Phillips Marjorie A, Jessen Bart A, Lu Ying, Qin Qin, Stevens Mary E, Rice Robert H
Department of Environmental Toxicology, University of California, Davis, CA 95616-8588, USA.
BMC Dermatol. 2004 Apr 5;4:2. doi: 10.1186/1471-5945-4-2.
TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment.
In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes.
In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity.
A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures.
转谷氨酰胺酶1（TGM1）是一种使成熟角质形成细胞的角质包膜发生交联的酶。TGM1基因的适当表达对于角质形成细胞的正常功能至关重要，因为失活突变会导致使人衰弱的皮肤病——板层状鱼鳞病。TGM1也在鳞状化生中表达，这是维生素A缺乏或毒性损伤在某些上皮中导致的结果，可能会引发肿瘤形成。了解该基因在正常和异常分化状态下的调控机制可能有助于改善疾病的诊断和治疗。
通过将不同长度的启动子DNA与报告基因融合，并将DNA注射到小鼠胚胎中以产生转基因动物，研究了TGM1基因表达的体内需求。通过蛋白质免疫印迹法和免疫组织化学法确定报告基因的表达。通过将逐渐缩短或突变的启动子DNA转染到培养的角质形成细胞中，进一步确定转录重要远端区域。
对由TGM1启动子驱动的报告转基因进行体内分析发现，1.6千碱基而非1.1千碱基的DNA足以赋予组织特异性和细胞层特异性表达。同一区域负责在因维生素A缺乏而发生鳞状化生的组织中报告基因的表达。在转染试验中被确定为重要转录元件的远端启动子AP1位点或近端启动子CRE位点的突变，并未阻止适当的表达。在培养的角质形成细胞中使用电泳迁移率变动分析（EMSA）和转染试验进一步寻找转录元件，在-1.6至-1.4千碱基之间的转录活性区域中鉴定出两个Sp1元件。虽然单独突变任何一个Sp1位点或AP1位点的影响都很小，但所有三个位点的突变几乎消除了所有转录活性。
TGM1基因启动子的一个远端区域，包含AP1和Sp1结合位点，在进化上是保守的，负责转基因小鼠和转染的角质形成细胞培养物中的高水平表达。
