Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells

Abstract

Background: Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2^V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2^V617F mutation and its effects on downstream signaling pathways in cMPNs.

Methods: Specimens of Jak2^V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes.

Results: Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2^V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2^V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2^V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest.

Conclusions: Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2^V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

Keywords: Cdkn1a; Classical myeloproliferative neoplasms; Dnmt3a; GAS; Jak2V617F; Stat5a.

Fig. 1

Aberrant activation of the JAK-STAT pathway represses Dnmt3a transcription and translation via Stat5a. A. The expression level of Jak2, p-Jak2, Stat5a, p-Stat5a and Dnmt3a in wild-type and Jak2^V617F BaF3 cells. Full-length blot images are presented in Supplementary Fig. 1. B. Transcription level of DNMT3a in bone marrow mononuclear cells from JAK2^V617F MPN patients and normal controls. C. p-Stat5a and Dnmt3a of Jak2^V617F Baf3 cells incubated with AZ960. Full-length blot images are presented in Supplementary Fig. 2. D. p-Stat5a and Dnmt3a of Jak2^V617F Baf3 cells incubated with LY2784544. Full-length blot images are presented in Supplementary Fig. 3. E. In Jak2^V617F BaF3 cells, the protein expression level of Dnmt3a after Stat5a was knocked down. Full-length blot images are presented in Supplementary Fig. 4. F. The transcriptional level of Dnmt3a in Jak2^V617F Baf3 cells with or without LY2784544 incubation

Fig. 2

Dnmt3a expression is repressed by Stat5a and Stat5a is the upstream regulator of Dnmt3a. A. The expression levels of p-STAT5a and DNMT3a proteins in human myeloid tumor cell lines. Full-length blot images are presented in Supplementary Fig. 5. B. The expression levels of p-Stat5a and Dnmt3a proteins in murine myeloid tumor cell lines. Full-length blot images are presented in Supplementary Fig. 6. C. In wild type BaF3 cells, the protein expression level of Dnmt3a after Stat5a was knocked down. Full-length blot images are presented in Supplementary Fig. 7. D. In wild type BaF3 cells, the protein expression level of Dnmt3a after overexpression of Stat5a. Full-length blot images are presented in Supplementary Fig. 8. E & F. In Jak2^V617F BaF3 cells, the protein expression of Stat5a after knockdown or overexpression of Dnmt3a. Full-length blot images are presented in Supplementary Fig. 9 and Supplementary Fig. 10. G & H. In wild-type BaF3 cells, the protein expression of Stat5a after knockdown or overexpression of Dnmt3a. Full-length blot images are presented in Supplementary Fig. 11 and Supplementary Fig. 12

Fig. 3

Aberrant activation of Stat5a downregulates the promoter transcriptional activity of Dnmt3a. A. Segments of the promoter region of the Dnmt3a gene. B & C. Incubation with LY2784544 significantly increased the fluorescence intensity of 293 T cells transfected with the P4 region luciferase plasmid. D. The P4 region promoter transcriptional activity was strongest after 3 h of LY2784544 incubation. E. Stat5a was overexpressed in 293 T cells, and IL-3 was added to simulate the abnormal activation and phosphorylation of Stat5a. Full-length blot images are presented in Supplementary Fig. 13. F. Overexpression and activation of Stat5a inhibited the transcriptional activity of the P4 region promoter. G. The effect of LY2784544 on transcriptional activity of the Dnmt3a promoter with or without mutated GAS

Fig. 4

Stat5a downregulates Dnmt3a protein expression by directly binding to the Dnmt3a promoter. A. The schema of the 8 regions of ChIP analysis; B & C. Stat5a protein occupied several regions (ChIP1, ChIP6 and ChIP7) of the Dnmt3a promoter. Full-length blot images are presented in Supplementary Fig. 14

Fig. 5

Knockdown or overexpression of Dnmt3a in Jak2^V617F BaF3 cells significantly affects cell proliferation capacity. A. Soft agar clone formation of Jak2^V617F BaF3 cells; B. Soft agar clone formation of Dnmt3a-overexpressing Jak2^V617F BaF3 cells; C. Soft agar clone formation of Dnmt3a-knockdown Jak2^V617F BaF3 cells; D. Cell proliferation level of Jak2^V617F BaF3 cells with Dnmt3a overexpression or with Dnmt3a knockdown

Fig. 6

Dnmt3a regulates the cell cycle of Jak2^V617F BaF3 cells by regulating Cdkn1a expression. A. Cell cycle distribution of Jak2^V617F BaF3 cells. B. Cell cycle distribution of Dnmt3a-knockdown Jak2^V617F BaF3 cells. C. Cell cycle distribution of Dnmt3a-overexpressing Jak2^V617F BaF3 cells. D. Cell cycle distribution statistics of each group (A-C) of Jak2^V617F BaF3 cells

Fig. 7

The expression level of Cdkn1a is downregulated in Jak2^V617F cells. A. The expression level of Cdkn1a was significantly suppressed in Jak2^V617F BaF3 cells compared with control BaF3 cells. Full-length blot images are presented in Supplementary Fig. 15. B. The transcription level of Cdkn1a was significantly suppressed in Jak2^V617F BaF3 cells compared with control BaF3 cells. C. The transcription level of Cdkn1a is lower in JAK2^V617F cMPN patients than that in normal controls

Fig. 8

Dnmt3a regulates the cell cycle of Jak2^V617F Baf3 cells through Cdkn1a. A & B. Dnmt3a induced Cdkn1a expression in Jak2^V617F BaF3 cells. Full-length blot images are presented in Supplementary Fig. 16 and Supplementary Fig. 17. Inhibitor: LY2784544 incubation. C. Jak2^V617F Baf3 cells with Dnmt3a overexpression and with both Dnmt3a overexpression and Cdkn1a knockdown were established. Full-length blot images are presented in Supplementary Fig. 18. D. Cell cycle distribution of wild-type BaF3 cells. E. Cell cycle distribution of Jak2^V617F BaF3 cells. F. Cell cycle distribution of Jak2^V617F BaF3 cells with Dnmt3a overexpression. G. Cell cycle distribution of Jak2^V617F BaF3 cells with both Dnmt3a overexpression and Cdkn1a knockdown. H. Cell cycle distribution statistics of each group of Jak2^V617F BaF3 cells

Fig. 9

Dnmt3a negatively regulates miR-17-5p and miR-17-5p represses Cdkn1a expression in Jak2^V617F BaF3 cells. A. The expression level of miR-17-5p in Jak2^V617F BaF3 cells and control BaF3 cells. B. The verification of the knockdown and overexpression effect of Dnmt3a in Jak2^V617F BaF3 cells. Full-length blot images are presented in Supplementary Fig.19. C. The expression level of miR-17-5p in Jak2^V617F BaF3 cells with Dnmt3a knockdown and overexpression. D. The verification of the effect of transfection of miR-17-5p mimic and inhibitor in Jak2^V617F BaF3 cells. E & F. The transcription and expression of Cdkn1a in Jak2^V617F BaF3 cells transfected with miR-17-5p mimic or inhibitor. Full-length blot images are presented in Supplementary Fig.19. G. Graphic abstract of the present study