Ki-67 gene expression

Ki-67 serves as a prominent cancer marker. We describe how expression of the MKI67 gene coding for Ki-67 is controlled during the cell cycle.MKI67 mRNA and Ki-67 protein are maximally expressed in G2 phase and mitosis. Expression is dependent on two CHR elements and one CDE site in the MKI67 promoter. DREAM transcriptional repressor complexes bind to both CHR sites and downregulate the expression in G0/G1 cells. Upregulation of MKI67 transcription coincides with binding of B-MYB-MuvB and FOXM1-MuvB complexes from S phase into G2/M. Importantly, binding of B-MYB to the two CHR elements correlates with loss of CHR-dependent MKI67 promoter activation in B-MYB-knockdown experiments. In knockout cell models, we find that DREAM/MuvB-dependent transcriptional control cooperates with the RB Retinoblastoma tumor suppressor. Furthermore, the p53 tumor suppressor indirectly downregulates transcription of the MKI67 gene. This repression by p53 requires p21/CDKN1A. These results are consistent with a model in which DREAM, B-MYB-MuvB, and FOXM1-MuvB together with RB cooperate in cell cycle-dependent transcription and in transcriptional repression following p53 activation.

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Fig: Ki-67 expression on the mRNA and protein levels reaches a maximum in G2/M: A Expression of MKI67/Mki67 mRNA over the course of one cell cycle. Human T98G (blue), mouse NIH3T3 (gray), and human hTERT-BJ cells (yellow) were arrested in G0 by serum starvation for 72 h. To stimulate the cells to re-enter the cell cycle, FCS was added to the medium. Cells were collected every 3 h after serum restimulation. Each sample was divided into three aliquots for RNA and protein extraction, and for flow cytometry analyses. Relative mRNA levels fromMKI67/Mki67 genes were quantified using real-time RT-qPCR. Mean values ± SD of two technical replicates for each time point are given. The maximum fold-change in MKI67/Mki67 mRNA levels was calculated for each cell line. Approximations of cell cycle phase distribution for hTERT-BJ cells were deduced from flow cytometry analyses in C and are indicated above the graphs. B Protein levels of Ki-67 in hTERT-BJ cells used in A, B, and C over the course of one cell cycle were analyzed by western blot analysis. One representative experiment from three biological replicates is shown. KIF23 and β-actin proteins served as cell cycle and loading controls, respectively. Approximate cell cycle phases are indicated. C Flow cytometry analyses of propidium iodide (PI) staining of hTERT-BJ cells analyzed in A and B. Quantification of Ki-67 protein expression of two biological replicates is displayed in Suppl. Fig. A Expression of MKI67/Mki67 mRNA over the course of one cell cycle. Human T98G (blue), mouse NIH3T3 (gray), and human hTERT-BJ cells (yellow) were arrested in G0 by serum starvation for 72 h. To stimulate the cells to re-enter the cell cycle, FCS was added to the medium. Cells were collected every 3 h after serum restimulation. Each sample was divided into three aliquots for RNA and protein extraction, and for flow cytometry analyses. Relative mRNA levels fromMKI67/Mki67 genes were quantified using real-time RT-qPCR. Mean values ± SD of two technical replicates for each time point are given. The maximum fold-change in MKI67/Mki67 mRNA levels was calculated for each cell line. Approximations of cell cycle phase distribution for hTERT-BJ cells were deduced from flow cytometry analyses in C and are indicated above the graphs. B Protein levels of Ki-67 in hTERT-BJ cells used in A, B, and C over the course of one cell cycle were analyzed by western blot analysis. One representative experiment from three biological replicates is shown. KIF23 and β-actin proteins served as cell cycle and loading controls, respectively. Approximate cell cycle phases are indicated. C Flow cytometry analyses of propidium iodide (PI) staining of hTERT-BJ cells analyzed in A and B. Quantification of Ki-67 protein expression of two biological replicates is displayed in Suppl. Fig. 

In conclusion, we present mechanisms howMKI67 gene expression followed by Ki-67 protein synthesis is controlled during the cell cycle and upon induction of DNA damage, as well as upon p53 activation.

Uxa, S., Castillo-Binder, P., Kohler, R. et al. Ki-67 gene expression. Cell Death Differ (2021). https://doi.org/10.1038/s41418-021-00823-x

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