Abstract
Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. Disruption of normal imprinting leads to abnormal embryogenesis, certain inherited diseases, and is associated with various cancers. In the context of screening for the gene(s) responsible for the alteration of phenotype in cyclophilin A knockdown (CypA-KD) P19 cells, we observed a silent paternally expressed gene, Peg3. Treatment of CypA-KD P19 cells with the DNA demethylating agent 5-aza-dC reversed the silencing of Peg3 biallelically. Genomic bisulfite sequencing and methylation-specific PCR revealed DNA hypermethylation in CypA-KD P19 cells, as the normally unmethylated paternal allele acquired methylation that resulted in biallelic methylation of Peg3. Chromatin immunoprecipitation assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 and MBD2 binding on the cytosine-guanine dinucleotide (CpG) islands of Peg3. Our results indicate that DNA hypermethylation on the paternal allele and allele-specific acquisition of histone methylation leads to silencing of Peg3 in CypA-KD P19 cells. This study is the first demonstration of the epigenetic function of CypA in protecting the paternal allele of Peg3 from DNA methylation and inactive histone modifications.
Highlights
A number of human-inherited diseases linked to faulty methylation pathways and exhibiting abnormal development include Rett, immunodeficiency, centromeric heterochromatin instability, and facial anomalies, and X-linked ␣ thalassemia/ mental retardation syndromes [7,8,9]
Chromatin immunoprecipitation (ChIP) assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 (Dnmt1) and MBD2 binding on the CpG islands of Peg3 in cyclophilin A knockdown (CypA-KD) P19 cells
Our data demonstrate the epigenetic function of Cyclophilin A (CypA), which protects the paternal allele of Peg3 from DNA methylation and inactive histone modifications
Summary
CypA, cyclophilin A; ChIP, chromatin immunoprecipitation; CsA, cyclosporine A; EC, embryonal carcinoma; HDAC, histone deacetylase; QRT, quantitative real-time; WT, wild type; KD, knockdown; KO, knockout; RNAi, RNA interference; Dnmt, DNA methyltransferase. Role of Cyclophilin A in Epigenetics ing that CypA affects gene expression by physically interacting with HDAC [22]. In screening for the gene(s) responsible for the alteration of phenotype in CypA-KD P19 cells [21], we observed a silent paternally expressed gene, Peg. Chromatin immunoprecipitation (ChIP) assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 (Dnmt1) and MBD2 binding on the CpG islands of Peg in CypA-KD P19 cells. Our data demonstrate the epigenetic function of CypA, which protects the paternal allele of Peg from DNA methylation and inactive histone modifications
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