Novel DNA methylation markers of PRRSV-specific antibodies and their intergenerational transmission from pregnant sows to piglets

The main strategy for preventing porcine reproductive and respiratory syndrome (PRRS) is vaccination.
However, current commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines have limited effectiveness and may even cause infections in pigs.
The identification of stable molecular markers associated with immune responses to PRRSV vaccination in pigs provides a new approach for PRRS prevention.
DNA methylation, the most stable epigenetic molecular marker related to PRRSV vaccination, has not been investigated.
In the current research, we used whole genome bisulfite sequencing (WGBS) to investigate DNA methylation in pregnant sows that received PRRSV vaccination and their piglets with high and low PRRSV-specific antibody levels.
By performing methylation data analysis and basing on our previous transcriptomic studies, we identified several differentially methylated genes (DMGs) that are involved in the pathways of inflammatory and immune responses.
Among the DMGs, ISG15, MX1, SERPINE1, GNG11 and IFIT3 were common hub genes in the two generations.
MX1 and GNG11 were located in quantitative trait loci related with PRRSV antibody titer and PRRSV susceptibility, respectively.
These results suggest that PRRSV vaccination in sows induces DNA methylation changes in genes and DNA methylation changes occur through intergenerational transmission.
The novel DNA methylation markers and target genes observed in our study provide new insights into the molecular mechanisms of immune responses to PRRSV vaccination across two pig generations.

Mitochondrial DNA Methylation Misleads Global DNA Methylation Detected by Antibody-Based Methods

Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria.
This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected.
However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA.
Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen.
Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells.
Experiments were performed to distinguish methylation between mtDNA and ncDNA.
Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in an overestimation of the quantitative level of nuclear methylation.

PP2AC Phospho-Tyr307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2AC Modifications Including Leu309 Methylation

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry.
Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags.
We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase.
Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.

Anti-endothelial cell antibody rich sera from rheumatic heart disease patients induces proinflammatory phenotype and methylation alteration in endothelial cells.

Rheumatic heart disease (RHD) is a major cause of cardiovascular morbidity and mortality in developing nations like India.
RHD commonly affects the mitral valve which is lined by a single layer of endothelial cells (ECs). The role of ECs in mitral valve damage during RHD is not well elucidated.
Here, an anti-endothelial cell antibody from RHD patients has been used to stimulate the ECs (HUVECs and HMVECs). ECs proinflammatory phenotype with increased expression of TNFα, IL-6, IL-8, IFNγ, IL-1β, ICAM1, VCAM1, E-selectin, laminin B, and vimentin was documented in both ECs.
The promoter hypomethylation of various key inflammatory cytokines (TNFα, IL-6, and IL-8), integrin (ICAM1) associated with leukocyte transendothelial migration, and extracellular matrix genes (vimentin, and laminin) were also observed.
Further, the in-vitro data was in accordance with ex-vivo observations which correlated significantly with the etiological factors such as smoking, socioeconomic status, and housing.
Thus, the study sheds light on the role of ECs in RHD which is a step forward in the elucidation of disease pathogenesis.

Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA.
However, the specificity of antibodies within these experiments has not been systematically studied.
Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methyl forms (me1, me2, and me3, with each ascribed distinct biological functions).
We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methyl form paradigms.
Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes.
Our results illustrate how poor antibody specificity contributes to the “reproducibility crisis,” demonstrating the need for rigorous, platform-appropriate validation.

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