br Results br Discussion br STAR Methods
Acknowledgments The authors thank Drs. Michael Wegner, Ron Waclaw, and Ed Hurlock for critical comments. We thank Drs. Lihui Tsai and Jiang Wu for CHD8 and ASH2L-expressing vectors, respectively, Dr. Yueh-Chiang Hu for generating transgenic lines, and Jinglong Xu for technical support. This study was funded in part by grants from the US National Institutes of Health R01NS072427 and R01NS075243 to Q.R.L., the National Multiple Sclerosis Society (RG1508) to Q.R.L., the CHARGE Syndrome Foundation to Q.R.L. and C.Z., the National Natural Science Foundation of China 81720108018 to W.Z., the National Multiple Sclerosis Society (NMSS RG-1501-02851) to C.P. and Q.R.L., and the Fondation pour l'Aide à la Recherche sur la Sclérose en Plaques (ARSEP, 2014, 2015, 2017) to C.P.
Introduction Carcinogenesis and metabolic disorder have been reported to be caused by epigenomic alterations , . In other words, diseases are caused by qualitative changes, which are not supposed to occur normally, but have occurred regardless, due to the genome in healthy NECA being epigenetically modified by genes or environmental factors. The development process is characterized by dynamic epigenetic alterations by complicated networks, giving rise to patterns of tissue-specific gene expression. Different epigenetic alterations in cells with identical genomes result in each individual acquiring unique identity. On the other hand, inadequate or inappropriate epigenetic alterations can result in birth defects . Modifications of histones, include acetylation, methylation, and phosphorylation, which are among the main mechanisms of epigenetic alterations and are highly preserved in eukaryotes , , . The manifold combinations of these modifications not only activate/deactivate gene expression but also regulate subtle differences in the duration and strength of gene expression, thus contributing to the diversity of cells , , . Histone modification, particularly, methylation of the lysine residues of histone H3, is known to regulate gene expression. SETDB1, a histone methyltransferase, is an enzyme that methylates the lysine 9 residue of the histone H3 protein (H3K9). Tri-methylation of H3K9, which is located in the euchromatin region, negatively regulates gene expression . Since conventional knockout mice of Setdb1 gene die at the implantation stage , its tissue specific roles remain unknown. For this reason, researchers are now using conditional knockout mice based on various Cre lines. Setdb1 was knocked out only in mesenchymal cells by using the Prx1-Cre line. The knockout resulted in accelerated chondrocyte hypertrophy and a decrease in the number of mature chondrocytes constituting the epiphyseal plates, which in turn led to abnormal endochondral ossification and deformed bone structure . This suggested that epigenetic regulation plays an important role in proliferation and differentiation of chondrocytes. Meckel's cartilage plays a crucial role as a supportive tissue for mandible formation during the embryogenesis period. The properties of chondrocytes in Meckel's cartilage differ from those of chondrocytes derived from mesenchymal cells that are involved in the process of endochondral ossification . In the early embryonic stage of the mandible, Meckel's cartilage forms an aggregation of cells near the first molar tooth. It then elongates in an anteroposterior direction, eventually taking the shape of a transparent pole-like structure. Endochondral ossification at its distal and proximal ends generates the alveolar bone of the mandibular anterior tooth as well as the malleus and incus, respectively. The chondrocytes in the central major part of Meckel's cartilage, on the other hand, differentiate into hypertrophic chondrocytes, stop differentiating further and degenerate eventually . Bone morphogenic protein (BMP) signals are responsible for essential functions in each of the endochondral ossification processes, such as differentiation from undifferentiated mesenchymal cells to chondrocytes, proliferation of chondrocytes, and their hypertrophy , , . The overexpression of BMPs expedites the proliferation of chondrocytes. This leads to a dramatic increase in the proportion of chondrocytes, resulting in abnormal bone structure and size . In mice, where BMP receptors were knocked out only in chondrocytes, the formation of cartilage was almost completely inhibited, with endochondral ossification deforming the resulting bone structure . In a mouse model, where both SMAD1 and SMAD5 were knocked out only in chondrocytes, displayed severe achondroplasia, suggesting that SMAD-mediated BMP signals are indispensable for cartilage differentiation during endochondral ossification . To further examine the roles of BMP signals in Meckel's cartilage, Wang et al. used a system in which BMP signals are stronger than normal by either suppressing the expression of Noggin or overexpressing BMPR1A. This resulted in the suppression of the denaturation of Meckel's cartilage and endochondral ossification, indicating, as above, that BMP signals probably play pivotal role in endochondral ossification and Meckel's cartilage itself .