In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Krüppel-like C2H2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.

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.

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.