Gene Regulation And Function Of Cartilage

软骨的基因调控和功能

• 批准号: 6966450
• 负责人: Yoshihiko Yamada
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966450
• 关键词: bone development; cartilage; cell differentiation; chondrocytes; cleft palate; craniofacial; dental development; ectoderm; gene induction /repression; genetic regulation

This project covers not only cartilage but also tooth and craniofacial development. Cartilage is a highly specialized connective tissue with distinct morphological and biochemical characteristics. Cartilage contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. When cartilage formation is impaired, skeletal malformation of the limbs, craniofacial bones, and appendicular skeleton occurs. Chondrocytes are the major cell type in cartilage and produce large amounts of cartilage-specific matrix molecules, such as type XI and type II collagen, and aggrecan. Cartilage formation is initiated by mesenchymal cell condensation to form primordial cartilage followed by chondrocyte differentiation. These include resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As a final step in endochondral bone formation, hypertrophic cartilage is invaded by blood vessels and osteoprogenitor cells, and the calcified cartilage is subsequently replaced by bone. Thus, spatial and temporal regulation of chondrocyte differentiation is essential in determining the length and width of skeletal components. Hormones and vitamins affect chondrocyte differentiation and maturation by regulating the transcription of genes. Our objective is to define the mechanisms for activating and repressing chondrocyte-specific genes and to elucidate the molecular basis of cartilage development and chondrocyte differentiation. We previously described the identification of a 24 bp sequence in the alpha 2 type XI collagen gene (Col11a2) promoter, which is able to switch promoter activity from neuronal tissues to cartilage in transgenic mice. We subsequently isolated a protein factor we named NT2, which bound to the 24 bp sequence. NT2 is a C2H2-type zinc finger protein consisting of a Kruppel-associated box (KRAB), a potent DNA-binding-dependent repression module. We found that NT2 functions as a negative regulator of Col11a2. We demonstrated by chromatin immunoprecipitation assays that NT2 forms a complex of these heterochromatin- associated proteins at the 24 bp region of the Col11a2 promoter in NIH3T3 cells, in which Col11a2 is repressed but NT2 is expressed. These results suggest that repression of Col11a2 in NIH3T3 cells is caused by heterochromatin formation. The heterochromatin can be nucleated by the 24 bp sequence, which is recognized by the zinc finger domain of NT2. The KRAB domain of NT2 then recruits KAP1 via the KRAB domain followed by the association of other proteins including histone-modifying enzymes. Our results suggest the importance of both positive and negative transcriptional gene regulation for cartilage gene expression during chondrogenesis. In our the Oral and Craniofacial Genome Anatomy Project (OC-GAP), our goal is to discover and characterize previously unknown genes to help understand how tooth and craniofacial tissues develop and to define the molecular defects underlying anomalies of these tissues or oral cancer. Craniofacial birth defects with anomalies of the mouth, neck, and head are of major public concern. We identified a cDNA clone for epiprofin, which is preferentially expressed in the tooth, by differential hybridization using DNA microarrays from an E19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Kruppel-Like Factor (KLF) family containing three characteristic C2H2-type zinc-finger motifs. The SP/KLF family consists of more than 17 proteins in mice, and has unique features including a DNA-binding domain with three tandem C2H2-type (Kruppel-like) zinc-finger motifs at the C-terminus and a transcriptional regulatory domain at the N-terminus. Except for its 5? terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of KLF14/SP6, whhhich was previously identified in EST databases and GenBank by a zinc-finger DNA-binding domain search. This sequence difference is due to differences in the assignment oof the location of exon 1. Epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblasts, and also hair follicle matrix epithelium. In addition, epiprofin mRNA is transiently expressed in cells of the apical ectodermal ridge (AER) in developing limb. We found by transfection of an epiprofin expression vector that epiprofin is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein, expressed by proliferating epithelial cells of tooth, hair follicle, and limb that may function in the development of these tissues by regulating cell growth. Because epiprofin is expressed primarily in tissues of ectodermal origin, ectodermal dysplasia may be a candidate human genetic disorder caused by mutations of the epiprofin gene.

这个项目不仅包括软骨，还包括牙齿和颅面发育。软骨是一种高度特化的结缔组织，具有独特的形态和生化特征。软骨含有广泛的细胞外基质，并提供机械强度以抵抗关节中的压缩。在发育过程中，软骨作为大多数骨骼生长和发育的模板。当软骨形成受损时，四肢、颅面骨和无骨骨骼的骨骼畸形发生。软骨细胞是软骨中的主要细胞类型，并产生大量的软骨特异性基质分子，如XI型和II型胶原蛋白以及聚集蛋白聚糖。软骨形成是由间充质细胞凝聚形成原始软骨，然后由软骨细胞分化启动的。这些包括静止、增殖、肥大前和肥大软骨细胞。作为软骨内骨形成的最后一步，肥大的软骨被血管和骨祖细胞侵入，钙化的软骨随后被骨取代。因此，软骨细胞分化的空间和时间调节在决定骨骼组成部分的长度和宽度方面是必不可少的。激素和维生素通过调节基因转录影响软骨细胞的分化和成熟。我们的目标是确定激活和抑制软骨细胞特异性基因的机制，并阐明软骨发育和软骨细胞分化的分子基础。我们之前描述了α 2型XI胶原基因（Col 11 a2）启动子中24 bp序列的鉴定，该序列能够将启动子活性从转基因小鼠的神经元组织切换到软骨。我们随后分离出一种蛋白因子，我们命名为NT 2，它与24 bp序列结合。NT 2是一种C2 H2型锌指蛋白，由Kruppel相关盒（KRAB）组成，KRAB是一种有效的DNA结合依赖性阻遏模块。我们发现NT 2作为Col 11 a2的负调节剂发挥作用。我们通过染色质免疫沉淀试验证明，在NIH 3 T3细胞中，NT 2在Col 11 a2启动子的24 bp区域形成这些异染色质相关蛋白的复合物，其中Col 11 a2被抑制，但NT 2被表达。这些结果表明，在NIH 3 T3细胞中Col 11 a2的抑制是由异染色质形成引起的。异染色质可以通过24 bp序列成核，该序列被NT 2的锌指结构域识别。NT 2的KRAB结构域然后通过KRAB结构域招募KAP 1，随后与包括组蛋白修饰酶在内的其他蛋白质结合。我们的研究结果表明，在软骨形成过程中，软骨基因表达的积极和消极的转录基因调控的重要性。 在我们的口腔和颅面基因组解剖项目（OC-GAP）中，我们的目标是发现和表征以前未知的基因，以帮助了解牙齿和颅面组织如何发育，并确定这些组织或口腔癌异常的分子缺陷。颅面出生缺陷与口腔、颈部和头部的异常是公众关注的主要问题。我们确定了一个cDNA克隆epiprofin，这是优先在牙齿中表达，通过差异杂交使用DNA微阵列从E19.5小鼠磨牙cDNA文库。序列分析显示，该cDNA编码Kruppel样因子（KLF）家族的一员，该家族包含三个特征性的C2 H2型锌指基序。SP/KLF家族在小鼠中由超过17种蛋白质组成，并且具有独特的特征，包括在C-末端具有三个串联C2 H2型（Kruppel样）锌指基序的DNA结合结构域和在N-末端的转录调节结构域。除了5？表普罗芬mRNA序列的末端序列与KLF 14/SP 6的预测序列几乎相同，该序列之前通过锌指DNA结合结构域搜索在EST数据库和GenBank中鉴定过。这种序列差异是由于外显子1的位置分配的差异。Epiprofin mRNA由增殖的牙上皮、分化的成牙本质细胞以及毛囊基质上皮表达。此外，epiprofin mRNA在发育肢体的顶端外胚层嵴（AER）细胞中瞬时表达。我们发现通过转染epiprofin表达载体，epiprofin定位于细胞核中并促进细胞增殖。因此，epiprofin是一种高度细胞和组织特异性的核蛋白，由牙齿、毛囊和肢体的增殖上皮细胞表达，其可以通过调节细胞生长在这些组织的发育中起作用。由于表普罗芬主要在外胚层来源的组织中表达，外胚层发育不良可能是由表普罗芬基因突变引起的候选人类遗传性疾病。