REGULATION OF A HUMAN EPIDERMAL KERATIN GENE - SEQUENCES AND NUCLEAR FACTORS INVOLVED IN KERATINOCYTE-SPECIFIC TRANSCRIPTION
REGULATION OF A HUMAN EPIDERMAL KERATIN GENE - SEQUENCES AND NUCLEAR FACTORS INVOLVED IN KERATINOCYTE-SPECIFIC TRANSCRIPTION
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DOI：
10.1101/gad.4.11.1985
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发表时间：
1990-11-01
影响因子：
10.5
通讯作者：
FUCHS, E
中科院分区：
文献类型：
--
作者：
LEASK, A;ROSENBERG, M;FUCHS, E
角质形成细胞是人体的主要细胞类型，在表皮中，角质形成细胞具有未来基因靶向和药物治疗的潜力。尽管角质形成细胞在细胞生物学和医学中很重要，但人们对角质形成细胞特异性基因表达的分子机制知之甚少。在这里，我们首次详细描述了控制在角质形成细胞中特异性表达的人类基因表达的序列和因子。使用与两个报告基因之一偶联的人K14角蛋白基因的5''上游序列，我们检查了在培养的人角质形成细胞和转基因小鼠表皮的有丝分裂活性基底角质形成细胞中表达K14所必需和充分的序列。我们证明了位于 hK14 基因转录起始位点 5'' 处的远端和近端元件的存在，当与 TATA 盒元件结合时，它们似乎协同作用以驱动角质形成细胞特异性表达。我们详细检查了近端区域。在使用 CAT 测定将转录激活元件缩小到 110 bp 以内后，我们证明了该结构域内存在与 10 bp 回文结构 5''-GCCTGCAGGC-3'' 结合的角质形成细胞核因子。通过甲基化干扰分析，我们鉴定了对因子结合重要的G残基，并表明这些G残基的点突变不仅阻断因子结合，而且导致hK14-CAT基因的转录活性降低。该因子在角质形成细胞中最为丰富，在一些简单的上皮细胞系中表达水平较低，并且在成纤维细胞或淋巴瘤细胞中未检测到。此外，10 bp 序列与角质形成细胞中表达的其他几个基因的 5'' 上游序列相似，并且这些基因中的至少一个（人类 K1 基因）包含与 hK14 近端元件竞争结合因子的序列。总的来说，我们的数据表明，我们鉴定的序列和核因子可能参与体外和体内控制角质形成细胞特异性表达。
The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5'' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5'' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assay to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5''-GCCTGCAGGC-3'', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5'' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.