JARID1B represses the osteogenic potential of human periodontal ligament mesenchymal cells.

BACKGROUND: Here, we evaluated whether the histone lysine demethylase 5B (JARID1B), is involved in osteogenic phenotype commitment of periodontal ligament cells (PDLCs), by considering their heterogeneity for osteoblast differentiation. MATERIALS AND METHODS: Epigenetic, transcriptional, and protein levels of a gene set, involved in the osteogenesis, were investigated by performing genome-wide DNA (hydroxy)methylation, mRNA expression, and western blotting analysis at basal (without osteogenic induction), and at the 3rd and 10th days of osteogenic stimulus, in vitro, using PDLCs with low (l) and high (h) osteogenic potential as biological models. RESULTS: h-PDLCs showed reduced levels of JARID1B, compared to l-PDLCs, with significant inversely proportional correlations between RUNX2 and RUNX2/p57. Epigenetically, a significant reduction in the global H3K4me3 content was observed only in h-PDLCs. Immunoblotting data reveal a significant reduction in the global H3K4me3 content, at 3 days of induction only in h-PDLCs, while an increase in the global H3K4me3 content was observed at 10 days for both PDLCs. Additionally, positive correlations were found between global H3K4me3 levels and JARID1B gene expression. CONCLUSIONS: Altogether, our results show the crucial role of JARID1B in repressing PDLCs osteogenic phenotype and this claims to pre-clinical protocols proposing JARID1B as a potential therapeutic target.

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs.

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p < 0.05); conversely, SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs.

Osteogenic Commitment of Human Periodontal Ligament Cells Is Predetermined by Methylation, Chromatin Accessibility and Expression of Key Transcription Factors.

Periodontal ligament stem cells (PDLCs) can be used as a valuable source in cell therapies to regenerate bone tissue. However, the potential therapeutic outcomes are unpredictable due to PDLCs' heterogeneity regarding the capacity for osteoblast differentiation and mineral nodules production. Here, we identify epigenetic (DNA (hydroxy)methylation), chromatin (ATAC-seq) and transcriptional (RNA-seq) differences between PDLCs presenting with low (l) and high (h) osteogenic potential. The primary cell populations were investigated at basal state (cultured in DMEM) and after 10 days of osteogenic stimulation (OM). At a basal state, the expression of transcription factors (TFs) and the presence of gene regulatory regions related to osteogenesis were detected in h-PDLCs in contrast to neuronal differentiation prevalent in l-PDLCs. These differences were also observed under stimulated conditions, with genes and biological processes associated with osteoblast phenotype activated more in h-PDLCs. Importantly, even after the induction, l-PDLCs showed hypermethylation and low expression of genes related to bone development. Furthermore, the analysis of TFs motifs combined with TFs expression suggested the relevance of SP1, SP7 and DLX4 regulation in h-PDLCs, while motifs for SIX and OLIG2 TFs were uniquely enriched in l-PDLCs. Additional analysis including a second l-PDLC population indicated that the high expression of OCT4, SIX3 and PPARG TFs could be predictive of low osteogenic commitment. In summary, several biological processes related to osteoblast commitment were activated in h-PDLCs from the onset, while l-PDLCs showed delay in the activation of the osteoblastic program, restricted by the persistent methylation of gene related to bone development. These processes are pre-determined by distinguishable epigenetic and transcriptional patterns, the recognition of which could help in selection of PDLCs with pre-osteoblastic phenotype.

Genome-wide DNA (hydroxy) methylation reveals the individual epigenetic landscape importance on osteogenic phenotype acquisition in periodontal ligament cells.

BACKGROUND: Mesenchymal cells' biology has been an important investigative tool to maximize bone regeneration through tissue engineering. Here we used mesenchymal cells from periodontal ligament (PDLCs) with high (h-) and low (l-) osteogenic potential, isolated from different donors, to investigate the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential. METHODS: Genome-wide and gene-specific DNA (hydroxy) methylation, mRNA expression and immunofluorescence analysis were carried out in h- and l-PDLCs at DMEM (non-induced to osteogenesis) and OM (induced-3rd and 10th days of osteogenic differentiation) groups in vitro. RESULTS: Genome-wide results showed distinct epigenetic profile among PDLCs with most of the differences on 10th day of OM; DMEMs showed higher concentrations (xOM) of differentially methylated probes in gene body, intronic and open sea (3rd day), increasing this concentration in TSS200 and island regions, at 10 days. At basal levels, h- and l-PDLCs showed different transcriptional profiles; l-PDLCs demonstrated higher levels of NANOG/OCT4/SOX2, BAPX1, DNMT3A, TET1/3, and lower levels of RUNX2 transcripts, confirmed by NANOG/OCT4 and RUNX2 immunofluorescence. After osteogenic induction, the distinct transcriptional profile of multipotentiality genes was maintained among PDLCs. In l-PDLCs, the anti-correlation between DNA methylation and gene expression in RUNX2 and NANOG indicates methylation could play a role in modulating both transcripts. CONCLUSIONS: Epigenetic and transcriptional distinct profiles detected at basal levels among PDLCs were maintained after osteogenic induction. We cannot discard the existence of a complex that represses osteogenesis, suggesting the individual donors' characteristics have significant impact on the osteogenic phenotype acquisition.