Gene Regulation And Function Of Cartilage

该项目不仅包括软骨，还包括牙齿和颅面发育。软骨是一种高度专业化的结缔组织，具有明显的形态和生化特征。软骨含有广泛的细胞外基质，并提供抵抗关节压缩的机械强度。在发育过程中，软骨是大多数骨骼生长和发育的模板。当软骨形成受损时，就会出现四肢、头面部骨骼和附件骨骼的骨骼畸形。软骨细胞是软骨中的主要细胞类型，能产生大量的软骨特异性基质分子，如XI型和II型胶原，以及聚集素。软骨形成是由间充质细胞凝聚形成原始软骨，然后软骨细胞分化而形成的。这些细胞包括静止期、增殖期、增生期前期和增生期软骨细胞。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袭，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成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。我们的目标是明确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。我们曾报道过在α2型XI胶原基因(Col11a2)启动子中有一个24bp的序列，它能够在转基因小鼠中将启动子的活性从神经元组织切换到软骨组织。随后，我们分离到了一个蛋白因子，命名为NT2，它与24bp的序列结合。NT2是一种C2H2型锌指蛋白，由Kruppel相关框(KRAb)组成，KRAb是一个有效的DNA结合依赖的阻遏模块。我们发现NT2对COL11a2具有负性调节作用。我们通过染色质免疫沉淀分析证明，在NIH3T3细胞中，NT2在Col11a2启动子的24bp区域形成了这些异染色质相关蛋白的复合体，其中Col11a2被抑制，而NT2被表达。这些结果表明，COL11a2在NIH3T3细胞中的抑制是由异染色质形成引起的。异染色质可被NT2的锌指结构域识别的24bp序列所核化。然后，NT2的KRAB结构域通过KRAB结构域招募KAP1，随后与包括组蛋白修饰酶在内的其他蛋白质结合。我们的结果表明，在软骨形成过程中，正转录基因和负转录基因对软骨基因表达的调节都很重要。 在我们的口腔和颅面基因组解剖计划(OC-GAP)中，我们的目标是发现和鉴定以前未知的基因，以帮助了解牙齿和颅面组织如何发育，并确定这些组织异常或口腔癌潜在的分子缺陷。有口腔、颈部和头部异常的头面部出生缺陷是公众主要关注的问题。我们利用E19.5小鼠磨牙c DNA文库中的DNA微阵列，通过差异杂交，鉴定了一个在牙齿中优先表达的表普洛林的cDNA克隆。序列分析表明，该基因编码KRUPEL样因子(KLF)家族中的一个成员，含有三个C2H2型锌指基序。在小鼠中，SP/KLF家族由17种以上的蛋白质组成，具有独特的功能，包括一个DNA结合域，在C-末端有三个串联的C2H2型(Kruppel-like)锌指基序，在N-末端有一个转录调节结构域。除了它的5个？该序列与先前在EST数据库和GenBank中通过锌指DNA结合区搜索得到的KLF14/SP6的预测序列几乎完全相同。这种序列差异是由于外显子1的位置分配不同所致。Epiproin mRNA在增殖的牙齿上皮、分化的成牙本质细胞和毛囊基质上皮中表达。此外，在发育中的肢体顶端外胚层脊(AER)的细胞中瞬时表达了表没食子素。我们通过转染表没食子素表达载体发现，表没食子素定位于细胞核，并促进细胞增殖。因此，表普洛林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，可能通过调节细胞生长而在这些组织的发育中发挥作用。由于表普洛芬主要在外胚层起源的组织中表达，外胚层发育不良可能是由表普洛林基因突变引起的一种候选人类遗传病。

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.