Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes
We previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2021-08-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1002/2211-5463.13252 |
| _version_ | 1797787909785387008 |
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| author | Xiefan Fang Ryan Poulsen Lu Zhao Jingjing Wang Scott A. Rivkees Christopher C. Wendler |
| author_facet | Xiefan Fang Ryan Poulsen Lu Zhao Jingjing Wang Scott A. Rivkees Christopher C. Wendler |
| author_sort | Xiefan Fang |
| collection | DOAJ |
| description | We previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1 is essential for embryonic development, during which it is involved in regulating cardiomyocyte DNA methylation and gene expression. We used siRNA to knock down DNMT1 expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining and multielectrode array were, respectively, utilized to evaluate cardiomyocyte growth and electrophysiology. RNA sequencing (RNA‐Seq) and multiplex bisulfite sequencing were, respectively, performed to examine gene expression and promoter methylation. At 72 h post‐transfection, reduction of DNMT1 expression decreased the number and increased the size of embryonic cardiomyocytes. Beat frequency and the amplitude of field action potentials were decreased by DNMT1 siRNA. RNA‐Seq analysis identified 801 up‐regulated genes and 494 down‐regulated genes in the DNMT1 knockdown cells when compared to controls. Pathway analysis of the differentially expressed genes revealed pathways that were associated with cell death and survival, cell morphology, cardiac function, and cardiac disease. Alternative splicing analysis identified 929 differentially expressed exons, including 583 up‐regulated exons and 308 down‐regulated exons. Moreover, decreased methylation levels were found in the promoters of cardiac genes Myh6, Myh7, Myh7b, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb, mef2c, mef2d, Camta2, Cdkn1A, and Cdkn1C. Of these 13 genes, 6 (Myh6, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb) and 1 (Cdkn1C) had increased or decreased gene expression, respectively. Altogether, these data show that DNMT1 is important in embryonic cardiomyocytes by regulating DNA methylation, gene expression, gene splicing, and cell function. |
| first_indexed | 2024-03-13T01:28:07Z |
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| id | doaj.art-66be8a155d704192813463ad529cae02 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-03-13T01:28:07Z |
| publishDate | 2021-08-01 |
| publisher | Wiley |
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| series | FEBS Open Bio |
| spelling | doaj.art-66be8a155d704192813463ad529cae022023-07-04T10:16:03ZengWileyFEBS Open Bio2211-54632021-08-011182364238210.1002/2211-5463.13252Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytesXiefan Fang0Ryan Poulsen1Lu Zhao2Jingjing Wang3Scott A. Rivkees4Christopher C. Wendler5Department of Pediatrics Child Health Research Institute College of Medicine University of Florida Gainesville FL USADepartment of Pediatrics Child Health Research Institute College of Medicine University of Florida Gainesville FL USACharles River Laboratories, Inc. Reno NV USACharles River Laboratories, Inc. Reno NV USADepartment of Pediatrics Child Health Research Institute College of Medicine University of Florida Gainesville FL USADepartment of Pediatrics Child Health Research Institute College of Medicine University of Florida Gainesville FL USAWe previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1 is essential for embryonic development, during which it is involved in regulating cardiomyocyte DNA methylation and gene expression. We used siRNA to knock down DNMT1 expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining and multielectrode array were, respectively, utilized to evaluate cardiomyocyte growth and electrophysiology. RNA sequencing (RNA‐Seq) and multiplex bisulfite sequencing were, respectively, performed to examine gene expression and promoter methylation. At 72 h post‐transfection, reduction of DNMT1 expression decreased the number and increased the size of embryonic cardiomyocytes. Beat frequency and the amplitude of field action potentials were decreased by DNMT1 siRNA. RNA‐Seq analysis identified 801 up‐regulated genes and 494 down‐regulated genes in the DNMT1 knockdown cells when compared to controls. Pathway analysis of the differentially expressed genes revealed pathways that were associated with cell death and survival, cell morphology, cardiac function, and cardiac disease. Alternative splicing analysis identified 929 differentially expressed exons, including 583 up‐regulated exons and 308 down‐regulated exons. Moreover, decreased methylation levels were found in the promoters of cardiac genes Myh6, Myh7, Myh7b, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb, mef2c, mef2d, Camta2, Cdkn1A, and Cdkn1C. Of these 13 genes, 6 (Myh6, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb) and 1 (Cdkn1C) had increased or decreased gene expression, respectively. Altogether, these data show that DNMT1 is important in embryonic cardiomyocytes by regulating DNA methylation, gene expression, gene splicing, and cell function.https://doi.org/10.1002/2211-5463.13252DNA methylationDNMT1embryonic cardiomyocytegene expression |
| spellingShingle | Xiefan Fang Ryan Poulsen Lu Zhao Jingjing Wang Scott A. Rivkees Christopher C. Wendler Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes FEBS Open Bio DNA methylation DNMT1 embryonic cardiomyocyte gene expression |
| title | Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| title_full | Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| title_fullStr | Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| title_full_unstemmed | Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| title_short | Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| title_sort | knockdown of dna methyltransferase 1 reduces dna methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes |
| topic | DNA methylation DNMT1 embryonic cardiomyocyte gene expression |
| url | https://doi.org/10.1002/2211-5463.13252 |
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