MECHANISM OF TRANSGLUTAMINASE TYPE I REGULATION BY HOXA7

HOXA7 调节 I 型转谷氨酰胺酶的机制

• 批准号: 6374768
• 负责人: PETER T LA CELLE
• 金额: $ 8.36万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374768
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; binding sites; cell differentiation; cell growth regulation; cell line; cell proliferation; cofactor; developmental genetics; gel mobility shift assay; gene expression; genetic regulatory element; homeobox genes; human tissue; immunoprecipitation; keratinocyte; polymerase chain reaction; protein binding; protein glutamine gamma glutamyltransferase; protein protein interaction; regulatory gene; tissue /cell culture; transcription factor; transfection; western blottings

The inability to control proliferation and differentiation of epidermal keratinocytes is a major obstacle in wound healing following trauma or surgery, and in the treatment of diseases involving abnormal epidermal differentiation, from carcinomas to psoriasis. The long-range goal of this research is to identify the mechanisms underlying the genetic regulatory pathways that specify the spectrum of proteins and the resulting cellular characteristics of particular stages of keratinocyte differentiation. To do this, we must identify the trans-acting factors and cis-acting elements that regulate differentiation-specific epidermal genes, such as transglutaminase type 1 (TGM1). The highly conserved homeobox (Hox) family of transcription factors can control cell identity and differentiation, and aberrant Hox regulation can lead to developmental abnormality or disease. Many Hox proteins recognize a common DNA sequence mainly via a small number of contacts in vitro, suggesting that additional mechanisms must underlie their diverse in vivo target specificity. Recent findings indicate that cooperative Hox interaction with cofactors can increase DNA specificity and affinity. We have identified the HOXA7 cDNA in a differentiating keratinocyte library through specific interaction with the KD-enhancer, that confers keratinocyte-specific activation to E6/E7 HPV-16 promoter. Sequence analysis indicates that KD-enhancer homology is present in the TGM1 5' promoter region (K3), which contains a Hox DNA recognition core element, as well as a binding site 90 percent identical to a known binding site for heterodimers of Hox and Pbx, the mammalian homolog of the Drosophila extradenticle. By transient transfection, HOXA7 suppresses transcriptional activity of K3 in neonatal keratinocytes, but strongly transactivates K3 in the epidermoid carcinoma cell line ME180. We propose that HOXA7 differentially regulates the keratinocyte differentiation marker gene TGM1 in neonatal keratinocytes and ME180, through interaction with co-factors, allowing cell-specific suppression or activation of the same gene. We will test this hypothesis by 1) determining the cis-acting elements in the TGM1 upstream regulatory region (K3) important in HOXA7 transactivation in neonatal keratinocytes and ME180 in vivo, and in HOXA7 binding in vitro, 2) identifying the functional domain of HOXA7 necessary for activation or repression of TGM1 in the two cell types in vivo, and for K3 binding in vitro, and 3) characterizing nuclear cofactors that form complexes with HOXA7 and K3 DNA. This study will begin to elucidate molecular mechanisms involved in HOX regulation of keratinocyte proliferation and differentiation, that in the long term may lead to new methods for controlling keratinocyte growth in re-epithelialization of wounds, and in treatment of cancers and other hyper-proliferative and disease.

无法控制表皮角质形成细胞的增殖和分化是创伤或手术后伤口愈合的主要障碍，以及涉及异常表皮分化的疾病，从癌症到牛皮癣。 这项研究的远距离目标是确定指定蛋白质光谱的遗传调节途径的机制以及角质形成细胞分化的特定阶段的所得细胞特征。 为此，我们必须确定调节分化特异性表皮基因的跨作用因子和顺式作用元件，例如转谷氨酰胺酶1型（TGM1）。 高度保守的同型转录因子家族可以控制细胞的身份和分化，异常HOX调节会导致发育异常或疾病。 许多HOX蛋白主要通过少量的体外接触识别常见的DNA序列，这表明其他机制必须构成其在体内靶标特异性上的多样化。 最近的发现表明，合作HOX与辅因子的相互作用可以提高DNA特异性和亲和力。 我们通过与KD-Enhancer的特定相互作用来确定了分化角质形成细胞文库中的Hoxa7 cDNA，该相互作用赋予了角质形成细胞特异性激活，以E6/E7 HPV-16启动子。序列分析表明，KD-Enhancer同源性存在于TGM1 5'启动子区域（K3）中，该区域包含HOX DNA识别核心元素，以及与HOX和PBX异二聚体相同的结合位点，与Hox和PBX的已知结合位点相同，Drosopophila脑外壳的哺乳动物同源物。 通过瞬时转染，HOXA7抑制了K3在新生儿角质形成细胞中的转录活性，但在表皮类癌细胞系ME180中强烈反式激活K3。 我们建议HOXA7通过与副因子的相互作用，允许对同一基因的细胞特异性抑制或激活，从而调节了新生儿角质形成细胞和ME180中角质形细胞分化标记基因TGM1。 We will test this hypothesis by 1) determining the cis-acting elements in the TGM1 upstream regulatory region (K3) important in HOXA7 transactivation in neonatal keratinocytes and ME180 in vivo, and in HOXA7 binding in vitro, 2) identifying the functional domain of HOXA7 necessary for activation or repression of TGM1 in the two cell types in vivo,对于在体外的K3结合，以及3）表征与Hoxa7和K3 DNA形成复合物的核辅助因子。 这项研究将开始阐明参与角质形成细胞增殖和分化的HOX调节的分子机制，从长远来看，这可能会导致新方法在伤口重新上皮化以及癌症和其他高增生性和疾病的治疗中控制角质形成细胞的生长。