Zhenhui Li

Comparison of the Genome-Wide DNA Methylation Profiles between Fast-Growing and Slow-Growing Broilers

Introduction Growth traits are important in poultry production, however, little is known for its regulatory mechanism at epigenetic level. Therefore, in this study, we aim to compare DNA methylation profiles between fast- and slow-growing broilers in order to identify candidate genes for chicken growth. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation pattern in high and low tails of Recessive White Rock (WRRh; WRRl) and that of Xinhua Chickens (XHh; XHl) at 7 weeks of age. The results showed that the average methylation density was the lowest in CGIs followed by promoters. Within the gene body, the methylation density of introns was higher than that of UTRs and exons. Moreover, different methylation levels were observed in different repeat types with the highest in LINE/CR1. Methylated CGIs were prominently distributed in the intergenic regions and were enriched in the size ranging 200–300 bp. In total 13,294 methylated genes were found in four samples, including 4,085 differentially methylated genes of WRRh Vs. WRRl, 5,599 of XHh Vs. XHl, 4,204 of WRRh Vs. XHh, as well as 7,301 of WRRl Vs. XHl. Moreover, 132 differentially methylated genes related to growth and metabolism were observed in both inner contrasts (WRRh Vs. WRRl and XHh Vs. XHl), whereas 129 differentially methylated genes related to growth and metabolism were found in both across-breed contrasts (WRRh Vs. XHh and WRRl Vs. XHl). Further analysis showed that overall 75 genes exhibited altered DNA methylation in all four contrasts, which included some well-known growth factors of IGF1R, FGF12, FGF14, FGF18, FGFR2, and FGFR3. In addition, we validate the MeDIP-seq results by bisulfite sequencing in some regions. Conclusions This study revealed the global DNA methylation pattern of chicken muscle, and identified candidate genes that potentially regulate muscle development at 7 weeks of age at methylation level.

The transient expression of miR-203 and its inhibiting effects on skeletal muscle cell proliferation and differentiation

Previous studies have shown that miR-203 is a skin-specific microRNA (miRNA) with a profound role in skin cell differentiation. However, emerging microarray and deep sequencing data revealed that miR-203 is also expressed in embryonic skeletal muscle and myoblasts. In this study, we found that miR-203 was transiently upregulated in chicken embryos on days 10 to 16 (E10–E16) and was sharply downregulated and even not expressed after E16 in chicken embryonic skeletal muscle. Histological profiles and weight variations of embryo skeletal muscle revealed that miR-203 expression is correlated with muscle development. In vitro experiments showed that miR-203 exhibited downregulated expression during myoblast differentiation into myotubes. miR-203 overexpression inhibited myoblast proliferation and differentiation, whereas its loss-of-function increased myoblast proliferation and differentiation. During myogenesis, miR-203 can target and inhibit the expression of c-JUN and MEF2C, which were important for cell proliferation and muscle development, respectively. The overexpression of c-JUN significantly promoted myoblast proliferation. Conversely, knockdown of c-JUN by siRNA suppressed myoblast proliferation. In addition, the knockdown of MEF2C by siRNA significantly inhibited myoblast differentiation. Altogether, these data not only suggested that the expression of miR-203 is transitory during chicken skeletal muscle development but also showed a novel role of miR-203 in inhibiting skeletal muscle cell proliferation and differentiation by repressing c-JUN and MEF2C, respectively.

MicroRNA-23b Promotes Avian Leukosis Virus Subgroup J (ALV-J) Replication by Targeting IRF1.

Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3′-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1.

Integrated Analysis of Long Non-coding RNAs (LncRNAs) and mRNA Expression Profiles Reveals the Potential Role of LncRNAs in Skeletal Muscle Development of the Chicken

Long non-coding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation. However, little is currently known about the mechanisms by which they regulate skeletal muscle development in the chicken. In this study, we used RNA sequencing to profile the leg muscle transcriptome (lncRNA and mRNA) at three stages of skeletal muscle development in the chicken: embryonic day 11 (E11), embryonic day 16 (E16), and 1 day after hatching (D1). In total, 129, 132, and 45 differentially expressed lncRNAs, and 1798, 3072, and 1211 differentially expressed mRNAs were identified in comparisons of E11 vs. E16, E11 vs. D1, and E16 vs. D1, respectively. Moreover, we identified the cis- and trans-regulatory target genes of differentially expressed lncRNAs, and constructed lncRNA-gene interaction networks. In total, 126 and 200 cis-targets, and two and three trans-targets were involved in lncRNA-gene interaction networks that were constructed based on the E11 vs. E16, and E11 vs. D1 comparisons, respectively. The comparison of the E16 vs. D1 lncRNA-gene network comprised 25 cis-targets. We determined that lncRNA target genes are potentially involved in cellular development, and cellular growth and proliferation using Ingenuity Pathway Analysis. The gene networks identified for the E11 vs. D1 comparison were involved in embryonic development, organismal development and tissue development. The present study provides an RNA sequencing based evaluation of lncRNA function during skeletal muscle development in the chicken. Comprehensive analysis facilitated the identification of lncRNAs and target genes that might contribute to the regulation of different stages of skeletal muscle development.

Proteomic Analysis of Chicken Skeletal Muscle during Embryonic Development

Embryonic growth and development of skeletal muscle is a major determinant of muscle mass, and has a significant effect on meat production in chicken. To assess the protein expression profiles during embryonic skeletal muscle development, we performed a proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ) in leg muscle tissues of female Xinghua chicken at embryonic age (E) 11, E16, and 1-day post hatch (D1). We identified 3,240 proteins in chicken embryonic muscle and 491 of them were differentially expressed (fold change ≥ 1.5 or ≤ 0.666 and p < 0.05). There were 19 up- and 32 down-regulated proteins in E11 vs. E16 group, 238 up- and 227 down-regulated proteins in E11 vs. D1 group, and 13 up- and 5 down-regulated proteins in E16 vs. D1 group. Protein interaction network analyses indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis, muscle contraction, and oxidative phosphorylation. Integrative analysis of proteome and our previous transcriptome data found 189 differentially expressed proteins that correlated with their mRNA level. The interactions between these proteins were also involved in muscle contraction and oxidative phosphorylation pathways. The lncRNA-protein interaction network found four proteins DMD, MYL3, TNNI2, and TNNT3 that are all involved in muscle contraction and may be lncRNA regulated. These results provide several candidate genes for further investigation into the molecular mechanisms of chicken embryonic muscle development, and enable us to better understanding their regulation networks and biochemical pathways.

DNA methylome in spleen of avian pathogenic Escherichia coli-challenged broilers and integration with mRNA expression.

Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV.

Genome-wide association study of aggressive behaviour in chicken

In the poultry industry, aggressive behaviour is a large animal welfare issue all over the world. To date, little is known about the underlying genetics of the aggressive behaviour. Here, we performed a genome-wide association study (GWAS) to explore the genetic mechanism associated with aggressive behaviour in chickens. The GWAS results showed that a total of 33 SNPs were associated with aggressive behaviour traits (P < 4.6E-6). rs312463697 on chromosome 4 was significantly associated with aggression (P = 2.10905E-07), and it was in the intron region of the sortilin-related VPS10 domain containing receptor 2 (SORCS2) gene. In addition, biological function analysis of the nearest 26 genes around the significant SNPs was performed with Ingenuity Pathway Analysis. An interaction network contained 17 genes was obtained and SORCS2 was involved in this network, interacted with nerve growth factor (NGF), nerve growth factor receptor (NGFR), dopa decarboxylase (L-dopa) and dopamine. After knockdown of SORCS2, the mRNA levels of NGF, L-dopa and dopamine receptor genes DRD1, DRD2, DRD3 and DRD4 were significantly decreased (P < 0.05). In summary, our data indicated that SORCS2 might play an important role in chicken aggressive behaviour through the regulation of dopaminergic pathways and NGF.

LncRNA-Six1 Encodes a Micropeptide to Activate Six1 in Cis and Is Involved in Cell Proliferation and Muscle Growth.

Long non-coding RNAs (lncRNAs) play important roles in epigenetic regulation of skeletal muscle development. In our previous RNA-seq study (accession number GSE58755), we found that lncRNA-Six1 is an lncRNA that is differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. In this study, we have further demonstrated that lncRNA-Six1 is located 432 bp upstream of the gene encoding the protein Six homeobox 1 (Six1). A dual-luciferase reporter assay identified that lncRNA-Six1 overlaps the Six1 proximal promoter. In lncRNA-Six1, a micropeptide of about 7.26 kDa was found to play an important role in the lncRNA-Six1 in cis activity. Overexpression of lncRNA-Six1 promoted the mRNA and protein expression level of the Six1 gene, while knockdown of lncRNA-Six1 inhibited Six1 expression. Moreover, tissue expression profiles showed that both the lncRNA-Six1 and the Six1 mRNA were highly expressed in chicken breast tissue. LncRNA-Six1 overexpression promoted cell proliferation and induced cell division. Conversely, its loss of function inhibited cell proliferation and reduced cell viability. Similar effects were observed after overexpression or knockdown of the Six1 gene. In addition, overexpression or knockdown of Six1 promoted or inhibited, respectively, the expression levels of muscle-growth-related genes, such as MYOG, MYHC, MYOD, IGF1R, and INSR. Taken together, these data demonstrate that lncRNA-Six1 carries out cis-acting regulation of the protein-encoding Six1 gene, and encodes a micropeptide to activate Six1 gene, thus promoting cell proliferation and being involved in muscle growth.

Circular RNAs are abundant and dynamically expressed during embryonic muscle development in chickens

Abstract The growth and development of skeletal muscle is regulated by proteins as well as non-coding RNAs. Circular RNAs (circRNAs) are universally expressed in various tissues and cell types, and regulate gene expression in eukaryotes. To identify the circRNAs during chicken embryonic skeletal muscle development, leg muscles of female Xinghua (XH) chicken at three developmental time points 11 embryo age (E11), 16 embryo age (E16) and 1 day post hatch (P1) were performed RNA sequencing. We identified 13,377 circRNAs with 3,036 abundantly expressed and most were derived from coding exons. A total of 462 differentially expressed circRNAs were identified (fold change > 2; q-value < 0.05). Parental genes of differentially expressed circRNAs were related to muscle biological processes. There were 946 exonic circRNAs have been found that harbored one or more miRNA-binding site for 150 known miRNAs. We validated that circRBFOX2s promoted cell proliferation through interacted with miR-206. These data collectively indicate that circRNAs are abundant and dynamically expressed during embryonic muscle development and could play key roles through sequestering miRNAs as well as other functions.

MiR-34b-5p Suppresses Melanoma Differentiation-Associated Gene 5 (MDA5) Signaling Pathway to Promote Avian Leukosis Virus Subgroup J (ALV-J)-Infected Cells Proliferaction and ALV-J Replication

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that has a similar replication cycle to multiple viruses and therefore can be used as a model system for viral entry into host cells. However, there are few reports on the genes or microRNAs (miRNAs) that are responsible for the replication of ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of miR-34b-5p was significantly upregulated in ALV-J-infected chicken spleens compared to non-infected chicken spleens, but melanoma differentiation-associated gene 5 (MDA5) had the opposite expression pattern. In this study, a dual-luciferase reporter assay showed that MDA5 is a direct target of miR-34b-5p. In vitro, overexpression of miR-34b-5p accelerated the proliferation of ALV-J-infected cells by inducing the progression from G2 to S phase and it promoted cell migration. Ectopic expression of MDA5 inhibited ALV-J-infected cell proliferation, the cell cycle and cell migration, and knockdown of MDA5 promoted proliferation, the cell cycle and migration. In addition, during ALV-J infections, MDA5 can detect virus invasion and it triggers the MDA5 signaling pathway. MDA5 overexpression can activate the MDA5 signaling pathway, and thus it can inhibit the mRNA and protein expression of the ALV-J env gene and it can suppress virion secretion. In contrast, in response to the knockdown of MDA5 by small interfering RNA (siRNA) or an miR-34b-5p mimic, genes in the MDA5 signaling pathway were significantly downregulated (P < 0.05), but the mRNA and protein expression of ALV-J env and the sample-to-positive ratio of virion in the supernatants were increased. This indicates that miR-34b-5p is able to trigger the MDA5 signaling pathway and affect ALV-J infections. Together, these results suggest that miR-34b-5p targets MDA5 to accelerate the proliferation and migration of ALV-J-infected cells, and it promotes ALV-J replication, via the MDA5 signaling pathway.