Search for differentially methylated regions in ancient and modern genomes.

Currently, active research is focused on investigating the mechanisms that regulate the development of various pathologies and their evolutionary dynamics. Epigenetic mechanisms, such as DNA methylation, play a significant role in evolutionary processes, as their changes have a faster impact on the phenotype compared to mutagenesis. In this study, we attempted to develop an algorithm for identifying differentially methylated regions associated with metabolic syndrome, which have undergone methylation changes in humans during the transition from a hunter-gatherer to a sedentary lifestyle. The application of existing whole-genome bisulfite sequencing methods is limited for ancient samples due to their low quality and fragmentation, and the approach to obtaining DNA methylation profiles differs significantly between ancient hunter-gatherer samples and modern tissues. In this study, we validated DamMet, an algorithm for reconstructing ancient methylomes. Application of DamMet to Neanderthal and Denisovan genomes showed a moderate level of correlation with previously published methylation profiles and demonstrated an underestimation of methylation levels in the reconstructed profiles by an average of 15-20 %. Additionally, we developed a new Python-based algorithm that allows for the comparison of methylomes in ancient and modern samples, despite the absence of methylation profiles in modern bone tissue within the context of obesity. This analysis involves a two-step data processing approach, where the first step involves the identification and filtration of tissue-specific methylation regions, and the second step focuses on the direct search for differentially methylated regions in specific areas associated with the researcher's target condition. By applying this algorithm to test data, we identified 38 differentially methylated regions associated with obesity, the majority of which were located in promoter regions. The pipeline demonstrated sufficient efficiency in detecting these regions. These results confirm the feasibility of reconstructing DNA methylation profiles in ancient samples and comparing them with modern methylomes. Furthermore, possibilities for further methodological development and the implementation of a new step for studying differentially methylated positions associated with evolutionary processes are discussed.

DNA Methylation: Genomewide Distribution, Regulatory Mechanism and Therapy Target.

DNA methylation is the most important epigenetic modification involved in the regulation of transcription, imprinting, establishment of X-inactivation, and the formation of a chromatin structure. DNA methylation in the genome is often associated with transcriptional repression and the formation of closed heterochromatin. However, the results of genome-wide studies of the DNA methylation pattern and transcriptional activity of genes have nudged us toward reconsidering this paradigm, since the promoters of many genes remain active despite their methylation. The differences in the DNA methylation distribution in normal and pathological conditions allow us to consider methylation as a diagnostic marker or a therapy target. In this regard, the need to investigate the factors affecting DNA methylation and those involved in its interpretation becomes pressing. Recently, a large number of protein factors have been uncovered, whose ability to bind to DNA depends on their methylation. Many of these proteins act not only as transcriptional activators or repressors, but also affect the level of DNA methylation. These factors are considered potential therapeutic targets for the treatment of diseases resulting from either a change in DNA methylation or a change in the interpretation of its methylation level. In addition to protein factors, a secondary DNA structure can also affect its methylation and can be considered as a therapy target. In this review, the latest research into the DNA methylation landscape in the genome has been summarized to discuss why some DNA regions avoid methylation and what factors can affect its level or interpretation and, therefore, can be considered a therapy target.

Kaiso Gene Knockout Promotes Somatic Cell Reprogramming.

Reprogramming of somatic cells is associated with overcoming the established epigenetic barrier. Key events in this process are changes in the DNA methylation landscape and histone modifications. Studying the factors affecting epigenetic plasticity will allow not only to reveal the principles underlying cell reprogramming but also to find possible ways to influence this process. Kaiso transcription factor is one of the protein interpreters of methylated DNA. By binding to methylated DNA, Kaiso attracts corepressor complexes affecting chromatin structure. In this work, we showed that the Kaiso gene knockout contributes to more efficient somatic reprogramming by affecting both cell proliferation and DNA methylation. The proposed mechanisms for the increase in the efficiency of somatic reprogramming associated with the Kaiso gene knockout is a decrease in the methylation level of the Oct4 promoter region in mouse embryonic fibroblasts before reprogramming.

[CRISPR/Cas9-editing-based modeling of hypoxia in renal cancer cells].

Uncontrolled growth in the cell mass of malignant tumors induces intensive angiogenesis. However, the demands of the cancer cells for nutrients and oxygen remain only partially met. Hypoxia is a process that accompanies malignant transformation and evokes changes in the DNA methylation profile in solid tumors. To a certain extent, these changes, including the hypermethylation of tumor suppressor gene promoters, are related to the decrease in the activity of Tet proteins under the conditions of oxygen and free radical deficit. Stabilization, accumulation, and nuclear translocation of the transcription factor HIF1α are the key molecular events in hypoxia. We modified the clear-cell renal cancer cell line Caki1 to stabilize the HIF1α protein and characterized a model cell line that will enable the studies of the mechanisms of changes of the DNA methylation level at a constant activity of Tet proteins and a gene transcription profile characteristic of hypoxia. The CRISPR/Cas9 DNA editing system was used to edit the VHL gene. The mutant VHL protein contained a disrupted alpha-helix at the C-terminus and could not participate in the molecular pathway of proteasomal degradation of the HIF1α factor; therefore, the latter accumulated in the nucleus and activated the specific target genes. An analysis of gene transcription revealed the induction of hypoxia-associated genes in the modified cell line. The developed Сaki-1/VHLmut model can be used to discriminate between the effects evoked by oxygen-suppressed hydroxylases of the Tet family and other hypoxia-associated mechanisms of DNA methylation/demethylation.

Epigenetics of Ancient DNA.

Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences.

[Bifunctional role of domain zinc fingers of methyl-DNA-binding protein Kaiso].

DNA methylation in mammals is one of the major epigenetic mark that associates with inactive chromatin state. Methyl-DNA-binding proteins bind methylated DNA and silence gene transcription by recruiting repression complexes. Kaiso is one of the methyl-DNA-binding proteins. It has a domain structure: N-terminal BTB/POZ domain involved in protein-protein interaction and C-terminal zinc-fingers of C2H2 type that bind methylated DNA (mCGmCG) or nonmethylated - TCCTGCNA. Here we show that Kaiso interacts with p120 catenin through zinc finger 2 and 3. This interaction has dual consequences. Firstly, binding to p120 inhibits nuclear import of Kaiso that results in most of Kaiso-p120 complexes becoming cytoplasmic. And secondly, bound p120 makes impossible interaction of the zinc fingers with methylated DNA. These modes of Kaiso modulation by p120 can open attractive perspectives in linking events on cell membrane and changes in nuclear gene expression.

[Regulation of human oct-1 gene transcription involves two promotors]. / Reguliatsiia transkriptsii gena oct-1 cheloveka s uchastiem dvukh promotorov.

Transcription initiation of human Oct-1 transcription factor-encoding gene involves two promoters, 1U and 1L, located at a substantial distance (about 100 kb) apart. The structure of these promoters and the adjacent sequences is different. Specifically, the 1U sequence is GC-rich, while the 1L sequence is AT-rich. Correspondingly, more than 25 GC-rich Sp1 cis-elements were localized within the 1U region, while in the 1L sequence nearly equal amount of homeo-specific NTAATNN sites along with two ATGCAAAT octamers were found. Analysis of transfection of recombinant plasmids, carrying the promoter fragments with or without enhancer indicated that expression from the 1L promoter was tissue-specific. In nonlymphoid HEK293 cells efficiency of transcription from the 1U promoter was several times higher than that from the 1L promoter. Another expression pattern was observed at transfection of the same constructs into Raji lymphoid cells. In this case the level of transcription from the L promoter (fragment L2) at the presence of external enhancer was higher than that from the fragments containing the 1U promoter. It was shown that the distal regions of 1U and 1L were capable of silencing activity. In Raji cells enhancer completely overcomes the activity of U silencer, but only partly overcomes the activity of L silencer. Our data on the interaction of two promoters with the enhancer and silencer in different cell types point to fine tissue-specific regulation of the oct-1 gene expression, especially in lymphatic cells.

[Spliced oct-1 gene mRNA isoforms with untranslated exons and a deletion in the region coding for the POU-specific domain]. / Splaisirovannye subformy mRNK gena oct-1, soderzhashchie netransliruemye ékzony i deletsiiu v oblasti POU-spetsificheskogo domena.

Transcription factor Oct-1 is involved in expression regulation of housekeeping genes, in lymphocyte differentiation, and in the immune response. Tissue-specific oct-1 mRNA isoforms are known to be expressed in lymphoid cells. Four new mouse isoforms were identified. Of these, two were tissue-specific (oct-1R alpha and oct-1R beta) and contained exon 1L. The oct-1R alpha was shown to contain an additional fragment, which corresponds to an exon located in the 3'-region of mouse otf-1. No homolog was found in human OTF-1. The oct-1R alpha isoform proved to lack an exon coding for a fragment of the POU domain. This deletion results in a loss of the first helix of the domain, and the mutant protein is devoid of affinity for octamer ATGCAAAT. Two other mRNA isoforms, oct-1d and oct-1e, were shown to contain untranslated regions between exons 1U and 2. The regions correspond to exons 1i and 2i located between exons 1U and 1L in the 5'-region of the mouse oct-1 gene. Human OTF-1 was not found to contain exon 1i. On evidence of these and published data, it was assumed that a set of oct-1 isoforms is present in the cell, reflecting the complexity of expression regulation of oct-1 and the multiplicity of its functions.

[Subforms of transcription factor Oct-1 synthesized in lymphocytes]. / Subformy faktora transkriptsii Oct-1, sinteziruemye v limfotsitakh.

Transcription factor Oct-1 is ubiquitous, participating in expression of the cell housekeeping genes as well as in differentiation of lymphocytes and activation of transcription of immunoglobulin genes in B cells. A new tissue-specific form of Oct-1 (Oct-1L) was found in lymphoid cells of bone marrow, lymph nodes, spleen, thymus, as well as in the cell lines of B and T lymphocytes at different stages of differentiation. This isoform was not found in embryonal and nonlymphoid tissues and cell lines. The complete structure of the oct-1L mRNA was determined, which generally corresponds to the structure of mouse Oct-1b. The difference between oct-1L and isoforms oct-a, b, and c functioning in all cells is replacement of the long first 5'-terminal exon (the 1U exon) encoding 21 amino acid residues with a short one encoding 10 amino acids in the isoforms Oct-1L and Oct-1R. Interestingly, attempts to find mature oct-1 mRNA simultaneously containing the 1L and 1U exons were unsuccessful. The parallel synthesis of "ubiquitous" isoforms Oct-1a, b, and c as well as tissue-specific Oct-1L and Oct-1R in lymphocytes and their precursors may be due either to a high demand of these cells for Oct-1 or to selective participation of different Oct-1 isoforms in regulation of the housekeeping genes and genes involved in the B and T cell differentiation and synthesis of imminoglobulins.

Tissue-specific isoforms of the ubiquitous transcription factor Oct-1.

The ubiquitously expressed transcription factor Oct-1 is a member of the POU protein family. It is involved in the activation of snRNA promoters and some mRNA promoters (e.g., promoters and enhancers of genes for histone H2B and immunoglobulins). In this work we have cloned and sequenced a new Oct-1 isoform, named Oct-1L. Both Oct-1L mRNA and Oct-1R mRNA (cloned earlier) are expressed in lymphocytes, but not in any other cell line tested. This is the first report of tissue-specific Oct-1 gene expression. Both these forms differ from the ubiquitously expressed Oct-1 isoforms in the N-termini. They are probably generated by alternative splicing and/or alternative initiation of transcription. The latter is confirmed by the localization of transcription start points upstream of exons 1L (lymphocyte-specific) and IU (ubiquitously expressed). We assume that tissue-specific expression of Oct-1L and Oct-1R in lymphocytes and their structural differences from the ubiquitously expressed Oct-1 isoforms may be related to B and T cell differentiation and/or expression of the immunoglobulin genes.