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

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 cellsgrew 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 clonesthrough 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.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells

“…We produced the FLAG-tagged acetylation-mimicking mutants of mouse Trim28, namely K255Q, K267Q, K290Q, and K305Q, as well as acetylation-defective mutants K255R, K267R, K290R, and K305R. These constructs were expressed in TRIM28 knockout human leukemia K562 cells [ 33 ] to test their interaction with Gal4 DNA-binding domain (Gal4 DBD )-KRAB. In an immunoprecipitation analysis, only K305Q had impaired interaction with recombinant Gal4 DBD -KRAB ( Figure 1 a).…”

“…Interestingly, the megakaryocytic repressor IKAROS family transcription factors IKZF2 and IKZF3 and the paternally imprinted genes PEG3 and DLK1 were upregulated, and the embryonic and fetal globin repressor SOX6 , the maternally imprinted gene MEG3 , and melanoma antigen family members MAGEC1, MAGEC2, MAGEB1 , and MAGEA1 were downregulated ( Table S1 ). Previously, we had demonstrated that both SOX6 and MAGEC2 were downregulated in TRIM28 -KO cells, which were analyzed by cDNA microarray [ 33 ]. To further verify the up- or downregulation of these genes, we performed RT-qPCR ( Figure 3 d), which confirmed the changes in the expression of specific genes identified by RNA-seq of TRIM28 -K304Q cells.…”

“…Fibronectin was also highly upregulated in K304Q cells but not in TRIM28 -KO cells ( Figure 4 b), which might explain the attached phenotype of TRIM28 -K304Q cells ( Figure S3C ). We also detected the induction of epsilon ( HBE ) and gamma ( HBG ) globin genes in K304Q cells ( Figure 4 c), which was increased in TRIM28 -KO cells [ 33 ].…”

TRIM28-Mediated Excessive Oxidative Stress Induces Cellular Senescence in Granulosa Cells and Contributes to Premature Ovarian Insufficiency In Vitro and In Vivo

“…The tripartite motif-containing protein superfamily (TRIMs) are involved in diverse biological processes and play a crucial role in cancer therapy [ 26 , 27 , 28 , 29 , 30 ]. Recent evidence suggests that TRIM28 regulates the post-translational modification of proteins and participates in autophagy [ 31 , 32 , 33 ].…”