Differential expression and methylation patterns of NFATC1, NADSYN1 and JAK3 gene in equine chondrocytes expanded in monolayer culture.

Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcript abundance and performed DNA bisulfite sequencing of upstream located elements. It seems that anabolic-like effects of chondrogenic differentiation were observed in form of NFATC1 and NADSYN1 upregulation in chondrocytes at the earlier stages of passaging whereas JAK3 upregulation at the 11th passage was the sign of chondrocytes dedifferentiation. Summarizing the inversely correlated DNA methylation and expression patterns in NFATC1 and JAK3 locus might be relevant for cellular dedifferentiation during chondrocyte expansion in monolayer. Obtained results are supportive for further studies on the role of encoded proteins in regenerative biology of articular cartilage using in vitro expanded chondrocytes.

Short communication: Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci.

Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.

Structural and functional analysis of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene in response to infection with coagulase-negative and coagulase-positive staphylococci.

Splice variants of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene have been identified, and differences in the expression of this gene have been demonstrated at the mRNA level in the mammary glands of healthy and mastitis-infected dairy cows. At the same time, significant associations have been found between a deletion in the SLAM7 gene exon, the occurrence of different splice variants, and the occurrence of mastitis in one group of dairy cows. An expression study was conducted on 40 Polish Holstein-Friesian dairy cows of the Black and White variety (group I). Milk samples were taken for microbiological analysis 2 d before slaughter and examined for the presence of bacteria. Immediately after slaughter, mammary tissue samples were taken and divided into 3 groups according to the health status of the mammary gland: healthy (without pathogenic bacteria in milk), coagulase-negative staphylococci (CNS), and coagulase-positive staphylococci (CPS). Based on different SLAMF7 gene DNA fragments, 2 alternative variants of this gene (V1 and V2) and complete gene expression were identified. Separate analyses performed for each isoform showed that the health status of the cow was strongly associated with the expression level of individual variants. The highest expression was detected for the SLAMF7 complete amplicon in healthy cows, and in the CNS and CPS cows the expression of this variant was also higher than V1 and V2. Sanger sequencing was applied to detect the polymorphism/indel variant in the second exon of the SLAMF7 gene probably having the greatest effect on the protein structure and function of SLAMF7. Two genotypes were detected: AA (wild-type) and AB (insertion A). In healthy cows, the frequency of homozygotes AA was higher than the heterozygotes, whereas in the infected animals, the genotypic distribution was the opposite. An association analysis between the identified polymorphism and production traits-including somatic cell count, as well as lactose, protein, and casein content and yield as indicators of subclinical mastitis occurrence-was performed on the group II cows (166 Polish Holstein-Friesian dairy cows). Unfortunately, due to the low number of AB animals, no relationship was demonstrated between genotype in the second exon and the health status of cows. Additionally, the difference in the percentage of SLAMF7-targeted DNA methylation between the groups of animals was not significant, with an average of ∼66 to 68%.

Methylation Marks of Blood Leukocytes of Native Hucul Mares Differentiated in Age.

Horses are one of the longest-living species of farm animals. Advanced age is often associated with a decrease in body condition, dysfunction of immune system, and late-onset disorders. Due to this, the search for new solutions in the prevention and treatment of pathological conditions of the advanced age of horses is desirable. That is why the identification of aging-related changes in the horse genome is interesting in this respect. In the recent years, the research on aging includes studies of age-related epigenetic effects observed on the DNA methylation level. We applied reduced representation bisulfite sequencing (RRBS) to uncover a range of age DMR sites in genomes of blood leukocytes derived from juvenile and aged horses of native Hucul breed. Genes colocated with age-related differentially methylated regions (age DMRs) are the members of pathways involved in cellular signal transduction, immune response, neurogenesis, differentiation, development, and cancer progression. A positive correlation was found between methylation states and gene expression in particular loci from our data set. Some of described age DMR-linked genes were also reported elsewhere. Obtained results contribute to the knowledge about the molecular basis of aging of equine blood cells.

Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse.

We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-lines. The general decline of transcript abundance of marker loci was detected in passage 11 which is the sign of dedifferentiation of cultivated chondrocytes in prolonged monolayer culture. Passages 13 and 14 were characterized by the upregulation of a number of genes, possibly due to the heterogeneity of developed cell lines at this stage of the culture. Instead, gradual increase of methylation percent at particular CpG sites of RUNX3 locus was associated with the growing number of passage. This finding led us to the conclusion that epigenetic alterations better describe the stage of cultivated chondrocytes.

DNA methylation patterns of the S100A14, POU2F3 and SFN genes in equine sarcoid tissues.

Genetic and epigenetic alterations in the equine sarcoid, a locally invasive skin tumour of equids, are still poorly characterized. Numerous studies have provided reliable evidence for the relationship between the development of cancer and the loss of function of a number of tumour suppressor genes. In the present study, we assessed methylation levels in the promoter region of SFN, S100A14 and POU2F3 genes in sarcoid samples to clarify whether DNA methylation may be associated with previously identified changes in the expression level of these genes during the course of tumour progression. Using bisulfite sequencing and clone sequencing, we detected that lesional samples had a significantly higher rate of DNA methylation in the analyzed S100A14A region than the corresponding normal skin tissue. A frequent methylation of the SFN and POU2F3 promoter sequences were observed in both the tumour samples and the control skin tissues. Further studies are needed to evaluate the role of aberrant methylation in sarcoid progression and to understand the mechanisms involved in reduced expression of SFN, S100A14 and POU2F3 genes in the lesional tissues.

Comparative analysis of DNA methylation patterns of equine sarcoid and healthy skin samples.

OBJECTIVE: In this study, for the first time we report the genome-wide DNA methylation profile of skin tumour in horses and describe differentially methylated genomic regions (DMRs) with respect to healthy skin. MATERIALS & METHODS: The comparative analysis of DNA methylation patterns detected using Reduced Representation Bisulfite Sequencing (RRBS) technique, allowed identification of 136 regions showing differential methylation between sarcoid and normal skin tissue. RESULTS: Most of the identified DMRs were short fragments, less than 1 kb in size, located in the intergenic regions. Among identified DMRs there were also regions located within genes directly or indirectly related with oncogenesis. We additionally validated 9 CpG sites showing hypomethylation and 9 CpG sites that were hypermethylated in lesional sample, confirming the identified changes in the DNA methylation. CONCLUSION: Knowledge on the changes taking place in the process of DNA methylation may provide a basis for the development of new alternative diagnostic or therapeutic approaches to equine sarcoids.

Transcriptome analysis of equine sarcoids.

Equine sarcoids are the most commonly detected skin tumours in Equidae. In the present research, a comparative transcriptomic analysis was performed which aimed at looking inside a tumour biology and identification of the expression profile as a potential source of cancer specific genes useful as biomarkers. We have used Horse Gene Expression Microarray data from matched equine sarcoids and tumour-distant skin samples. In total, 901 significantly differentially expressed genes (DEGs) between lesional and healthy skin samples have been identified (fold change ≥ 2; P < 0.05). The large subset of DEGs, with decreased expression, was associated with a suppression of malignant transformation, whereas several overexpressed genes were involved in the processes associated with growth and progression of a tumour or immune system activity. Our results, as a first to date, showed comprehensive transcriptome analysis of skin tumour in horses and pinpointed significant pathways and genes related with oncogenesis processes.

Age-related methylation profiles of equine blood leukocytes in the RNASEL locus.

Methylation profiles across three CpG islands of the RNASEL gene were determined in blood leukocyte samples of Anglo-Arabian and Hucul horses. Bisulfite sequencing revealed hypomethylated state of the RNASEL promoter coinciding with methylated CpG island placed inside the gene. Several CpG sites were identified for which the methylation state was influenced by DNA polymorphism. Two of them showed monoallelic methylation. One of the CpG sites revealed functional polymorphism. A number of partially methylated CpG sites have been observed in the promoter area of RNASEL, which were used for the comparison of breed- and age-related effects. Clone bisulfite sequencing of blood leukocyte samples collected at different ages from particular individuals of AA and HC breeds and, also, BSPCR sequencing of 50 samples of juvenile and old AA and HC horses revealed increased methylation in particular CpG sites during aging. The age-related heterogeneity of white blood cells was hypothesized as being one of the potential causes of observed variability of methylation profiles in the RNASEL promoter.

[Microsatellite markers polymorphism in the breeding nutria (Myocastor coypus) population in Poland].

The aim of the research was to establish a microsatellite panel to determine the genetic diversity within the breeding nutria population in Poland. In the study, 92 animals representing six color forms were used. Ten fluorescently labeled microsatellite markers were investigated by multicolored capillary electrophoresis. All the microsatellites were polymorphic. The average heterozygosity observed among the population was 41%. The mean number of alleles per locus was 9.2. The average heterozygosity observed in the whole population was lower than expected. This implies that the nutria population deviates from the Hardy­Weinberg equilibrium. Low M values (from 0.078 to 0.545) of the Garza­Williamson index reveal a reduction of genetic variation in the investigated population and suggest that the breeding nutria population is remnant.

Genome-wide characteristics of copy number variation in Polish Holstein and Polish Red cattle using SNP genotyping assay.

Copy number variation (CNV), which results from deletions or amplifications of large fragments of genomic DNA, is widespread in mammalian genomes and apart from its potential pathogenic effect it is considered as a source of natural genetic diversity. In cattle populations, this kind of genetic variability remains still insufficiently elucidated and studies focusing on the detection of new structural genomic variants in different cattle populations may contribute to a better understanding of cattle breeds' diversity and genetic basis of production traits. In this study, by using BovineSNP50 assay and cnvPartition algorithm we identified CNVs in two different cattle breeds: Holstein (859 animals) and Polish Red (301). In Holstein cattle we found 648 CNVs which could be reduced to 91 non-redundant variable genomic regions (CNVRs) covering in total 168.6 Mb of the genomic sequence. In Polish Red cattle we detected 62 CNVs, localized in 37 variable regions encompassing 22.3 Mb of the sequence, corresponding to 0.89 % of the autosomal genome. Within the regions we identified 1,192 unique RefSeq genes which are engaged in a variety of biological processes. High concordance of the regions' distribution was found between the studied breeds, however copy number variants seemed to be more common in Holstein cattle. About 26 % of the regions described in this study could be classified as newly identified. The results of this study will broaden the knowledge of CNVs in genomes of cattle of different breeds and will provide foundations for further research aiming to identify a relationship between this type of genetic variation and phenotypic traits.

Epigenetic structure and the role of polymorphism in the shaping of DNA methylation patterns of equine OAS1 locus.

DNA methylation patterns and their relation with genetic polymorphisms were determined in the equine OAS1 locus. Genetic variants of OAS1 were previously found to be associated with susceptibility to West Nile virus infections in horses. The subject of the study were white blood cells of 13 juvenile and 13 old horses from AA and HC breed and a set of solid tissues from a single adult horse. The aim was to determine the degree of variation of CpG methylation profiles with concern for tissue type, horse breed and age. Results of direct BSPCR and cloned BSPCR sequencing revealed that all of determined CpG islands (CGIs) were hypermethylated in exception to CGI covering OAS1 promoter and exon 1. One of intragenic CGIs displayed variability of methylation patterns across eight tissue types. The variability of particular sub-types of white blood cells between AA and HC horses were considered as the possible cause of interbreed differences of methylation levels. Comparison of sequence variability between converted and unconverted DNAs of both horse breeds showed polymorphisms of CpG sites to be the source of monoallelic methylation in exception to the polymorphic CpGs located in the OAS1 promoter. Two of them are new polymorphic variants in the OAS1 promoter region. Application of methylation data in conjunction with genetic variation detected at the OAS1 locus might be useful to deepen the knowledge about mechanisms underlying immunity to viral infections in the horse.

IHH gene polymorphism among three horse breeds and its application for association test in horses with osteochondrosis.

Genetic polymorphism of IHH gene were investigated in Angloarabian, Polish Coldblood and Polish Halfbred horses with the inclusion of a group of Polish Halfbreds affected by osteochondrosis. IHH is a good candidate gene for association study of developmental disorders mainly affecting skeleton development. DNA sequence spanning IHH gene annotated in the horse genome and its putative promoter were investigated using SANGER sequencing. Analysis of genetic variability at polymorphic sites in the IHH gene body and the promoter region confirmed genetic differences between warmblood and coldblood horse breeds. A test for allelic and genotypic association at particular SNP sites revealed no association with osteochondrosis in investigated group of Polish Halfbreds. It was concluded that participation of different warmblood breeds in pedigrees of Polish Halfbreds make it difficult to search for genetic variants being associated with this complex disorder in this breed. IHH gene polymorphism investigated among three different horse populations would be valuable for further studies on equine bone developmental disorders.

A case of an intersex horse with 63,X/64,XX/65,XX,del(Y)(q?) karyotype.

Cytogenetic and molecular genetic studies of an intersex horse have been carried out. The investigated animal had overall male body conformation; however, its external genitalia consisted of incompletely developed vulva and penis. The X and Y chromosome painting probes detected three cell lines in the examined horse: 63,X, 64,XX and 65,XX with a fragment of a Y chromosome (del Y). The DNA analysis with the PCR and PCR/RFLP methods showed absence of SRY,AMELY and ZFY genes as well as of six Y microsatellite markers (YM2, YP9, YJ10, YE1, YH12, and YA16). These results suggest that the Y chromosome fragment detected in the investigated animal was the result of a deletion of a euchromatic fragment comprising the above-mentioned markers.