Hypermethylation of tumor suppressors and other aberrations of DNA methylation in tumors play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions including hypoxia. The response to hypoxia is mainly achieved through activation of the transcription program associated with HIF1a transcription factor. VHL inactivation by genetic or epigenetic events, which also induces aberrant activation of HIF1a, is the most common driver event for renal cancer. With whole-genome bisulfite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We show that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate—a metabolite that inhibits DNA demethylation by Tet enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in IDH wild type cells. Key points Inactivation of VHL gene leads to genome hypermethylation in kidney cancer cells. The DNA methylation phenotype can be rescued by endogenous expression of wild-type VHL. DNA hypermethylation can be attributed to the accumulation of a Tet inhibitor 2-hydroxyglutarate The accumulation of 2-hydroxyglutarate in IDH wild-type cells is explained by gene expression changes in key metabolic enzymes (malate dehydrogenase MDH and 2-hydroxyglutrarate dehydrogenase L2HGDH).
In mammalian intestines, Notch signaling plays a critical role in mediating cell fate decisions; it promotes the absorptive (or enterocyte) cell fate, while concomitantly inhibiting the secretory cell fate (i.e. goblet, Paneth and enteroendocrine cells). We recently reported that intestinal-specific Kaiso overexpressing mice (Kaiso Tg ) exhibited chronic intestinal inflammation and had increased numbers of all three secretory cell types, hinting that Kaiso might regulate Notch signaling in the gut. However, Kaiso's precise role in Notch signaling and whether the Kaiso Tg secretory cell fate phenotype was linked to Kaiso-induced inflammation had yet to be elucidated. Intestines from 3-month old Non-transgenic and Kaiso Tg mice were "Swiss" rolled and analysed for the expression of Notch1, Dll-1, Jagged-1, and secretory cell markers by immunohistochemistry and immunofluorescence. To evaluate inflammation, morphological analyses and myeloperoxidase assays were performed on intestines from 3-month old Kaiso Tg and control mice. Notch1, Dll-1 and Jagged-1 expression were also assessed in stable Kaiso-depleted colon cancer cells and isolated intestinal epithelial cells using real time PCR and western blotting. To assess Kaiso binding to the DLL1, JAG1 and NOTCH1 promoter regions, chromatin immunoprecipitation was performed on three colon cancer cell lines. Here we demonstrate that Kaiso promotes secretory cell hyperplasia independently of Kaiso-induced inflammation. Moreover, Kaiso regulates several components of the Notch signaling pathway in intestinal cells, namely, Dll-1, Jagged-1 and Notch1. Notably, we found that in Kaiso Tg mice intestines, Notch1 and Dll-1 expression are significantly reduced while Jagged-1 expression is increased. Chromatin immunoprecipitation experiments revealed that Kaiso associates with the DLL1 and JAG1 promoter regions in a methylation-dependent manner in colon carcinoma cell lines, suggesting that these Notch ligands are putative Kaiso target genes. Here, we provide evidence that Kaiso's effects on intestinal secretory cell fates precede the development of intestinal inflammation in Kaiso Tg mice. We also demonstrate that Kaiso inhibits the expression of Dll-1, which likely contributes to the secretory cell phenotype observed in our transgenic mice. In contrast, Kaiso promotes Jagged-1 expression, which may have implications in Notch-mediated colon cancer progression.
Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate – a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.
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 Kru ¨ppel-like C 2 H 2 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.
Метилирование является общим для всех позвоночных организмов процессом, одна из основных функций которого заключается в том, чтобы фиксировать транскрипционно неактивное состояние генов. Это может происходить, в частности, благодаря специальным белкам, которые специфично узнают метилированные районы ДНК и привлекают к ним белковые комплексы, репрессирующие транскрипцию. Белок Каизо является одним из таких специализированных белков и содержит два функциональных домена: N-концевой домен BTB/POZ и три “цинковых пальца” С2Н2-типа на С-конце, которыми он связывается с метилированной ДНК и привлекает к ней репрессионные комплексы за счет взаимодействия BTB/POZ-домена с корепрессором NCoR. В некоторых клеточных линиях позвоночных ортологи Каизо взаимодействуют с катенином р120, который, хотя и является преимущественно цитоплазматическим белком, иногда обнаруживается в ядре. Цель настоящего исследования определение параметров взаимодействия белков Каизо и катенина р120, а также изучение функциональных последствий образования такого белкового комплекса с точки зрения транскрипционного контроля метилированных генов. Мы установили, что второй и третий “цинковые пальцы” белка Каизо необходимы и достаточны для взаимодействия с катенином р120. При взаимодействии с катенином р120 происходит два молекулярных события, которые приводят к инактивации Каизо как транскрипционного фактора. Во-первых, катенин р120 маскирует сигнал ядерной локализации Каизо, и Каизо в составе комплекса с р120 мигрирует из ядра в цитоплазму; и, во-вторых, связывание с р120 делает невозможным взаимодействие Каизо с метилированной ДНК. Таким образом, С-концевой домен белка Каизо выполняет две важные функции: связывание с ДНК и взаимодействие с цитоплазматическим белком катенином р120. Подробное изучение данного взаимодействия позволит определить новые механизмы ядерно-цитоплазматических сигнальных путей в клетках млекопитающих.
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
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.
