Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression
Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression
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DOI：
10.1111/j.1432-0436.2000.660405.x
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发表时间：
2000-12-01
期刊：
影响因子：
2.9
通讯作者：
Williams, J
中科院分区：
文献类型：
--
作者：
Ceccarelli, A;Zhukovskaya, N;Williams, J
网柄藻的ecmB基因在进入柄管的前柄细胞和辅助柄细胞结构如基盘中的顶点都表达。茎管特异性表达受启动子近端帽位点内的序列元件调节，即茎管（ST）启动子区。Dd-STATa是STAT转录因子家族的成员，与ST启动子区的元件结合，在进入茎管之前抑制转录。我们已经确定了一个激活的DNA序列元件，位于远端的阻遏元件，这是必要的和足够的表达内的茎管。我们已经映射到一个28个核苷酸的区域（28聚体）内，我们已经确定了一个含GA的序列元件，是所需的有效的基因转录激活剂。在体外结合试验中，Dd-STATa蛋白与28聚体结合，并且结合依赖于含GA的序列。然而，ecmB基因在Dd-STATa无效突变体中表达，因此Dd-STATa不能负责在体内激活28-mer。相反，我们在Dd-STATa无效突变体的核提取物中鉴定了不同的28-mer结合活性，该CA结合活性的活性在野生型提取物中被Dd-STATa蛋白的高亲和力结合在很大程度上掩盖。我们建议，除了通过结合到两个已知的阻遏物位点所施加的长距离阻遏之外，Dd-STATa通过与该假定的激活剂直接竞争结合到GA序列来抑制转录。
The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this CA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.