EXPRESSION, STRUCTURE AND FUNCTION OF FILAGGRIN

丝聚合蛋白原是表达的主要结构蛋白 在表皮的颗粒层中。它是一种多蛋白前体 由 10、11 或 12 个丝聚蛋白重复序列组成，其邻接 短接头序列。在终末分化过程中，这 前体被切割成单个丝聚蛋白分子，其基于 在体外实验中，被认为参与 角蛋白中间体的聚集和特异性排列 完全分化的、死亡的、角化细胞层中的细丝 表皮。在早期的研究中，我们探索了 丝聚合蛋白原基因的表达并表征了其 近端启动子。 AP1 位点及其同源结合 c-fos 和 c-jun 蛋白赋予该基因角质形成细胞特异性表达， 与邻近的 Sp1、ets-like 和 NF-KB 元素相协调。我们 发现了一种新的 ets 转录蛋白 调节多种晚期表达的功能 表皮中的分化基因，包括聚丝蛋白原。 此外，莫拉索和合作者的研究已经确定了 同源域转录因子，称为 Distal-less 3 (Dlx3)， 它在激活表达中起着核心作用 聚丝蛋白原（以及表皮的其他结构蛋白） 角质层的形成所必需的。表皮期间 发育时，Dlx3 的表达仅限于 分化的（基底上）细胞，主要是颗粒细胞， 小鼠表皮分层。功能获得实验 异位驱动 Dlx3 在基底细胞中的表达 使用基础特异性角蛋白 5 启动子的转基因小鼠，结果 严重异常的表皮表型导致围产期 由于无法形成功能性角质层而具有致命性， 它构成皮肤的水屏障并防止 脱水。重要的是，转基因表皮中的基底细胞 停止增殖并表达晚期分化表皮 标记物如兜甲蛋白和聚丝蛋白原。符合这些 结果，我们在近端确定了 Dlx3 的结合位点 聚丝蛋白原基因的启动子。 Dlx3 的重要性 结构的图案化和发展源自 胚胎发生过程中的上皮-间质相互作用（即 DLX3 突变的影响证实了牙齿、头发） 常染色体显性遗传患者的基因 毛牙骨（TDO）综合征。我们目前 开发 TDO 综合征转基因模型 应该可以让我们更好的理解Dlx3的功能 以及与 TDO 相关的病理学的分子基础。 该项目的主要重点现在已更改为 了解调节早期的分子机制 表皮分化阶段，利用 Dlx3 的作用 丝聚合蛋白基因表达系统。表皮已经 用作研究细胞过程的优秀模型 分化是因为细胞在分化过程中形成分层结构 发育，每个地层都可以通过形态轻松识别 和具体标记。当前研究的目的是： 确定 Dlx3 在调节晚期分化中的作用 以聚丝蛋白原为范例的结构基因；确定 Dlx3 作用和控制的信号通路 转录；并通过小鼠分析Dlx3的功能作用 TDO综合征模型。使用基因打靶研究 Dlx3 函数 以并行的方式理解函数的作用 Dlx3，对该基因进行了靶向删除， 表型分析已完成。针对性删除 DLX3基因导致胚胎发育停滞 第 9.5-10 天，与胎盘严重衰竭有关 进行适当的形态发生。无法评估 Dlx3 功能丧失对表皮分化的影响，因为 胚胎死亡发生的时间明显早于胚胎死亡的发生 表皮分层（E15.5）。在大量的目标中 导致胚胎发生早期死亡的基因突变， 其中一些是由于胎盘发育缺陷引起的 通过四倍体聚集实验（即 Mash-2、Ets-2）拯救。 拯救 Dlx3 -/- 胚胎中的滋养层缺陷 四倍体聚集实验已启动以确定是否 在这种情况下，无效动物是可存活的或呈现出表型 Dlx3的具体结构在全文中表达 胚胎发育，特别关注分层 表皮。最近，Hart 和 合作者报告说，遗传性基因异常 Tricho-Dento-Osseous 综合征 (TDO) 是一种四 人类DLX3基因中的核苷酸缺失，立即 同源域编码区的下游。这种综合症 作为常染色体显性遗传，包括 牙齿、头发和面部骨骼异常。小鼠杂合子 对于 Dlx3 中断（？淘汰？项目），没有显示任何此类 异常，表明 TDO 综合征占主导地位 是由于非功能性异聚复合物的形成 涉及截短的 Dlx3 蛋白，而不是 单倍体不足。我们已经培育出转基因小鼠作为 TDO 综合征模型。确切的突变存在于 报道人类家族被引入小鼠DLX3基因 并在 Dlx3 启动子区域的调控下驱动 这是之前已经描述过的特征。对这些小鼠的分析将 帮助理解Dlx3的结构、突变的影响 关于蛋白质：蛋白质相互作用，以及 DNA 结合和/或 Dlx3 转录能力的激活导致缺陷 已知表达 Dlx3 的几个结构中。 分化角质形成细胞中 Dlx3 表达的调节 为了确定 Dlx3 被激活的信号通路 在表皮中，我们研究了培养的小鼠角质形成细胞 体外。

Profilaggrin is a major structural protein expressed in the granular layer of the epidermis. It is a polyprotein precursor consisting 10, 11 or 12 filaggrin repeats which are adjoined by a short linker sequences. During terminal differentiation, this precursor is cleaved into individual filaggrin molecules, which based on in vitro experiments, are thought to be involved in the aggregation and specific alignment of the keratin intermediate filaments in the fully differentiated, dead, cornified cell layers of the epidermis. In earlier studies, we have explored the regulation of expression of the profilaggrin gene and have characterized its proximal promoter. An AP1 site and its cognate binding c-fos and c-jun proteins confer keratinocyte-specific expression to the gene, in concert with neighboring Sp1, ets-like, and NF-KB elements. We have discovered a novel ets transcription protein that seems to function in the regulation of expression of several late differentiation genes in the epidermis, including profilaggrin. Moreover, studies by Morasso and collaborators have identified a homeodomain transcription factor, known as Distal-less 3 (Dlx3), which plays a central role in activating the expression of profilaggrin (and other structural proteins of the epidermis) that are necessary for the formation of the cornified layer. During epidermal development, the expression of Dlx3 is restricted to the differentiated (suprabasal) cells, predominantly the granular cells, of the mouse stratified epidermis. Gain-of-function experiments by ectopically driving the expression of Dlx3 in the basal cells of transgenic mice using the basal-specific keratin 5 promoter, resulted in a severely abnormal epidermal phenotype leading to perinatal lethality because of the inability to form a functional cornified layer, that constitutes the water-barrier of the skin and prevents dehydration. Importantly, the basal cells in the transgenic epidermis ceased to proliferate and expressed late differentiation epidermal markers such as loricrin and profilaggrin. Consistent with these findings, we identified a binding site for Dlx3 in the proximal promoter of the profilaggrin gene. The importance of Dlx3 in the patterning and development of structures derived from epithelial-mesenchymal interactions during embryogenesis (i.e. tooth, hair) is corroborated by the effects of mutations in the DLX3 gene in patients with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome. We are presently developing a transgenic model for the TDO syndrome, which should allow us to obtain a better understanding of Dlx3 function and the molecular basis for the pathologies associated with TDO. The major focus of this project has now changed to an understanding the molecular mechanisms that regulate the early stages of epidermal differentiation, using the effects of the Dlx3 system on expression of the profilaggrin gene. Epidermis has been used as an excellent model for studying the process of cellular differentiation because the cells form a stratified structure during development, with each strata being easily identified by morphology and specific markers. The aims of the current studies are to: determine the role of Dlx3 in the regulation of late differentiation structural genes using profilaggrin as the paradigm; determine the signaling pathway through which Dlx3 acts and controls transcription; and analyze functional role of Dlx3 through mouse model of TDO syndrome. Use of gene targeting to study Dlx3 function In a parallel approach to understand the functional role of Dlx3, a targeted deletion of the gene was performed and the analysis of the phenotype has been completed. Targeted deletion of the DLX3 gene results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). Of the large number of targeted mutagenesis of genes that lead to early death in embryogenesis, several of those due to defects in placenta development have been rescued by tetraploid aggregation experiments (i.e. Mash-2, Ets-2). Rescue of the trophoblast defect in the Dlx3 -/- embryos by tetraploid aggregation experiments has been initiated to determine if in this context, the null animals are viable or present a phenotype in the specific structures were Dlx3 is expressed throughout embryonic development, with special interest in the stratified epidermis. Generating mouse models for TDO Recently, Hart and collaborators reported that the genetic abnormality in the hereditary disease Tricho-Dento-Osseous syndrome (TDO) was a four nucleotide deletion in the human DLX3 gene, immediately downstream from the homeodomain coding region. This syndrome is inherited as an autosomal dominant trait, and includes abnormalities in the teeth, hair and facial bones. Mice heterozygous for the Dlx3 disruption (?knockout? project) do not show any such abnormalities, suggesting that the dominance of the TDO syndrome is due to formation of nonfunctional heteromeric complexes involving the truncated Dlx3 protein, as opposed to haploinsufficiency. We have generated transgenic mice to serve as a model for the TDO syndrome. The exact mutation present in the reported human families was introduced in the mouse DLX3 gene and driven under the regulatory control of a Dlx3 promoter region that has been previously characterized. Analyses of these mice will help to understand the structure of Dlx3, the effect of the mutation on the protein:protein interactions, and on the DNA binding and/or activation of transcription capability of Dlx3 that results in defects in several of the structures were Dlx3 is known to be expressed. Regulation of Dlx3 expression in differentiating keratinocytes In order to determine the signaling pathway by which Dlx3 is activated in the epidermis, we have studied mouse keratinocytes cultivated in vitro.