Dataset Information

Stage-specific requirement for METTL3-dependent m⁶A modification during dental pulp stem cell differentiation.

ABSTRACT:

Background

N⁶-methyladenosine (m⁶A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m⁶A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m⁶A methylation in dental pulp stem cell (DPSC) differentiation.

Methods

m⁶A RNA immunoprecipitation sequencing was utilized to assess the m⁶A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m⁶A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3'-RACE and poly(A) tail length assays.

Results

We characterized a dynamic m⁶A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3' untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m⁶A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m⁶A deposition in the 3' UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m⁶A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m⁶A-tagged NOG by shortening the poly(A) tail length in the differentiated stage.

Conclusions

Our results address an essential role of dynamic m⁶A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.

SUBMITTER: Luo H

PROVIDER: S-EPMC9756505 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Publications

Stage-specific requirement for METTL3-dependent m<sup>6</sup>A modification during dental pulp stem cell differentiation.

Luo Haiyun H Liu Wenjing W Zhou Yachuan Y Zhang Yanli Y Wu Junrong J Wang Ruolan R Shao Longquan L

Journal of translational medicine 20221216 1

<h4>Background</h4>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m<sup>6</sup>A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m<sup>6</sup>A methylation in dental pulp stem cell (DPSC) differentiation.<h4>Meth ...[more]

PMID: 36527141

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Project description:BackgroundThe dentinogenesis differentiation of dental pulp stem cells (DPSCs) is controlled by the spatio-temporal expression of differentiation related genes. RNA N6-methyladenosine (m6A) methylation, one of the most abundant internal epigenetic modification in mRNA, influences various events in RNA processing, stem cell pluripotency and differentiation. Methyltransferase like 3 (METTL3), one of the essential regulators, involves in the process of dentin formation and root development, while mechanism of METTL3-mediated RNA m6A methylation in DPSC dentinogenesis differentiation is still unclear.MethodsImmunofluorescence staining and MeRIP-seq were performed to establish m6A modification profile in dentinogenesis differentiation. Lentivirus were used to knockdown or overexpression of METTL3. The dentinogenesis differentiation was analyzed by alkaline phosphatase, alizarin red staining and real time RT-PCR. RNA stability assay was determined by actinomycin D. A direct pulp capping model was established with rat molars to reveal the role of METTL3 in tertiary dentin formation.ResultsDynamic characteristics of RNA m6A methylation in dentinogenesis differentiation were demonstrated by MeRIP-seq. Methyltransferases (METTL3 and METTL14) and demethylases (FTO and ALKBH5) were gradually up-regulated during dentinogenesis process. Methyltransferase METTL3 was selected for further study. Knockdown of METTL3 impaired the DPSCs dentinogenesis differentiation, and overexpression of METTL3 promoted the differentiation. METTL3-mediated m6A regulated the mRNA stabiliy of GDF6 and STC1. Furthermore, overexpression of METTL3 promoted tertiary dentin formation in direct pulp capping model.ConclusionThe modification of m6A showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated m6A regulated in dentinogenesis differentiation through affecting the mRNA stability of GDF6 and STC1. METTL3 overexpression promoted tertiary dentin formation in vitro, suggesting its promising application in vital pulp therapy (VPT).

Project description:BackgroundDifferentiating dental pulp stem cells (DPSCs) into odontoblasts is a critical process for tooth self-repair and dentine‒pulp engineering strategies in the clinic. However, the mechanism underlying the regulation of DPSC odontoblastic differentiation remains largely unknown. Here, we demonstrated that BCL-2 interacting protein 3 (BNIP3)-dependent mitophagy is associated with importin subunit beta-1 (KPNB1)-activating transcription factor 4 (ATF4), which promotes DPSC odontoblastic differentiation.MethodsThe key genes involved in DPSC odontogenic differentiation were identified via bioinformatics. Stable silencing or overexpression of BNIP3 was performed to investigate its impact on DPSC differentiation in vitro (n ≥ 3). To explore the role of BNIP3 in vivo, tooth root fragments loaded with the hydrogel-transfected DPSC complex were implanted into nude mice (n ≥ 6). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) polymerase chain reaction (PCR) were conducted to explore the binding site of ATF4 to the BNIP3 promoter (n ≥ 3). Mitochondrial function experiments were performed to investigate the impact of ATF4-BNIP3 on mitochondria (n ≥ 3). Immunoprecipitation (IP) mass spectrometry (MS) was used to investigate the interaction between ATF4 and its binding protein, KPNB1. Plasmids containing wild-type (WT)/mutant (MUT)-nuclear localization signal (NLS) forms of ATF4 were constructed to determine the specific amino acid residues recognized by KPNB1 and their effects on DPSC odontoblastic differentiation (n ≥ 3).ResultsCompared with those in the control group, the levels of autophagy and mitophagy, especially BNIP3-dependent mitophagy, were greater in the DPSC odontoblastic differentiation group (P < 0.05). Genetic silencing or overexpression of BNIP3 demonstrated that BNIP3 expression was positively correlated with the transition of DPSCs into odontoblasts both in vitro and in vivo (P < 0.05). ATF4 regulates the expression of BNIP3 by directly binding to approximately -1292 to -1279 bp and approximately -1185 to -1172 bp within the BNIP3 promoter region, which is associated with mitophagy and mitochondrial reactive oxygen species (mtROS) levels (P < 0.05). Moreover, ATF4 increased mitophagy, mitochondrial function, and cell differentiation potential via BNIP3 (P < 0.05). Mechanistically, KPNB1 is a novel interacting protein of ATF4 that specifically recognizes amino acids (aa) 280-299 within ATF4 to control its translocation into the nucleus and subsequent transcription and differentiation processes (P < 0.05).ConclusionsWe reported that the critical role of KPNB1/ATF4/BNIP3 axis-dependent mitophagy could provide new cues for the regeneration of the dental pulp‒dentin complex in DPSCs.

Dataset Information

Stage-specific requirement for METTL3-dependent m⁶A modification during dental pulp stem cell differentiation.

Background

Methods

Results

Conclusions

Publications

Stage-specific requirement for METTL3-dependent m<sup>6</sup>A modification during dental pulp stem cell differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation.

Background

Methods

Results

Conclusions

Publications

Stage-specific requirement for METTL3-dependent m<sup>6</sup>A modification during dental pulp stem cell differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Stage-specific requirement for METTL3-dependent m⁶A modification during dental pulp stem cell differentiation.