Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.

Clear cell renal cell carcinoma (ccRCC), accounting for 80–90% of renal malignancies, is frequently driven by VHL inactivation—either through mutation or promoter hypermethylation—resulting in constitutive HIF2$\alpha$ activation and pseudohypoxic signaling. VHL gene inactivation is a hallmark of von Hippel–Lindau syndrome, a hereditary disorder predisposing patients to ccRCC and other tumors, underscoring its central role in disease pathogenesis. While VHL dysfunction promotes aggressive tumor phenotypes, the therapeutic potential of VHL restoration remains underexplored. Here, using the Cas9 induced VHL-mutation in the Caki-1 cell line model, we demonstrate that VHL inactivation augments hypoxia-like pathways and enhances anaerobic glycolysis. Rescue of functional VHL reversed these activation patterns and modulated the expression of genes associated with angiogenesis. Using single cell transcriptomics, we show that the VHL-positive and -negative Caki-1 cells are characterized with different proportions of benign and aggressive cells as seen by analysis of specific gene expression. Furthermore, the identified angiogenesis-related genes were linked to affect clinical outcomes in ccRCC patients, suggesting that VHL restoration may mitigate high-risk molecular features.

Understanding the phenotypic consequences of epigenetic variation and its role in adaptation remains a central challenge in evolutionary biology. Marine and freshwater sticklebacks provide a powerful system to study the interplay between genetic and epigenetic components of phenotypic plasticity that enables colonization of contrasting salinity habitats. Here, we used whole-genome bisulfite sequencing (WGBS) to characterize DNA methylation entropy—a measure of epigenetic stochasticity—in gill tissue from marine and freshwater ecotypes. We found that freshwater sticklebacks exhibit elevated methylation entropy in divergence islands (DIs), genomic regions known as hotspots of genetic divergence between marine and freshwater populations. Within DIs, we identified a subset of genes exhibiting concurrent increases in methylation entropy and transcriptional variance, including osmoregulatory candidates involved in growth modulation, cytoskeletal reorganization, metabolism, and extracellular matrix remodeling. Their linked variability suggests that they may act as “adaptation capacitors” facilitating phenotypic plasticity during salinity transitions. Exploratory enrichment analysis further revealed overrepresented epigenetic regulators within DIs, such as DNA demethylase TET1 and chromatin remodelers ARID5B and BPTF, indicating a potential regulatory basis by which these factors may convert genetic variation into epigenetic diversity. Collectively, our findings demonstrate that DIs are focal points of both genetic divergence and epigenetic heterogeneity, consistent with a model in which DIs may act as multi-layered genomic regions associated with adaptive responses to salinity change.

For evolutionary biology, the phenotypic consequences of epigenetic variations and their potential contribution to adaptation and diversification are pressing issues. Marine and freshwater sticklebacks represent an ideal model for studying both genetic and epigenetic components of phenotypic plasticity that allow fish to inhabit water with different salinity. Here, we applied single-cell genomics (scRNA-seq and scATAC-seq) and whole-genome bisulfite sequencing to characterize intercellular variability in transcription, the abundance of open chromatin regions, and CpG methylation level in gills of marine and freshwater stickleback morphs. We found little difference in overall transcriptional variance between the morphs but observed significant changes in chromatin openness variance. In addition, genomic divergence islands (DIs) coincided with regions of increased methylation entropy in freshwater fish. Moreover, analysis of transcription factor binding sites within DIs revealed that СTCF motifs around marker SNPs were significantly enriched within the region. Altogether, our data show that increased epigenetic variance accompanies the adaptation of marine sticklebacks to freshwater.

This study introduces a novel approach for noninvasive prenatal testing (NIPT) of chromosomal abnormalities, based on analysis of epigenetic features in circulating cell-free DNA (cfDNA). The core innovation of our method leverages fundamental differences in chromatin organization between maternal and fetal cells. Specifically, we focused on genomic regions that exhibit open chromatin configuration in maternal blood cells but remain tightly packed in fetal tissues (DNase I hypersensitive sites or DHSs). These epigenetic differences create distinct cfDNA fragmentation signatures that allow selective identification of fetal DNA within the maternal cfDNA pool. The study workflow comprised several key steps: performing genome-wide screening to identify differentially accessible chromatin regions, selecting the most informative markers using a machine learning algorithm, and targeted sequencing of the selected epigenetic markers using molecular barcodes. Subsequently, a LASSO regression model was constructed and validated. As a proof of concept, the study demonstrates the method’s efficacy in identifying trisomy 21 (Down syndrome), though the underlying principles can be readily adapted to other abnormalities. Complementing its robust performance, the technique offers practical advantages in terms of platform compatibility – the same epigenetic markers can be assessed using either next-generation sequencing or simpler, more cost-efficient methods like digital PCR. With further refinement, the approach could be extended to screen for additional aneuploidies (trisomies 13 and 18) and microdeletion syndromes. Therefore, this approach offers new opportunities for developing cost-effective testing systems suitable for widespread routine clinical implementation, combining high diagnostic accuracy with reduced analysis costs.