Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits
TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited <i>TRIM28</i>...
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MDPI AG
2022-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/12/6839 |
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| author | Yao-Jen Chang Zhifu Kang Jiayuan Bei Shu-Jen Chou Mei-Yeh Jade Lu Yu-Lun Su Sheng-Wei Lin Hsin-Hui Wang Steven Lin Ching-Jin Chang |
| author_facet | Yao-Jen Chang Zhifu Kang Jiayuan Bei Shu-Jen Chou Mei-Yeh Jade Lu Yu-Lun Su Sheng-Wei Lin Hsin-Hui Wang Steven Lin Ching-Jin Chang |
| author_sort | Yao-Jen Chang |
| collection | DOAJ |
| description | TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited <i>TRIM28</i> using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The <i>TRIM28</i> KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in <i>TRIM28</i> KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of <i>TRIM28</i> can induce miR-874 expression to downregulate <i>MAGEC2</i> mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in <i>TRIM28</i> KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells. |
| first_indexed | 2024-03-09T23:31:55Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T23:31:55Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-312f2ecf53ac479fb56f2b8b6461fc372023-11-23T17:07:42ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-06-012312683910.3390/ijms23126839Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta SubunitsYao-Jen Chang0Zhifu Kang1Jiayuan Bei2Shu-Jen Chou3Mei-Yeh Jade Lu4Yu-Lun Su5Sheng-Wei Lin6Hsin-Hui Wang7Steven Lin8Ching-Jin Chang9Institute of Biological Chemistry, Academia Sinica, Taipei 11529, TaiwanGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, TaiwanGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, TaiwanInstitute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, TaiwanBiodiversity Research Center, Academia Sinica, Taipei 11529, TaiwanGraduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, TaiwanInstitute of Biological Chemistry, Academia Sinica, Taipei 11529, TaiwanDepartment of Pediatrics, Division of Pediatric Immunology and Nephrology, Taipei Veterans General Hospital, Taipei 11217, TaiwanInstitute of Biological Chemistry, Academia Sinica, Taipei 11529, TaiwanInstitute of Biological Chemistry, Academia Sinica, Taipei 11529, TaiwanTRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited <i>TRIM28</i> using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The <i>TRIM28</i> KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in <i>TRIM28</i> KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of <i>TRIM28</i> can induce miR-874 expression to downregulate <i>MAGEC2</i> mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in <i>TRIM28</i> KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.https://www.mdpi.com/1422-0067/23/12/6839TRIM28CRISPR/Cas9ddPCRK562hemoglobin betaMAGEC2 |
| spellingShingle | Yao-Jen Chang Zhifu Kang Jiayuan Bei Shu-Jen Chou Mei-Yeh Jade Lu Yu-Lun Su Sheng-Wei Lin Hsin-Hui Wang Steven Lin Ching-Jin Chang Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits International Journal of Molecular Sciences TRIM28 CRISPR/Cas9 ddPCR K562 hemoglobin beta MAGEC2 |
| title | Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits |
| title_full | Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits |
| title_fullStr | Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits |
| title_full_unstemmed | Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits |
| title_short | Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits |
| title_sort | generation of trim28 knockout k562 cells by crispr cas9 genome editing and characterization of trim28 regulated gene expression in cell proliferation and hemoglobin beta subunits |
| topic | TRIM28 CRISPR/Cas9 ddPCR K562 hemoglobin beta MAGEC2 |
| url | https://www.mdpi.com/1422-0067/23/12/6839 |
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