The E3 ligase DTX2 inhibits RUNX1 function by binding its C terminus and prevents the growth of RUNX1-dependent
Taishi Yonezawa1, Hirotaka Takahashi2, Yangying Hao3
1Division of Molecular Oncology, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan.
Abstract:
Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome-independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1-interacting E3 ligase using a cell-free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C-terminal region. DTX2-induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1-induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth-inhibitory effect in RUNX1-dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome-independent manner.
Insights
DTX2 E3 ligase regulates the RUNX1 transcription factor through proteasome-independent ubiquitination, inhibiting its acetylation and activity. This finding offers new insights into RUNX1 regulation in leukemia.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Biology
Background:
- Transcription factor RUNX1 is crucial for blood cell development and leukemia.
- RUNX1 activity is regulated by posttranslational modifications like ubiquitination and acetylation.
- Proteasome-independent ubiquitination of RUNX1 remains poorly understood.
Purpose of the Study:
- To investigate the role of DTX2, a previously identified RUNX1-interacting E3 ligase, in regulating RUNX1.
- To elucidate the mechanism of RUNX1 ubiquitination and its functional consequences.
Main Methods:
- In vitro and ex vivo analyses were performed.
- Binding assays confirmed DTX2 interaction with RUNX1, RUNX2, and RUNX3.
- Ubiquitination, acetylation, and reporter gene assays were utilized.
- Cellular localization and proliferation assays were conducted.
Main Results:
- DTX2 binds to the C-terminal region of RUNX1.
- DTX2 induces RUNX1 ubiquitination without causing protein degradation.
- DTX2 inhibits RUNX1 acetylation and RUNX1-mediated transcriptional activation.
- DTX2 promotes RUNX1 cytoplasmic mislocalization and inhibits leukemia cell growth.
Conclusions:
- DTX2 modulates RUNX1 ubiquitination and acetylation in a proteasome-independent manner.
- DTX2's regulation of RUNX1 impacts its function and cellular localization.
- DTX2 exhibits potential as a therapeutic target for RUNX1-dependent leukemias.
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