Blobel G A, Simon M C, Orkin S H
Children's Hospital, Dana-Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1995 Feb;15(2):626-33. doi: 10.1128/MCB.15.2.626.
Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.
全能性小鼠胚胎干细胞（ES细胞）可在体外分化形成包含多种谱系造血细胞（包括红系细胞）的胚状体（EBs）。体外红系发育与体内观察到的情况相似。红系转录因子GATA-1基因被破坏的ES细胞在体内或体外均无法产生成熟的红系细胞。利用EB体外分化试验，测试了表达异源GATA结合蛋白的构建体纠正GATA-1缺陷型ES细胞发育缺陷的能力。此处呈现的结果表明，高度分化的鸡GATA-1能够在一定程度上挽救GATA-1缺陷，其程度与小鼠GATA-1（mGATA-1）相似，这是通过EBs的大小和形态、红细胞的存在以及珠蛋白基因表达来确定的。此外，通常在不同组织中表达的GATA-3和GATA-4，以及由与单纯疱疹病毒VP16转录激活域融合的GATA-1锌指组成的一种蛋白质，能够补偿GATA-1缺陷。其中mGATA-1的两个锌指被人GATA-3的锌指或真菌GATA因子AreA的单个锌指取代的嵌合分子，以及仅带有锌指区域的构建体，均显示出挽救活性。这些结果表明，mGATA-1的转录激活域及其锌指均未赋予其在体内作用的红系细胞特异性。相反，特异性似乎是通过决定GATA-1基因时空表达的顺式作用控制区域介导的。此外，我们的结果表明，锌指区域除了介导DNA结合外可能还具有生物学功能。
