Exploring the role of DNMT1 in dental papilla cell fate specification during mouse tooth germ development through integrated single-cell transcriptomics and bulk RNA sequencing

Article

作者: Okada, Hiroyuki ; Suzuki, Yutaka ; Seki, Masahide ; Chung, Ung-Il ; Eldeeb, Dahlia ; Ohba, Shinsuke ; Tanaka, Junichi ; Mishima, Kenji ; Hojo, Hironori

OBJECTIVESThis study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process.METHODSWe performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.RESULTSscRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.CONCLUSIONSDnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.

2024-04-30·ACS Nano

Dual-Responsive Nanomedicine Activates Programmed Antitumor Immunity through Targeting Lymphatic System

Article

作者: Huang, Jinsheng ; Yang, Shuguang ; Xiao, Hong ; Ren, Jie ; Li, Xiaoxia ; Liang, Simin ; Shuai, Xintao ; Han, Shisong

Effective antitumor immunotherapy depends on evoking a cascade of cancer-immune cycles with lymph nodes (LNs) as the initial sites for activating antitumor immunity, making drug administration through the lymphatic system highly attractive. Here, we describe a nanomedicine with dual responsiveness to pH and enzyme for a programmed activation of antitumor immune through the lymphatic system. The proposed nanomedicine can release the STING agonist diABZI-C2-NH₂ in the LNs' acidic environment to activate dendritic cells (DCs) and T cells. Then, the remaining nanomedicine hitchhikes on the activated T cells (PD-1⁺ T cells) through binding to PD-1, resulting in an effective delivery into tumor tissues owing to the tumor-homing capacity of PD-1⁺ T cells. The enzyme matrix metalloproteinase-2 (MMP-2) being enriched in tumor tissue triggers the release of PD-1 antibody (aPD-1) which exerts immune checkpoint blockade (ICB) therapy. Eventually, the nanomedicine delivers a DNA methylation inhibitor GSK-3484862 (GSK) into tumor cells, and then the latter combines with granzyme B (GZMB) to trigger tumor cell pyroptosis. Consequently, the pyroptotic tumor cells induce robust immunogenic cell death (ICD) enhancing the DCs maturation and initiating the cascading antitumor immune response. Study on a 4T1 breast tumor mouse model demonstrates the prominent antitumor therapeutic outcome of this nanomedicine through creating a positive feedback loop of cancer-immunity cycles including immune activation in LNs, T cell-mediated drug delivery, ICB therapy, and tumor cell pyroptosis-featured ICD.

2023-03-11·NAR Cancer

GSK-3484862 targets DNMT1 for degradation in cells

Article

作者: Santos, Margarida A ; Cheng, Xiaodong ; Zhang, Xing ; Chen, Qin ; Dai, Nan ; Corrêa, Ivan R ; Liu, Bigang ; Zeng, Yang ; Estecio, Marcos R ; Hwang, Jee Won ; Chen, Taiping

AbstractMaintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

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