Abstract
The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.
Highlights
In mammals, DNA methylation at the 5-position of cytosine residues occurs predominantly in the context of CpG dinucleotides and is generally associated with transcriptional repression.[1,2] It is essential for normal development and involved in various cellular processes, such as genomic imprinting, X chromosome inactivation, tissue-specific gene expression, and silencing of repetitive DNA.[3,4] While CpG sites are underrepresented throughout the genome and tend to be methylated, there are CpG-rich regions termed CpG islands (CGIs), which are generally unmethylated
Complementary to the overexpression of TET1, we performed the simultaneous knockdown of TET1, TET2, and TET3
Analysis of endogenous ten-eleven translocation (TET) expression levels relative to GAPDH expression revealed low basal levels of TET enzyme expression in HEK293 control cells ranging from 0.42% of GAPDH expression for TET1 and 0.36% for TET3 down to 0.09% for TET2 transcripts (Fig. S1)
Summary
DNA methylation at the 5-position of cytosine residues occurs predominantly in the context of CpG dinucleotides and is generally associated with transcriptional repression.[1,2] It is essential for normal development and involved in various cellular processes, such as genomic imprinting, X chromosome inactivation, tissue-specific gene expression, and silencing of repetitive DNA.[3,4] While CpG sites are underrepresented throughout the genome and tend to be methylated, there are CpG-rich regions termed CpG islands (CGIs), which are generally unmethylated They often cover transcriptional start sites and approximately 70% of annotated gene promoters are associated with a CGI.[5] Genomic methylation patterns are dynamic during development and disturbed in various diseases, such as cancer, imprinting-related diseases, and psychiatric disorders.[6,7,8,9] Methylation patterns are established by the de novo DNA methyltransferases DNMT3A and DNMT3B and are accurately maintained through cell division by the maintenance methyltransferase DNMT1.10 In contrast to its establishment, less is known about the enzymes and pathways involved in DNA demethylation. Reduced representation 5-hydroxymethylcytosine profiling[31] (RRHP) revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures, but that this oxidation was related to endogenous 5hmC levels
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