Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line.
DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2'-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil.
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Araújo
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil.
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Vasques
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil.
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Stabellini
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil.
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Krepischi
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil.
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Pereira
D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins; D000077209:Decitabine; D063185:Epigenetic Repression / Q000235:genetics*; D055786:Gene Knockout Techniques; D016678:Genome / Q000235:genetics*; D015894:Genome, Human / Q000187:drug effects; D018392:Genomic Imprinting / Q000235:genetics*; D045325:HCT116 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D046228:Microarray Analysis; D020641:Polymorphism, Single Nucleotide; D011506:Proteins / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D000097584:DNA Methyltransferase 3B
D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D004268:DNA-Binding Proteins; D000077209:Decitabine; D007335:Insulin-Like Growth Factor II; D011506:Proteins; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; D000097584:DNA Methyltransferase 3B; C094335:H19 long non-coding RNA; C035170:IGF2 protein, human; C432965:PEG10 protein, human; C077885:XIST non-coding RNA; C000619380:DNMT1 protein, human