Distinct Functions Are Implicated for the GATA-4, -5, and -6 Transcription Factors in the Regulation of Intestine Epithelial Cell Differentiation

Based on conserved expression patterns, three members of the GATA family of transcriptional regulatory proteins, GATA-4, -5, and -6, are thought to be involved in the regulation of cardiogenesis and gut development. Functions for these factors are known in the heart, but relatively little is understood regarding their possible roles in the regulation of gut-specific gene expression. In this study, we analyze the expression and function of GATA-4, -5, and -6 using three separate but complementary vertebrate systems, and the results support a function for these proteins in regulating the terminal-differentiation program of intestinal epithelial cells. We show that xGATA-4, -5, and -6 can stimulate directly activity of the promoter for the intestinal fatty acid-binding protein (xIFABP) gene, which is a marker for differentiated enterocytes. This is the first direct demonstration of a target for GATA factors in the vertebrate intestinal epithelium. Transactivation by xGATA-4, -5, and -6 is mediated at least in part by a defined proximal IFABP promoter element. The expression patterns for cGATA-4, -5, and -6 are markedly distinct along the proximal-distal villus axis. Transcript levels for cGATA-4 increase along the axis toward the villus tip; likewise, cGATA-5 transcripts are largely restricted to the distal tip containing differentiated cells. In contrast, the pattern of cGATA-6 transcripts is complementary to cGATA-5, with highest levels detected in the region of proliferating progenitor cells. Undifferentiated and proliferating human HT-29 cells express hGATA-6 but not hGATA-4 or hGATA-5. Upon stimulation to differentiate, the transcript levels for hGATA-5 increase, and this occurs prior to increased transcription of the terminal differentiation marker intestinal alkaline phosphatase. At the same time, hGATA-6 steady-state transcript levels decline appreciably. All of the data are consistent with evolutionarily conserved but distinct roles for these factors in regulating the differentiation program of intestinal epithelium. Based on this data, we suggest that GATA-6 might function primarily within the proliferating progenitor population, while GATA-4 and GATA-5 function during differentiation to activate terminal-differentiation genes including IFABP.

基于保守的表达模式，GATA家族转录调控蛋白的三个成员——GATA-4、-5和-6——被认为参与心脏发生（cardiogenesis）与肠道发育（gut development）的调控。此类因子在心脏中的功能已被明确阐释，但学界对其在肠道特异性基因表达调控中可能发挥的作用仍知之甚少。本研究采用三种独立且互为补充的脊椎动物实验模型体系，分析GATA-4、-5和-6的表达与功能，实验结果证实这些蛋白参与调控肠道上皮细胞的终末分化程序（terminal-differentiation program）。本研究证实，xGATA-4、-5和-6可直接激活肠道脂肪酸结合蛋白（intestinal fatty acid-binding protein, IFABP）基因的启动子活性，而该基因为分化肠上皮细胞（enterocytes）的标志物；这是首次直接证实脊椎动物肠道上皮中存在GATA因子的靶基因。xGATA-4、-5和-6的转录激活作用（transactivation）至少部分通过已明确的IFABP启动子近端元件介导。cGATA-4、-5和-6的表达模式沿绒毛近-远轴（proximal-distal villus axis）呈现显著差异：cGATA-4的转录本水平沿该轴向绒毛尖端逐渐升高；同理，cGATA-5的转录本主要局限于包含分化细胞的远端尖端区域。与之相反，cGATA-6转录本的表达模式与cGATA-5互为补充，其最高表达水平出现在增殖性祖细胞（proliferating progenitor cells）区域。未分化且处于增殖状态的人源HT-29细胞仅表达hGATA-6，而不表达hGATA-4或hGATA-5；当诱导细胞分化时，hGATA-5的转录本水平会升高，且这一变化早于终末分化标志物肠道碱性磷酸酶（intestinal alkaline phosphatase）转录水平的上调。与此同时，hGATA-6的稳态转录本（steady-state transcript）水平会显著下降。所有实验数据均支持，此类因子在调控肠道上皮分化程序中发挥了进化保守但功能各异的作用。基于上述实验结果，我们推测GATA-6主要在增殖性祖细胞群体中发挥功能，而GATA-4与GATA-5则在分化阶段激活包括IFABP在内的终末分化基因。