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

by Selleckchem | Dec 06 2023

Background: Despite 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.

Methods: Specimens 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.

Results: Firstly, 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.

Conclusions: Transcription 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.

Comments:

Title: **Role of Stat5a-Mediated Dnmt3a Regulation in Jak2V617F Positive Classical Myeloproliferative Neoplasms: Implications for Targeted Therapy**

**Abstract:**
Classical myeloproliferative neoplasms (cMPNs) are characterized by the Jak2V617F mutation, but the underlying molecular mechanisms driving these disorders remain incompletely understood. In this study, we investigated the regulatory relationship between Jak2V617F and Dnmt3a, a critical epigenetic regulator, in cMPNs. Our findings demonstrate that Dnmt3a is significantly downregulated in Jak2V617F positive cMPN samples. Mechanistically, we identified that aberrantly activated Stat5a binds to the Dnmt3a promoter, suppressing its transcription and translation. Specifically, we pinpointed a gamma-activated sequence (GAS) motif approximately 800 bp upstream of the Dnmt3a promoter as the site of Stat5a binding. Furthermore, our functional assays revealed that Dnmt3a modulation influenced cell proliferation and cell cycle distribution in Jak2V617F-BaF3 cells. Dnmt3a-mediated G0/G1 arrest was shown to occur through the miR-17-5p/Cdkn1a axis. Collectively, our results shed light on the Stat5a/Dnmt3a/miR-17-5p/Cdkn1a pathway as a potential therapeutic target for cMPNs, offering new avenues for targeted therapies in these disorders.

**Keywords:** Classical myeloproliferative neoplasms, Jak2V617F mutation, Dnmt3a, Stat5a, epigenetic regulation, miR-17-5p, Cdkn1a, targeted therapy.