Divine Joyce K, Staloch Lora J, Haveri Hanna, Jacobsen Christina M, Wilson David B, Heikinheimo Markku, Simon Theodore C
Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Nov;287(5):G1086-99. doi: 10.1152/ajpgi.00421.2003. Epub 2004 Jan 8.
Transcriptional regulation by GATA-4, GATA-5, and GATA-6 in intestine and liver was explored using a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene (Fabpl). An immunohistochemical survey detected GATA-4 and GATA-6 in enterocytes, GATA-6 in hepatocytes, and GATA-5 in neither cell type in adult animals. In cell transfection assays, GATA-4 or GATA-5 but not GATA-6 activated the Fabpl transgene solely through the most proximal of three GATA binding sites in the Fabpl promoter. However, all three factors activated transgenes constructed from each Fabpl site upstream of a minimal viral promoter. GATA factors interact with hepatic nuclear factor (HNF)-1alpha, and the proximal Fabpl GATA site adjoins an HNF-1 site. GATA-4, GATA-5, or GATA-6 bounded to HNF-1alpha in solution, and all cooperated with HNF-1alpha to activate the Fabpl transgene. Mutagenizing all Fabpl GATA sites abrogated transgene activation by GATA factors, but GATA-4 activated the mutagenized transgene in the presence of HNF-1alpha. These in vitro results suggested GATA/HNF-1alpha interactions function in Fabpl regulation, and in vivo relevance was determined with subsequent experiments. In mice, the Fabpl transgene was active in enterocytes and hepatocytes, a transgene with mutagenized HNF-1 site was silent, and a transgene with mutagenized GATA sites had identical expression as the native transgene. Mice mosaic for biallelic Gata4 inactivation lost intestinal but not hepatic Fabpl expression in Gata4-deficient cells but not wild-type cells. These results demonstrate GATA-4 is critical for intestinal gene expression in vivo and suggest a specific GATA-4/HNF-1alpha physical and functional interaction in Fabpl activation.
利用从大鼠肝脏脂肪酸结合蛋白基因（Fabpl）近端启动子构建的转基因，研究了GATA - 4、GATA - 5和GATA - 6在肠道和肝脏中的转录调控。免疫组织化学调查在成年动物的肠细胞中检测到GATA - 4和GATA - 6，在肝细胞中检测到GATA - 6，在这两种细胞类型中均未检测到GATA - 5。在细胞转染试验中，GATA - 4或GATA - 5而非GATA - 6仅通过Fabpl启动子中三个GATA结合位点中最近端的位点激活Fabpl转基因。然而，所有这三种因子都激活了由最小病毒启动子上游的每个Fabpl位点构建的转基因。GATA因子与肝细胞核因子（HNF）- 1α相互作用，并且Fabpl近端GATA位点毗邻一个HNF - 1位点。GATA - 4、GATA - 5或GATA - 6在溶液中与HNF - 1α结合，并且都与HNF - 1α协同激活Fabpl转基因。将所有Fabpl GATA位点诱变可消除GATA因子对转基因的激活作用，但在存在HNF - 1α的情况下，GATA - 4可激活诱变后的转基因。这些体外结果表明GATA/HNF - 1α相互作用在Fabpl调控中起作用，随后的实验确定了其体内相关性。在小鼠中，Fabpl转基因在肠细胞和肝细胞中具有活性，具有诱变HNF - 1位点的转基因无活性，而具有诱变GATA位点的转基因与天然转基因具有相同的表达。双等位基因Gata4失活的嵌合小鼠在Gata4缺陷细胞而非野生型细胞中失去了肠道Fabpl表达，但肝脏Fabpl表达未受影响。这些结果表明GATA - 4对体内肠道基因表达至关重要，并提示在Fabpl激活中存在特定的GATA - 4/HNF - 1α物理和功能相互作用。
