GENETIC-ANALYSIS OF A TRANSCRIPTIONAL ACTIVATION PATHWAY BY USING HEPATOMA-CELL VARIANTS
GENETIC-ANALYSIS OF A TRANSCRIPTIONAL ACTIVATION PATHWAY BY USING HEPATOMA-CELL VARIANTS
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DOI：
10.1128/mcb.14.11.7086
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发表时间：
1994-11-01
影响因子：
5.3
通讯作者：
FOURNIER, REK
中科院分区：
文献类型：
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作者：
BULLA, GA;FOURNIER, REK
一系列肝脏富集转录因子在激活许多肝脏基因的表达中起着重要作用。特别是，肝细胞核因子4 （HNF-4）是编码HNF-1基因的主要激活因子，而HNF-1本身激活了20多个肝脏基因的表达。为了从遗传学上剖析这一激活途径，我们制备了肝脏特异性α 1-抗胰蛋白酶（α 1AT）基因表达不足的体细胞变体，该基因需要HNF-1和HNF-4才能获得高水平的基因活性。这分两步完成。首先，制备了在α 1AT启动子控制下稳定表达两种可选择标记的肝癌转染物；其次，通过直接选择分离出不再表达任何一种转基因的变异亚系。在本报告中，我们证明了这些变体在HNF-4/HNF-1激活途径中含有缺陷。这些缺陷在反式中起作用，因为许多肝脏基因的表达受到影响，但在体细胞杂交种中变异表型是隐性的。通过对HNF-4或HNF-1异位表达的表型反应，可以区分出三种不同的变异类别。两个变异克隆表现出特异性的HNF-4表达缺陷，因为转染了HNF-4表达盒完全恢复了它们的肝脏表型。另一种细胞系激活了HNF-1，以响应HNF-4的强制表达，但下游基因的激活未能发生。一个克隆对HNF-1或HNF-4均无反应。利用这些变异，我们进一步证明了编码α 1AT、醛缩酶B和α纤维蛋白原的染色体基因在体内对HNF-1的激活有严格的要求，而其他肝脏基因不受HNF-1或HNF-4存在与否的影响。我们还提供了证据，证明存在一个自动调节回路，在这个回路中，HNF-1通过激活HNF-4来调节自身的表达。
A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.