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Published on: December 3, 2015
BCL6B-dependent suppression of ETV2 hampers endothelial cell differentiation
Zhonghao Li1, Wei Wu2, Qiushi Li1
1Department of Cardiology, Translational Research Center for Regenerative Medicine and 3D Printing Technologies, Guangdong Provincial Key Laboratory of Major Obstetric Diseases; Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510150, China.
Insights
B-cell CLL/lymphoma 6 member B (BCL6B) suppresses endothelial cell (EC) differentiation by inhibiting ETV2. This finding reveals a novel mechanism for regulating vascular development and offers potential therapeutic targets.
Area of Science:
- Molecular Biology
- Stem Cell Biology
- Vascular Biology
Background:
- BCL6B is a transcriptional repressor involved in various cellular processes.
- Its role in endothelial cell (EC) development was previously unknown.
- ETV2 is a known facilitator of EC differentiation.
Purpose of the Study:
- To investigate the role of BCL6B in EC differentiation.
- To elucidate the underlying mechanisms of BCL6B's function in EC development.
Main Methods:
- Overexpression and knockdown of BCL6B in human induced pluripotent stem cells (hiPSCs).
- RNA sequencing, qRT-PCR, and flow cytometry for gene expression and differentiation efficiency.
- In vitro tube formation assays and immunofluorescence for EC function and structure.
- Luciferase reporter and ChIP-PCR assays to determine BCL6B-ETV2 interaction.
Main Results:
- BCL6B overexpression suppressed EC differentiation and tube formation from hiPSCs.
- BCL6B knockdown improved EC differentiation.
- BCL6B directly binds to the ETV2 promoter, repressing its transcriptional activity.
- Overexpression of ETV2 rescued the inhibitory effects of BCL6B.
Conclusions:
- BCL6B inhibits EC differentiation and vessel organoid development.
- This inhibition occurs through the repression of ETV2 transcriptional activity.
- BCL6B represents a novel regulator of vascular development.
Background:
B-cell CLL/lymphoma 6 member B (BCL6B) operates as a sequence-specific transcriptional repressor within the nucleus, playing crucial roles in various biological functions, including tumor suppression, immune response, stem cell self-renew, and vascular angiogenesis. However, whether BCL6B is involved in endothelial cell (EC) development has remained largely unknown. ETS variant transcription factor 2 (ETV2) is well known to facilitate EC differentiation. This study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms.
Methods:
Doxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown were established and subjected to differentiate into ECs and vessel organoids (VOs). RNA sequencing analysis was performed to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pluripotency and vascular-specific marker genes expression. EC differentiation efficiency was determined by Flow cytometry analysis. The performance of EC was evaluated by in vitro Tube formation assay. The protein expression and the vessel-like structures were assessed using immunofluorescence analysis or western blot. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP)-PCR analysis were used to determine the regulatory relationship between BCL6B and ETV2.
Results:
Functional ECs and VOs were successfully generated from hiPSCs. Notably, overexpression of BCL6B suppressed while knockdown of BCL6B improved EC differentiation from hiPSCs. Additionally, the overexpression of BCL6B attenuated the capacity of derived hiPSC-ECs to form a tubular structure. Furthermore, compared to the control VOs, BCL6B overexpression repressed the growth of VOs, whereas BCL6B knockdown had little effect on the size of VOs. RNA sequencing analysis confirmed that our differentiation protocol induced landscape changes for cell/tissue/system developmental process, particularly vascular development and tube morphogenesis, which were significantly modulated by BCL6B. Subsequent experiments confirmed the inhibitory effect of BCL6B is facilitated by the binding of BCL6B to the promoter region of ETV2, led to the suppression of ETV2's transcriptional activity. Importantly, the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression.
Conclusions:
BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.
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