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1.
Epigenetics ; 17(8): 894-905, 2022 08.
Article in English | MEDLINE | ID: mdl-34494499

ABSTRACT

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.


Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Carcinoma, Renal Cell/genetics , DNA/metabolism , DNA Methylation , Humans , Hypoxia/genetics , Isocitrate Dehydrogenase , Kidney Neoplasms/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism
2.
Int J Mol Sci ; 22(19)2021 Sep 23.
Article in English | MEDLINE | ID: mdl-34638562

ABSTRACT

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.


Subject(s)
Gene Editing/methods , Zygote/metabolism , Animals , CRISPR-Cas Systems , DNA End-Joining Repair , DNA, Single-Stranded , Homologous Recombination , Mice , Microinjections/methods , Models, Animal , RNA, Guide, Kinetoplastida
3.
Int J Mol Sci ; 22(14)2021 Jul 15.
Article in English | MEDLINE | ID: mdl-34299205

ABSTRACT

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.


Subject(s)
DNA Methylation , Genomic Imprinting , Insulin-Like Growth Factor II/metabolism , Locus Control Region , RNA, Long Noncoding/genetics , Transcription Factors/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Editing , HEK293 Cells , Humans , Insulin-Like Growth Factor II/genetics , Promoter Regions, Genetic , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , DNA Methyltransferase 3B
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