Dual Chromatin and Cytoskeletal Remodeling by SETD2.

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.

The CH25H-CYP7B1-RORα axis of cholesterol metabolism regulates osteoarthritis.

Osteoarthritis-the most common form of age-related degenerative whole-joint disease1-is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling2,3. However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated4-6. Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes7,8. Here we show that the CH25H-CYP7B1-RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H-CYP7B1-RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis.

Molecular determinants for α-tubulin methylation by SETD2.

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2-Rpb1-interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.

Neuronal SETD2 activity links microtubule methylation to an anxiety-like phenotype in mice.

Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.

Understanding the Molecular Mechanisms Underlying the Pathogenesis of Arthritis Pain Using Animal Models.

Arthritis, including osteoarthritis (OA) and rheumatoid arthritis (RA), is the leading cause of years lived with disability (YLD) worldwide. Although pain is the cardinal symptom of arthritis, which is directly related to function and quality of life, the elucidation of the mechanism underlying the pathogenesis of pain in arthritis has lagged behind other areas, such as inflammation control and regulation of autoimmunity. The lack of therapeutics for optimal pain management is partially responsible for the current epidemic of opioid and narcotic abuse. Recent advances in animal experimentation and molecular biology have led to significant progress in our understanding of arthritis pain. Despite the inherent problems in the extrapolation of data gained from animal pain studies to arthritis in human patients, the critical assessment of molecular mediators and translational studies would help to define the relevance of novel therapeutic targets for the treatment of arthritis pain. This review discusses biological and molecular mechanisms underlying the pathogenesis of arthritis pain determined in animal models of OA and RA, along with the methodologies used.

Anterior Segmental Osteotomy Using Customized Spider-Plates Based on Computer-aided Surgery System.

Anterior segmental osteotomy (ASO) is considered the treatment modality of choice in patients with the bimaxillary dentoalveolar protrusion. However, this meticulous surgical technique accompanies a number of possible disadvantages. The considerable time required before, during, and after the operation, limited movement of the segment, damage of the mental nerve, loss of tooth vitality, loss of a tooth or teeth, or indeed total loss of the anterior segments are those that affect the result of the surgery. Recently, the authors have devised a computer-aided surgical simulation programme and fabricated the customized osteotomy guides and the spider-shaped plates based on the programme. They were then applied to a 28-year-old patient with the complaint of a bimaxillary dentoalveolar protrusion. This approach helped to overcome several problems related to ASO reported earlier.

The accuracy and stability of the maxillary position after orthognathic surgery using a novel computer-aided surgical simulation system.

BACKGROUND: Many reports have been published on orthognathic surgery (OGS) using computer-aided surgical simulation (CASS). The purpose of this study was to evaluate the accuracy of the maxillary repositioning and the stability of the maxilla in patients who underwent OGS using a newly developed CASS program, a customized osteotomy guide, and a customized miniplate. METHODS: Thirteen patients who underwent OGS from 2015 to 2017 were included. All patients underwent a bimaxillary operation. First, a skull-dentition hybrid 3D image was rendered by merging the cone beam computed tomography (CBCT) images with the dentition scan file. After virtual surgery (VS) using the FaceGide® program, patient-customized osteotomy guides and miniplates were then fabricated and used in the actual operation. To compare the VS with the actual surgery and postoperative skeletal changes, each reference point marked on the image was compared before the operation (T0) and three days (T1), four months (T2), and a year (T3) after the operation, and with the VS (Tv). The differences between ΔTv (Tv-T0) and ΔT1 (T1-T0) were statistically compared using tooth-based reference points. The superimposed images of Tv and T1 were also investigated at eight bone-based reference points. The differences between the reference points of the bone surface were examined to evaluate the stability of the miniplate on the maxilla over time. RESULTS: None of the patients experienced complications. There were no significant differences between the reference points based on the cusp tip between ΔTv and ΔT1 (p > 0.01). Additionally, there were no significant differences between the Tv and T1 values of the bone surface (p > 0.01). The mean difference in the bone surface between Tv and T1 was 1.01 ± 0.3 mm. Regarding the stability of the miniplate, there were no significant differences between the groups. The difference in the bone surface between T1 and T3 was - 0.37 ± 0.29 mm. CONCLUSIONS: VS was performed using the FaceGide® program, and customized materials produced based on the VS were applied in actual OGS. The maxilla was repositioned in almost the same manner as in the VSP plan, and the maxillary position remained stable for a year.

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes.

BACKGROUND: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. METHODS: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1ß- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. RESULTS: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1ß-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1ß- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. CONCLUSION: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

Quantification of histone modifications by parallel-reaction monitoring: a method validation.

Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C.

A 2-year follow-up of changes after bimaxillary surgery in patients with mandibular prognathism: 3-dimensional analysis of pharyngeal airway volume and hyoid bone position.

PURPOSE: The aims of this study were to use 3-dimensional cone-beam computed tomography (CBCT) to evaluate how the upper airway and hyoid bone position changed after orthognathic surgery in patients with skeletal Class III malocclusions and to analyze the relations among upper airway changes, the change in the position of the hyoid bone, and postsurgical stability. MATERIALS AND METHODS: CBCT scans were obtained from 15 patients with mandibular prognathism before surgery (T0), 6 months after surgery (T1), 1 year after surgery (T2), and 2 years after surgery (T3). Positional displacement of the hyoid bone was assessed using the coordinates at T0, T1, T2, and T3. In addition, the volume of each patient's pharyngeal airway was measured. Differences in CBCT scans at the established time points were determined by the Wilcoxon signed rank test. The Spearman correlation coefficient was used to determine the relations among changes in hyoid bone position, airway volume, and skeletal reference points. RESULTS: The hyoid bone moved backward at 6 months after surgery (T0 to T1), and the total volume of the pharyngeal airway was considerably decreased at the same time points. At 1 year after surgery (T1 to T2), although the hyoid moved more posteriorly and the total volume of the pharyngeal airway was decreased, the changes were not major. At 2 years after surgery, the hyoid bone moved anteriorly and the size of the upper pharyngeal airway was increased (T2 to T3). CONCLUSION: The hyoid bone moved posteriorly and the pharyngeal airway volume was decreased at 6 months after bimaxillary surgery. These measurements had a tendency to recover at 2 years postoperatively. The decrease in pharyngeal airway volume was not correlated with positional changes of the hyoid bone.