Coffman J A, Kirchhamer C V, Harrington M G, Davidson E H
Stowers Institute for Medical Research, California Institute of Technology, Pasadena 91125, USA.
Development. 1997 Dec;124(23):4717-27. doi: 10.1242/dev.124.23.4717.
The CyIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme.
紫球海胆的CyIIIa肌动蛋白基因仅在胚胎反口外胚层中进行转录，受一个2.3 kb顺式调控结构域的控制，该结构域包含一个在胚胎早期发育中控制表达的近端模块，以及一个在胚胎后期发育中控制表达的中间模块。先前的研究表明，反口外胚层分化时CyIIIa转录的急剧增加需要中间模块内的SpRunt-1靶位点，并且SpRunt-1靶位点附近的一个负调控区域对于防止口外胚层和造骨间充质中的异位转录是必需的。这个负调控区域包含转录因子myb家族的一个共有结合位点。体外DNA结合实验表明，囊胚期细胞核中的一种蛋白质与myb靶位点特异性相互作用。利用含有寡核苷酸替代的CyIIIa报告构建体进行的基因转移实验表明，该位点对于防止CyIIIa的异位表达既必要又充分。含有myb靶位点的合成寡核苷酸通过亲和层析从海胆胚胎核提取物中纯化一种蛋白质。这种蛋白质被针对进化保守myb结构域的特异性抗体免疫沉淀，并且从纯化蛋白质获得的氨基酸序列与myb结构域内的序列相同。序列信息被用于获得转录因子myb家族的紫球海胆成员SpMyb的cDNA克隆。通过中间模块内的相互作用，SpMyb发挥作用抑制口外胚层和造骨间充质中CyIIIa的激活。
