Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation, rapid target gene repression, and mouse viability.

GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability.

The proinflammatory cytokine TNFα induces DNA demethylation-dependent and -independent activation of interleukin-32 expression.

IL-32 is a cytokine involved in proinflammatory immune responses to bacterial and viral infections. However, the role of epigenetic events in the regulation of IL-32 gene expression is understudied. Here we show that IL-32 is repressed by DNA methylation in HEK293 cells. Using ChIP sequencing, locus-specific methylation analysis, CRISPR/Cas9-mediated genome editing, and RT-qPCR (quantitative RT-PCR) and immunoblot assays, we found that short-term treatment (a few hours) with the proinflammatory cytokine tumor necrosis factor α (TNFα) activates IL-32 in a DNA demethylation-independent manner. In contrast, prolonged TNFα treatment (several days) induced DNA demethylation at the promoter and a CpG island in the IL-32 gene in a TET (ten-eleven translocation) family enzyme- and NF-κB-dependent manner. Notably, the hypomethylation status of transcriptional regulatory elements in IL-32 was maintained for a long time (several weeks), causing elevated IL-32 expression even in the absence of TNFα. Considering that IL-32 can, in turn, induce TNFα expression, we speculate that such feedforward events may contribute to the transition from an acute inflammatory response to chronic inflammation.

Small molecules capable of activating DNA methylation-repressed genes targeted by the p38 mitogen-activated protein kinase pathway.

Regulation of gene expression by epigenetic modifications such as DNA methylation is crucial for developmental and disease processes, including cell differentiation and cancer development. Genes repressed by DNA methylation can be derepressed by various compounds that target DNA methyltransferases, histone deacetylases, and other regulatory factors. However, some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Chemical agents that can counteract the effects of epigenetic repression that is not regulated by DNA methyltransferases or histone deacetylases therefore may be of research interest. Here, we report the results of a high-throughput screen using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation. Seven hit compounds were identified that did not directly target bulk DNA methylation or histone acetylation. Analyzing the effect of these compounds on endogenous gene expression, we discovered that three of these compounds (compounds LX-3, LX-4, and LX-5) selectively activate the p38 mitogen-activated protein kinase (MAPK) pathway and derepress a subset of endogenous genes repressed by DNA methylation. Selective agonists of the p38 pathway have been lacking, and our study now provides critical compounds for studying this pathway and p38 MAPK-targeted genes repressed by DNA methylation.

Comparison of human dental tissue RNA extraction methods for RNA sequencing.

OBJECTIVE: The purpose of this study was to identify an efficient RNA extraction method for periodontal ligament (PDL) and dental pulp (DP) tissues to be used in RNA sequencing studies, given the increased use of these techniques in dental research and the lack of standard protocols. DESIGN: PDL and DP were harvested from extracted third molars. Total RNA was extracted with four RNA extraction kits. RNA concentration, purity and integrity were assessed by means of NanoDrop and Bioanalyzer and statistically compared. RESULTS: RNA from PDL was more likely to be degraded than that of DP. The TRIzol method yielded the highest RNA concentration from both tissues. All methods harvested RNA with A260/A280 close to 2.0 and with A260/A230 above 1.5, except for the A260/A230 from PDL obtained with the RNeasy Mini kit. For RNA integrity, the RNeasy Fibrous Tissue Mini kit yielded the highest RIN values and 28 S/18 S from PDL, while the RNeasy Mini kit obtained relatively high RIN values with an appropriate 28 S/18 S for DP. CONCLUSION: Significantly different results were obtained for PDL and DP when using the RNeasy Mini kit. The RNeasy Mini kit provided the highest RNA yields and quality for DP, while the RNeasy Fibrous Tissue Mini kit obtained the highest quality RNA from PDL.

Third molar position after completion of orthodontic treatment: a prospective follow-up.

OBJECTIVES: To prospectively follow up a previously reported sample, analyzing (1) changes in third molar (M3) position after completion of 2 different types of orthodontic treatment: (2) non-extraction treatment with (HG) vs without cervical headgear (non-HG) and (3) first or second premolar extractions (PM1-2) compared to a non-extraction group (NE). METHODS: A total of 474 patients were prospectively followed up. Panoramic radiographs were taken pre- (T1), post-treatment (T2) and at follow-up (T3). T3 records (a mean of three years after treatment) were available for 135 (HG vs non-HG) and 134 patients (PM1-2 vs NE), respectively. Angulation, vertical position, relation with the mandibular canal and mineralization status of M3 at T2 and T3 were statistically compared. RESULTS: The HG group presented more M3 with ideal vertical orientation at T3. In NE-cases, further improvement in angulation and orientation can be expected after debonding, as well as a deterioration in the relationship with the mandibular canal. Extractions accelerated upper M3 vertical eruption and PM2 extractions led to long-term larger lower retromolar spaces. CONCLUSIONS: The use of cervical headgear increased upper M3 uprighting three years after debonding, while little changes in M3 position were found after orthodontic treatment with extractions. However, PM2 extractions led to larger retromolar spaces and better M3 angulation in the long term.

Biomimetic Periodontal Ligament Transplantation Activated by Gold Nanoparticles Protects Alveolar Bone.

Stem cell therapy might be a promising method to stimulate alveolar bone regeneration, which is currently a major clinical challenge. However, its therapeutic features largely depend on pretreatment and transplantation preparation. Herein, a novel biomimetic periodontal ligament transplantation composed of human periodontal ligament stem cells (hPDLSCs) pretreated with gold nanocomplexes (AuNCs) and embedded in a type-I collagen hydrogel scaffold is developed to protect alveolar bone from resorption. AuNCs are readily absorbed by primary hPDLSCs, with limited cytotoxicity, and promote osteogenic differentiation of hPDLSCs effectively in vitro. In addition, the AuNCs-induced hPDLSCs are encapsulated with type-I collagen hydrogel scaffold to mimic their native physiological niche, and then are transplanted into a rat model of alveolar bone resorption. Both micro-computed tomography (micro-CT) and immunohistochemical assays demonstrate that alveolar bone loss is significantly prevented. Furthermore, the underlying therapeutic mechanism is elucidated, in which transplantation-activated osteogenesis is associated with autophagy, which enables bone remodeling and regeneration. This study provides critical insight into the role of PDLSCs in bone homeostasis and proposes an innovative AuNCs-based strategy for stem cell therapy in bone regeneration.

Acellular matrix hydrogel for repair of the temporomandibular joint disc.

Application of tissue-derived extracellular matrix (ECM) biomaterials in the repair of the temporomandibular joint (TMJ) disc is a promising approach for the treatment of disc abrasion and perforation, particularly for the young patient population. Although decellularized ECM (dECM) scaffolds preserve tissue-specific structures as well as biological and biomechanical properties, they require surgical implantation. To address this issue, we prepared porcine TMJ discs into decellularized ECM with serial detergent and enzyme treatments, and the TMJ disc-derived ECM was then processed into hydrogels via pepsin digestion. The decellularization efficiency was assessed by quantification of the DNA and matrix component contents. The fibrous ultrastructure of the hydrogel was observed by scanning electron microscopy (SEM). Rheological characterization and mechanical properties were measured. in vitro experiments with costal chondrocytes ensured the cellular proliferative capacity and compatibility in the injectable disc-derived ECM hydrogel. The results showed that a large amount of DNA (>95%) was removed after decellularization; but, the collagen was retained. SEM of the hydrogels demonstrated a multiaperture fiber ultrastructure. Rheological studies revealed a rapid gelation temperature (37°C) and injectable properties. The mechanical properties of the hydrogels were adjusted by changing the ECM concentration. The in vitro studies revealed that the hydrogels are not cytotoxic, but instead showed good cytocompatibility. The hydrogel also showed good injectability and degradability through an in vivo study. Overall, these results suggest the great potential of injectable disc-derived hydrogels for TMJ disc repair and regeneration applications.

Quantitative evaluation of retromolar space in adults with different vertical facial types.

OBJECTIVE: To investigate the differences in mandibular retromolar space among skeletal Class I subjects with different vertical divergence using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: A total of 123 skeletal Class I patients (aged 20-40 years) were categorized into hypodivergent, normodivergent, and hyperdivergent groups based on S-N/Go-Me and facial height index (FHI). Mandibular retromolar space was measured at four planes parallel to the occlusal plane along the sagittal line and molar cuspal line, respectively. The mandibular retromolar space was compared among the three vertical groups. RESULTS: The hyperdivergent group had a significantly smaller mandibular retromolar space compared with the other two groups, while the hypodivergent group had the largest retromolar space. In addition, the hyperdivergent group had a larger number of subjects whose roots contacted the lingual cortex of the mandibular body. CONCLUSIONS: The hyperdivergent group tends to exhibit the smallest mandibular retromolar space and highest risk of cortex contact. Clinicians should keep in mind that successful molar distalization requires sufficient retromolar space, especially for hyperdivergent subjects, which should be verified with CBCT.

Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.

Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues.

Genes are the instruction manuals of life and contain the information needed to build the building blocks that keep cells alive. To read these instructions, cells use specific signals that activate genes. The process, known as gene expression, is tightly controlled and for the most part, fairly stable. But gene expression can be modified in various ways. Epigenetics is a broad term for describing reversible changes made to genes to switch them on and off. Sometimes, certain genes even develop a kind of 'transcriptional memory' where over time, their expression is enhanced and speeds up with repeated activation signals. But this may also have harmful effects. For example, the signalling molecule called tumour necrosis factor α (TNF-α) is an essential part of the immune system. But it is also implicated in chronic inflammatory diseases, such as rheumatoid arthritis. In these conditions, cell signalling pathways triggering inflammation are overactive. One possibility is that TNF-α could be inducing the transcriptional memory of certain genes, amplifying their expression. But little is known about which fraction of genes exhibits transcriptional memory, and what differentiates memory genes from genes with stable expression. Here, Zhao et al. treated cells grown in the laboratory with TNF-α to investigate its role in transcriptional memory and find out what epigenetic features might govern the process. The experiments showed that mimicking a sustained inflammation by stimulating TNF-α, triggered a transcriptional memory in some genes, and enabled them to respond to much lower levels of TNF-α on subsequent exposure. Zhao et al. also discovered that genes tagged with methyl groups are more likely to show transcriptional memory when stimulated by TNF-α. However, they also found that these groups must be removed to consolidate any transcriptional memory. This work shows how TNF-α influences can alter the expression of certain genes. It also suggests that transcriptional memory, stimulated by TNF-α, may be a possible mechanism underlying chronic inflammatory conditions. This could help future research in identifying more genes with transcriptional memory.

Inhibition of Histone Deacetylation by MS-275 Alleviates Colitis by Activating the Vitamin D Receptor.

BACKGROUND: Ulcerative colitis [UC] is a common chronic inflammatory bowel disease without curative treatment. METHODS: We conducted gene set enrichment analysis to explore potential therapeutic agents for UC. Human colon tissue samples were collected to test H3 acetylation in UC. Both in vivo and in vitro colitis models were constructed to verify the role and mechanism of H3 acetylation modification in UC. Intestine-specific vitamin D receptor [VDR]-/- mice and VD [vitamin D]-deficient diet-fed mice were used to explore downstream molecular mechanisms accordingly. RESULTS: According to the Connectivity Map database, MS-275 [class I histone deacetylase inhibitor] was the top-ranked agent, indicating the potential importance of histone acetylation in the pathogenesis of UC. We then found that histone H3 acetylation was significantly lower in the colon epithelium of UC patients and negatively associated with disease severity. MS-275 treatment inhibited histone H3 deacetylation, subsequently attenuating nuclear factor kappa B [NF-κB]-induced inflammation, reducing cellular apoptosis, maintaining epithelial barrier function, and thereby reducing colitis activity in a mouse model of colitis. We also identified VDR as be a downstream effector of MS-275. The curative effect of MS-275 on colitis was abolished in VDR-/- mice and in VD-deficient diet-fed mice and VDR directly targeted p65. In UC patients, histone H3 acetylation, VDR and zonulin-1 expression showed similar downregulation patterns and were negatively associated with disease severity. CONCLUSIONS: We demonstrate that MS-275 inhibits histone deacetylation and alleviates colitis by ameliorating inflammation, reducing apoptosis, and maintaining intestinal epithelial barrier via VDR, providing new strategies for UC treatment.