Avian influenza has caused significant damage to the poultry industry globally. Consequently, efforts have been made to elucidate the disease mechanisms as well as the mechanisms of disease resistance. Here, by investigating two chicken breeds with distinct responses to avian influenza virus (AIV), Leghorn GB2 and Fayoumi M43, we compared their genome, methylation, and transcriptome differences. MX1, HSP90AB1, and HSP90B1 exhibited high degrees of genetic differentiation (FST) between the two species. Except for the MX1-involved direct anti-virus mechanism, we found that at the methylation and transcriptome levels, the more AIV-resistant breed, Fayoumi, exhibited less variation compared with Leghorn after AIV inoculation, which included change trends in differentially expressed regions, top-fold change genes with FDR-corrected p < 0.05, immune response related genes, and housekeeping genes. Fayoumi also showed better consistency regarding changes in methylation and changes at the transcriptome level. Our results suggest a homeostasis hypothesis for avian influenza resistance, with Fayoumi maintaining superior homeostasis at both the epigenetic and gene expression levels. Three candidate genes-MX1, HSP90AB1, and HSP90B1-showed genetic differentiation and altered gene expression, methylation, and protein expression, which merit attention in further functional studies.

Background

DNA cytosine methylation is an important epigenetic modification that has significant effects on a variety of biological processes in animals. Avian species hold a crucial position in evolutionary history. In this study, we used whole-genome bisulfite sequencing (MethylC-seq) to generate single base methylation profiles of lungs in two genetically distinct and highly inbred chicken lines (Fayoumi and Leghorn) that differ in genetic resistance to multiple pathogens, and we explored the potential regulatory role of DNA methylation associated with immune response differences between the two chicken lines.

Methods

The MethylC-seq was used to generate single base DNA methylation profiles of Fayoumi and Leghorn birds. In addition, transcriptome profiling using RNA-seq from the same chickens and tissues were obtained to interrogate how DNA methylation regulates gene transcription on a genome-wide scale.

Results

The general DNA methylation pattern across different regions of genes was conserved compared to other species except for hyper-methylation of repeat elements, which was not observed in chicken. The methylation level of miRNA and pseudogene promoters was high, which indicates that silencing of these genes may be partially due to promoter hyper-methylation. Interestingly, the promoter regions of more recently evolved genes tended to be more highly methylated, whereas the gene body regions of evolutionarily conserved genes were more highly methylated than those of more recently evolved genes. Immune-related GO (Gene Ontology) terms were significantly enriched from genes within the differentially methylated regions (DMR) between Fayoumi and Leghorn, which implicates DNA methylation as one of the regulatory mechanisms modulating immune response differences between these lines.

Conclusions

This study establishes a single-base resolution DNA methylation profile of chicken lung and suggests a regulatory role of DNA methylation in controlling gene expression and maintaining genome transcription stability. Furthermore, profiling the DNA methylomes of two genetic lines that differ in disease resistance provides a unique opportunity to investigate the potential role of DNA methylation in host disease resistance. Our study provides a foundation for future studies on epigenetic modulation of host immune response to pathogens in chickens.

The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.

Background

Mitochondrial dysfunction is a prominent hallmark of many sensory neuropathies. The purpose of this study was to assess the influence of mitochondrial DNA sequence variation on peripheral nerve function in the population-based Health, Aging, and Body Composition Study.

Methods

We investigated the role of common mitochondrial DNA variation (n = 1,580) and complete mitochondrial DNA sequences (n = 138) on peroneal motor nerve conduction velocity and amplitude, average vibration detection threshold, and monofilament sensitivity.

Results

Nominal associations among common mitochondrial DNA variants and haplogroups were identified but were not statistically significant after adjustment for multiple comparisons. Sequence-based approaches were used to identify aggregate variant associations across the 16S rRNA (weighted-sum, p = 2E-05 and variable threshold, p = 9E-06) for nerve conduction velocity. Several of these rare 16S variants occurred at or near sites with earlier disease associations and are also in close proximity to the peptidyl transferase center, which is the catalytic center of the 16S rRNA CONCLUSIONS: These results suggest that sequence variation related to mitochondrial protein synthesis/assembly is associated with peripheral nerve function and may provide insight into targets for intervention or new clinical strategies to preserve nerve function in late life.

The decline in activity energy expenditure underlies a range of age-associated pathological conditions, neuromuscular and neurological impairments, disability, and mortality. The majority (90%) of the energy needs of the human body are met by mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS is dependent on the coordinated expression and interaction of genes encoded in the nuclear and mitochondrial genomes. We examined the role of mitochondrial genomic variation in free-living activity energy expenditure (AEE) and physical activity levels (PAL) by sequencing the entire (~16.5 kilobases) mtDNA from 138 Health, Aging, and Body Composition Study participants. Among the common mtDNA variants, the hypervariable region 2 m.185G>A variant was significantly associated with AEE (p=0.001) and PAL (p=0.0005) after adjustment for multiple comparisons. Several unique nonsynonymous variants were identified in the extremes of AEE with some occurring at highly conserved sites predicted to affect protein structure and function. Of interest is the p.T194M, CytB substitution in the lower extreme of AEE occurring at a residue in the Qi site of complex III. Among participants with low activity levels, the burden of singleton variants was 30% higher across the entire mtDNA and OXPHOS complex I when compared to those having moderate to high activity levels. A significant pooled variant association across the hypervariable 2 region was observed for AEE and PAL. These results suggest that mtDNA variation is associated with free-living AEE in older persons and may generate new hypotheses by which specific mtDNA complexes, genes, and variants may contribute to the maintenance of activity levels in late life.

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.