Frudakis T N, Wilt F
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Dev Biol. 1995 Nov;172(1):230-41. doi: 10.1006/dbio.1995.0018.
The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.
后生动物胚胎中许多区域特异性基因的表达模式是通过一个活跃的负向空间调控过程来维持的。然而，在任何系统中，这种基因调控策略的机制都尚未得到充分理解。在这里，我们表明，含有紫海胆SM30-α基因调控序列的报告构建体，其表达模式与内源性SM30基因的表达模式一致，这主要是由于在该基因通常不表达的细胞谱系中存在活跃的转录抑制作用。对来自2600 bp上游区域的缺失构建体进行氯霉素乙酰转移酶分析表明，在-1628至-300区域存在抑制元件。原位杂交分析表明，当-1628至-300区域被删除时，表达的空间保真度会严重受损。在-1628至-300区域存在两个高度重复的序列基序，即(G/A/C)CCCCT和(T/C)(T/A/C)CTTTT(T/A/C)。通过凝胶迁移率变动实验对这些元件的代表进行了分析，发现它们能与粗核提取物中的蛋白质特异性相互作用。当含有任何一种序列元件的寡核苷酸与一个调控正确的报告基因共同注射作为潜在竞争者时，该报告基因会在不适当的细胞中表达。当含有两种序列元件的复合寡核苷酸与一个调控错误的报告基因融合时，该报告基因在不适当细胞中的表达会受到抑制。将复合寡核苷酸与含有单个组成元件的寡核苷酸进行比较表明，两种序列元件对于有效的空间调控都是必需的。因此，两种单个元件都是必需的，但只有包含两种元件的复合元件才足以作为组织特异性抑制元件发挥作用。
