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Published on: May 2, 2025
Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene
Yao-Jen Chang1, Zhifu Kang2, Jiayuan Bei2
1Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Abstract:
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 TRIM28 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 TRIM28 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 TRIM28 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 TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 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.
Insights
Knocking out TRIM28 in K562 leukemia cells slowed growth and induced erythroid differentiation. This revealed TRIM28’s role in regulating cell cycle, proliferation, and globin gene expression.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- TRIM28 is a scaffold protein involved in gene silencing and physiological processes like cell growth and differentiation.
- Understanding TRIM28's function in leukemia is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the role of TRIM28 in regulating cell proliferation and erythroid differentiation in K562 chronic myeloid leukemia cells.
- To elucidate the molecular mechanisms underlying TRIM28's control over these processes.
Main Methods:
- CRISPR/Cas9 gene editing was used to generate TRIM28 knockout (KO) K562 cell lines.
- Quantitative droplet digital PCR (ddPCR) and amplicon sequencing confirmed gene editing and assessed off-target effects.
- Transcriptome profiling (RNA sequencing) was performed to identify differentially expressed genes in wild-type versus TRIM28 KO cells.
Main Results:
- TRIM28 KO cells exhibited reduced proliferation and a tendency towards erythroid differentiation, indicated by red coloration.
- Transcriptome analysis revealed altered expression of cell cycle regulators (e.g., increased p21, decreased cyclin D2) and reduced melanoma antigen (MAGE) family expression.
- TRIM28 knockout induced miR-874, leading to downregulation of MAGEC2 mRNA.
- Embryonic epsilon-globin gene expression was significantly upregulated in TRIM28 KO cells, associated with SOX6 downregulation.
Conclusions:
- TRIM28 plays a significant role in regulating cell growth and erythroid differentiation in K562 leukemia cells.
- The study identified a regulatory network involving TRIM28, cell cycle genes, MAGE family, miR-874, and globin gene expression.
- These findings provide insights into TRIM28-mediated pathways in leukemia, potentially offering therapeutic targets.
