Abstract
BackgroundDespite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs.MethodsSpecimens of Jak2V617F 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.ResultsFirstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F 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 Jak2V617F 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 Jak2V617F-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.ConclusionsTranscription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F 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.
Highlights
Despite of the frequently reported DNA methyltransferase 3 alpha (Dnmt3a) abormality in classical myeloproliferative neoplasms patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation
The expression of DNMT3a in bone marrow mononuclear cells were detected by RT-quantitative reverse-transcription PCR (qPCR), and the results showed that DNMT3a was less transcribed in classical myeloproliferative neoplasms (cMPNs) patients with JAK2V617F mutation compared with the normal controls (Fig. 1B), which was consistent with the result observed in Jak2V617F BaF3 cells
LY2784544 incubation of 293 T cells transfected with the P1, P2, P3, R1, R2 and R3 regions of Dnmt3a promoter showed no significant change in relative fluorescence intensity (Fig. 3B and Fig. 3C). These results indicated that inhibition of the protein level of Signal transducer and activator of transcription 5a (Stat5a) significantly increased the transcriptional activity of the Dnmt3a promoter, and we hypothesized that promoter region of Dnmt3a might locate at P4, which was located in the region approximately 800 bp upstream of the first exon
Summary
Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. Jacquelin et al has reported that there is a synergistic carcinogenic effect in cMPNs driven by JAK2V617F mutation and DNMT3a deletion, which leads to the activation of inflammatory signals driven by hematopoietic stem and progenitor cell (HSPC) enhancers [6]. These above studies indicated that as well as the JAK2 gene, the DNMT3a gene is important in the pathogenesis of cMPNs, but their relationship in cMPNs is still a mystery. Few research has answered the question how the Dnmt3a is regulated by Jak2V617F mutation, and its effects on downstream signaling pathways in cMPNs still needs further research
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