.  Epigenetics and animal virus infections 
PMCID: PMC4349154  PMID: 25788901
histones; non-coding RNAs; epigenetics; virus infection; Methylation
13.  Genome-wide mapping of DNase I hypersensitive sites and association analysis with gene expression in MSB1 cells 
Frontiers in Genetics  2014;5:308.
DNase I hypersensitive sites (DHSs) mark diverse classes of cis-regulatory regions, such as promoters and enhancers. MSB-1 derived from chicken Marek's disease (MD) lymphomas is an MDV-transformed CD4+ T-cell line for MD study. Previously, DNase I HS sites were studied mainly in human cell types for mammalian. To capture the regulatory elements specific to MSB1 cells and explore the molecular mechanisms of T-cell transformation caused by MDV in MD, we generated high-quality of DHSs map and gene expression profile for functional analysis in MSB1 cell line. The total of 21,724 significant peaks of DHSs was identified from around 40 million short reads. DHSs distribution varied between chromosomes and they preferred to enrich in the gene-rich chromosomes. More interesting, DHSs enrichments appeared to be scarce on regions abundant in CpG islands. Besides, we integrated DHSs into the gene expression data and found that DHSs tended to enrich on high expressed genes throughout whole gene regions while DHSs did not show significant changes for low and silent expressed genes. Furthermore, the correlation of DHSs with lincRNAs expression was also calculated and it implied that enhancer-associated lincRNAs probably originated from enhancer-like regions of DHSs. Together, our results indicated that DNase I HS sites highly correlate with active genes expression in MSB1 cells, suggesting DHSs can be considered as markers to identify the cis-regulatory elements associated with chicken Marek's disease.
PMCID: PMC4195362  PMID: 25352859
DNase I; DHS; intergenic DHSs; MSB1; CpG islands; gene expressions; long non-coding RNAs; Marek's disease (MD)
14.  Genome wide CNV analysis reveals additional variants associated with milk production traits in Holsteins 
BMC Genomics  2014;15(1):683.
Milk production is an economically important sector of global agriculture. Much attention has been paid to the identification of quantitative trait loci (QTL) associated with milk, fat, and protein yield and the genetic and molecular mechanisms underlying them. Copy number variation (CNV) is an emerging class of variants which may be associated with complex traits.
In this study, we performed a genome-wide association between CNVs and milk production traits in 26,362 Holstein bulls and cows. A total of 99 candidate CNVs were identified using Illumina BovineSNP50 array data, and association tests for each production trait were performed using a linear regression analysis with PCA correlation. A total of 34 CNVs on 22 chromosomes were significantly associated with at least one milk production trait after false discovery rate (FDR) correction. Some of those CNVs were located within or near known QTL for milk production traits. We further investigated the relationship between associated CNVs with neighboring SNPs. For all 82 combinations of traits and CNVs (less than 400 kb in length), we found 17 cases where CNVs directly overlapped with tag SNPs and 40 cases where CNVs were adjacent to tag SNPs. In 5 cases, CNVs located were in strong linkage disequilibrium with tag SNPs, either within or adjacent to the same haplotype block. There were an additional 20 cases where CNVs did not have a significant association with SNPs, suggesting that the effects of those CNVs were probably not captured by tag SNPs.
We conclude that combining CNV with SNP analyses reveals more genetic variations underlying milk production traits than those revealed by SNPs alone.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-683) contains supplementary material, which is available to authorized users.
PMCID: PMC4152564  PMID: 25128478
Copy number variation (CNV); dPTA; Association; Milk production traits
15.  DNMT gene expression and methylome in Marek’s disease resistant and susceptible chickens prior to and following infection by MDV 
Epigenetics  2013;8(4):431-444.
Marek’s disease (MD) is characterized as a T cell lymphoma induced by a cell-associated α-herpesvirus, Marek’s disease virus type 1 (MDV1). As with many viral infectious diseases, DNA methylation variations were observed in the progression of MD; these variations are thought to play an important role in host-virus interactions. We observed that DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) were differentially expressed in chicken MD-resistant line 63 and MD-susceptible line 72 at 21 d after MDV infection. To better understand the role of methylation variation induced by MDV infection in both chicken lines, we mapped the genome-wide DNA methylation profiles in each line using Methyl-MAPS (methylation mapping analysis by paired-end sequencing). Collectively, the data sets collected in this study provide a more comprehensive picture of the chicken methylome. Overall, methylation levels were reduced in chickens from the resistant line 63 after MDV infection. We identified 11,512 infection-induced differential methylation regions (iDMRs). The number of iDMRs was larger in line 72 than in line 63, and most of iDMRs found in line 63 were overlapped with the iDMRs found in line 72. We further showed that in vitro methylation levels were associated with MDV replication, and found that MDV propagation in the infected cells was restricted by pharmacological inhibition of DNA methylation. Our results suggest that DNA methylation in the host may be associated with disease resistance or susceptibility. The methylation variations induced by viral infection may consequentially change the host transcriptome and result in diverse disease outcomes.
PMCID: PMC3674052  PMID: 23538681
DNA methylation; Marek’s disease; chicken; epigenetics; tumor; viral infection
16.  Co-Expression Analysis of Fetal Weight-Related Genes in Ovine Skeletal Muscle during Mid and Late Fetal Development Stages 
Background: Muscle development and lipid metabolism play important roles during fetal development stages. The commercial Texel sheep are more muscular than the indigenous Ujumqin sheep.
Results: We performed serial transcriptomics assays and systems biology analyses to investigate the dynamics of gene expression changes associated with fetal longissimus muscles during different fetal stages in two sheep breeds. Totally, we identified 1472 differentially expressed genes during various fetal stages using time-series expression analysis. A systems biology approach, weighted gene co-expression network analysis (WGCNA), was used to detect modules of correlated genes among these 1472 genes. Dramatically different gene modules were identified in four merged datasets, corresponding to the mid fetal stage in Texel and Ujumqin sheep, the late fetal stage in Texel and Ujumqin sheep, respectively. We further detected gene modules significantly correlated with fetal weight, and constructed networks and pathways using genes with high significances. In these gene modules, we identified genes like TADA3, LMNB1, TGF-β3, EEF1A2, FGFR1, MYOZ1, and FBP2 correlated with fetal weight.
Conclusion: Our study revealed the complex network characteristics involved in muscle development and lipid metabolism during fetal development stages. Diverse patterns of the network connections observed between breeds and fetal stages could involve some hub genes, which play central roles in fetal development, correlating with fetal weight. Our findings could provide potential valuable biomarkers for selection of body weight-related traits in sheep and other livestock.
PMCID: PMC4183924  PMID: 25285036
Serial expression analysis; WGCNA; fetal development stages; fetal weight.
17.  Genome-Wide Copy Number Variant Analysis in Inbred Chickens Lines With Different Susceptibility to Marek’s Disease 
G3: Genes|Genomes|Genetics  2013;3(2):217-223.
Breeding of genetically resistant chickens to Marek’s disease (MD) is a vital strategy to poultry health. To find the markers underlying the genetic resistance to MD, copy number variation (CNV) was examined in inbred MD-resistant and -susceptible chicken lines. A total of 45 CNVs were found in four lines of chickens, and 28 were potentially involved in immune response and cell proliferation, etc. Importantly, two CNVs related with MD resistance were transmitted to descendent recombinant congenic lines that differ in susceptibility to MD. Our findings may lead to better strategies for genetic improvement of disease resistance in poultry.
PMCID: PMC3564982  PMID: 23390598
CNV; disease resistance; Marek’s disease; chicken
18.  Marek’s disease virus infection induces widespread differential chromatin marks in inbred chicken lines 
BMC Genomics  2012;13:557.
Marek’s disease (MD) is a neoplastic disease in chickens caused by the MD virus (MDV). Successful vaccine development against MD has resulted in increased virulence of MDV and the understanding of genetic resistance to the disease is, therefore, crucial to long-term control strategies. Also, epigenetic factors are believed to be one of the major determinants of disease response.
Here, we carried out comprehensive analyses of the epigenetic landscape induced by MDV, utilizing genome-wide histone H3 lysine 4 and lysine 27 trimethylation maps from chicken lines with varying resistance to MD. Differential chromatin marks were observed on genes previously implicated in the disease such as MX1 and CTLA-4 and also on genes reported in other cancers including IGF2BP1 and GAL. We detected bivalent domains on immune-related transcriptional regulators BCL6, CITED2 and EGR1, which underwent dynamic changes in both lines as a result of MDV infection. In addition, putative roles for GAL in the mechanism of MD progression were revealed.
Our results confirm the presence of widespread epigenetic differences induced by MD in chicken lines with different levels of genetic resistance. A majority of observed epigenetic changes were indicative of increased levels of viral infection in the susceptible line symptomatic of lowered immunocompetence in these birds caused by early cytolytic infection. The GAL system that has known anti-proliferative effects in other cancers is also revealed to be potentially involved in MD progression. Our study provides further insight into the mechanisms of MD progression while revealing a complex landscape of epigenetic regulatory mechanisms that varies depending on host factors.
PMCID: PMC3505159  PMID: 23072359
Histone modifications; Thymus; Differential marks; Bivalent domain; Chromatin signature; Marek’s disease
19.  WaveSeq: A Novel Data-Driven Method of Detecting Histone Modification Enrichments Using Wavelets 
PLoS ONE  2012;7(9):e45486.
Chromatin immunoprecipitation followed by next-generation sequencing is a genome-wide analysis technique that can be used to detect various epigenetic phenomena such as, transcription factor binding sites and histone modifications. Histone modification profiles can be either punctate or diffuse which makes it difficult to distinguish regions of enrichment from background noise. With the discovery of histone marks having a wide variety of enrichment patterns, there is an urgent need for analysis methods that are robust to various data characteristics and capable of detecting a broad range of enrichment patterns.
To address these challenges we propose WaveSeq, a novel data-driven method of detecting regions of significant enrichment in ChIP-Seq data. Our approach utilizes the wavelet transform, is free of distributional assumptions and is robust to diverse data characteristics such as low signal-to-noise ratios and broad enrichment patterns. Using publicly available datasets we showed that WaveSeq compares favorably with other published methods, exhibiting high sensitivity and precision for both punctate and diffuse enrichment regions even in the absence of a control data set. The application of our algorithm to a complex histone modification data set helped make novel functional discoveries which further underlined its utility in such an experimental setup.
WaveSeq is a highly sensitive method capable of accurate identification of enriched regions in a broad range of data sets. WaveSeq can detect both narrow and broad peaks with a high degree of accuracy even in low signal-to-noise ratio data sets. WaveSeq is also suited for application in complex experimental scenarios, helping make biologically relevant functional discoveries.
PMCID: PMC3461018  PMID: 23029045
20.  Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array 
BMC Genomics  2012;13:376.
Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases.
In this study using the high density BovineHD SNP array, we performed high resolution CNV analyses on both Btau_4.0 and UMD3.1 with 674 animals of 27 cattle breeds. We first compared CNV results derived from these two different SNP array platforms on Btau_4.0. With two thirds of the animals shared between studies, on Btau_4.0 we identified 3,346 candidate CNV regions representing 142.7 megabases (~4.70%) of the genome. With a similar total length but 5 times more event counts, the average CNVR length of current Btau_4.0 dataset is significantly shorter than the previous one (42.7 kb vs. 205 kb). Although subsets of these two results overlapped, 64% (91.6 megabases) of current dataset was not present in the previous study. We also performed similar analyses on UMD3.1 using these BovineHD SNP array results. Approximately 50% more and 20% longer CNVs were called on UMD3.1 as compared to those on Btau_4.0. However, a comparable result of CNVRs (3,438 regions with a total length 146.9 megabases) was obtained. We suspect that these results are due to the UMD3.1 assembly's efforts of placing unplaced contigs and removing unmerged alleles. Selected CNVs were further experimentally validated, achieving a 73% PCR validation rate, which is considerably higher than the previous validation rate. About 20-45% of CNV regions overlapped with cattle RefSeq genes and Ensembl genes. Panther and IPA analyses indicated that these genes provide a wide spectrum of biological processes involving immune system, lipid metabolism, cell, organism and system development.
We present a comprehensive result of cattle CNVs at a higher resolution and sensitivity. We identified over 3,000 candidate CNV regions on both Btau_4.0 and UMD3.1, further compared current datasets with previous results, and examined the impacts of genome assemblies on CNV calling.
PMCID: PMC3583728  PMID: 22866901
Cattle genome; Breed; Copy number variation (CNV); Single nucleotide polymorphism (SNP)
21.  Histone Methylation Analysis and Pathway Predictions in Chickens after MDV Infection 
PLoS ONE  2012;7(7):e41849.
Marek's disease (MD) is a lymphoproliferative disease in chicken induced by Marek's disease virus (MDV). Although studies have focused on the genetic differences between the resistant and susceptible chicken, less is known about the role of epigenetic factors in MD. In this study, genome-wide histone modifications in the non-MHC-associated resistant and susceptible chicken lines were examined. We found that tri-methylation at histone H3 Lys4 (H3K4me3) enrichment is positively correlated with the expression of protein coding genes as well as microRNA (miRNA) genes, whereas tri-methylation at histone H3 Lys27 (H3K27me3) exhibits a negative correlation. By identifying line-specific histone modifications in MDV infection, we found unique H3K4me3 islands in the resistant chicken activated genes, which are related to immune response and cell adhesion. Interestingly, we also found some miRNAs from unique H3K27me3 patterns in the susceptible chickens that targeted genes involved in 5-hydroxytryptamine (5-HT)-receptor and adrenergic receptor pathways. In conclusion, dynamic line-specific histone modifications in response to MDV infection suggested that intrinsic epigenetic mechanisms may play a role in MD-resistance and -susceptibility.
PMCID: PMC3406056  PMID: 22848633
22.  Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes 
PLoS ONE  2012;7(6):e39094.
Gene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.
The genome-wide status of H3K27me3 modifications on blood lymphocytes in lactating Holsteins was performed via ChIP-Seq approach. Combined with digital gene expression (DGE) technique, the regulation effects of H3K27me3 on genes expressions were analyzed.
The ChIP-seq results showed that the peaks of H3K27me3 in cows lymphocytes were mainly enriched in the regions of up20K (∼50%), down20K (∼30%) and intron (∼28%) of the genes. Only ∼3% peaks were enriched in exon regions. Moreover, the highest H3K27me3 modification levels were mainly around the 2 Kb upstream of transcriptional start sites (TSS) of the genes. Using conjoint analysis with DGE data, we found that H3K27me3 marks tended to repress target genes expressions throughout whole gene regions especially acting on the promoter region. A total of 53 differential expressed genes were detected in third parity cows compared to first parity, and the 25 down-regulated genes (PSEN2 etc.) were negatively correlated with H3K27me3 levels on up2Kb to up1Kb of the genes, while the up-regulated genes were not showed in this relationship.
The first blueprint of bovine H3K27me3 marks that mediates gene silencing was generated. H3K27me3 plays its repressed role mainly in the regulatory region in bovine lymphocytes. The up2Kb to up1Kb region of the down-regulated genes in third parity cows could be potential target of H3K27me3 regulation. Further studies are warranted to understand the regulation mechanisms of H3K27me3 on somatic cell count increases and milk losses in latter parities of cows.
PMCID: PMC3386284  PMID: 22761725
23.  miRNA-dysregulation associated with tenderness variation induced by acute stress in Angus cattle 
miRNAs are a class of small, single-stranded, non-coding RNAs that perform post-transcriptional repression of target genes by binding to 3’ untranslated regions. Research has found that miRNAs involved in the regulation of many metabolic processes. Here we uncovered that the beef quality of Angus cattle sharply diversified after acute stress. By performing miRNA microarray analysis, 13 miRNAs were significantly differentially expressed in stressed group compared to control group. Using a bioinformatics method, 135 protein-coding genes were predicted as the targets of significant differentially expressed miRNAs. Gene Ontology (GO) term and Ingenuity Pathway Analysis (IPA) mined that these target genes involved in some important pathways, which may have impact on meat quality and beef tenderness.
PMCID: PMC3436641  PMID: 22958451
miRNA; Bovine; Beef tenderness; Stress
24.  Comprehensive Analysis of Gene-Environmental Interactions with Temporal Gene Expression Profiles in Pseudomonas aeruginosa 
PLoS ONE  2012;7(4):e35993.
To explore gene-environment interactions, based on temporal gene expression information, we analyzed gene and treatment information intensively and inferred interaction networks accordingly. The main idea is that gene expression reflects the response of genes to environmental factors, assuming that variations of gene expression occur under different conditions. Then we classified experimental conditions into several subgroups based on the similarity of temporal gene expression profiles. This procedure is useful because it allows us to combine diverse gene expression data as they become available, and, especially, allowing us to lay the regulatory relationships on a concrete biological basis. By estimating the activation points, we can visualize the gene behavior, and obtain a consensus gene activation order, and hence describe conditional regulatory relationships. The estimation of activation points and building of synthetic genetic networks may result in important new insights in the ongoing endeavor to understand the complex network of gene regulation.
PMCID: PMC3338772  PMID: 22558298
25.  Functional Genomic Analysis of Variation on Beef Tenderness Induced by Acute Stress in Angus Cattle 
Beef is one of the leading sources of protein, B vitamins, iron, and zinc in human food. Beef palatability is based on three general criteria: tenderness, juiciness, and flavor, of which tenderness is thought to be the most important factor. In this study, we found that beef tenderness, measured by the Warner-Bratzler shear force (WBSF), was dramatically increased by acute stress. Microarray analysis and qPCR identified a variety of genes that were differentially expressed. Pathway analysis showed that these genes were involved in immune response and regulation of metabolism process as activators or repressors. Further analysis identified that these changes may be related with CpG methylation of several genes. Therefore, the results from this study provide an enhanced understanding of the mechanisms that genetic and epigenetic regulations control meat quality and beef tenderness.
PMCID: PMC3332163  PMID: 22566754

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