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.

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.

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.

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus).

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.

Determination of the correlation between stallion's age and number of sex chromosome aberrations in spermatozoa.

The aim of this study was a cytogenetic analysis of stallions semen to find sex chromosome aberrations and to determine if there was an association between stallion's age and aberration frequency for the sex chromosomes. Sperm samples were collected from 22 stallions of various age from 3 to 23 years. Multicolour FISH was performed on each sample, using probes for the sex chromosomes and EGFR gene, localized on 4p12 in domestic horse. A total of 26199 sperm cells were analysed (from 1 070 to 1 532 per animal). Among the analysed cells, there were 50.318% with X chromosome, 48.543% with Y chromosome and 1.139% with aberrant chromosomes. The frequency of aberrations was: sex chromosomes nullisomy (0.466%), XY aneuploidy (0.454%), XX disomy (0.146%), YY disomy (0.041%), diploidy (0.024%) and trisomy XXY (0.008%). Additionally there was a correlation between the age of an animal and the frequency of sex chromosome aberration and a significant positive correlation between age and disomy of XY, XX, YY, trisomy of XXY, autosomal disomy was seen. A Correlation between the age of a stallion and the level of nullisomy was negative. The present study demonstrated that FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and might be very helpful for a breeder during a selection for the best stallion.

The mechanisms determining the nucleolar-organizing regions inactivation of domestic horse chromosomes.

Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with HpaII, silver staining (AgNOR) and C-banding technique (CBG). The obtained results suggest that variation in the number and size of silver deposits is related to the number of rDNA copies, DNA methylation and the localization of ribosomal DNA loci in telomeric regions. Moreover, we have found that chromosome pairs 28 and 31 are characterized by higher variation in the NORs number.

[Information sources on carcinogenic agents]. / Zródla informacji o czynnikach rakotwórczych.

This work comprises lists of publications and computer data bases available at the Scientific Information Department, The Nofer Institute of Occupational Medicine, Lodz, Poland, containing information on cancer risks resulting from exposure to carcinogenic agents. The following publications have been described: IARC Monographs on the Evaluation of Carcinogenic Risk to Humans; Official lists of carcinogens valid in individual countries; a periodical--Survey of compounds which have been tested for carcinogenic activity; a guide--The Annual Report on Carcinogens, a series on Carcinogens in the Occupational Environment, comprising Information Cards and Monographs; a guide on Carcinogenic Agents, and computer databases, including the INCAR, IRIS, IRPTC, RTECS CHEMINFO factual databases and the CANCER-CD, MEDLINE, TOXLINE, NIOSHTIC and MEDIP bibliographic (or reference) databases. The presented publications and databases may prove helpful in searching for data on identifying and preventing the adverse health effects of environmental and occupational exposure to carcinogens.