Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: Demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression
Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: Demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression
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DOI：
10.1210/me.2002-0064
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发表时间：
2002-09-01
影响因子：
--
通讯作者：
Chilton, BS
中科院分区：
文献类型：
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作者：
Hewetson, A;Hendrix, EC;Chilton, BS
RUSH-1 α（β）转录因子通过用兔子宫珠蛋白基因的85-bp区域（-170/-85）进行识别位点筛选来克隆。缺失分析表明，该区域是必不可少的催乳素（PRL）的行动，但结论是有限的大缺失的复杂性。使用循环扩增和靶标选择（CASTing）来鉴定RUSH结合位点（-126/-121）。将子宫内膜核蛋白与简并寡核苷酸池孵育，并与RUSH-1 α（β）抗体免疫沉淀。通过PCR扩增结合的DNA。在五轮CASTing后鉴定共有基序（MCWTDK），通过用RUSH-la特异性抗体和重组蛋白的CASTing进行验证，并用EMSA进行精制。解离速率常数（Kd = 0.1-1.0 nm; r = 0.99）显示高亲和力结合。染色质免疫沉淀证实了RUSH与转录活性子宫珠蛋白启动子的体内结合。CASTing还揭示了RUSH-GATA转录因子的相互作用。子宫内膜加塔-4表达是孕酮依赖性的（北方分析），并且优先定位于上皮（原位杂交）。虽然身体上与RUSH有关，但RUSH-DNA结合不需要子宫形式的加塔-4。定点突变和瞬时转染试验表明，RUSH基序介导的PRL的能力，以增加胆固醇依赖的子宫珠蛋白转录。RUSH是PRL增强胆固醇依赖性基因转录的机制的核心。
RUSH-1alpha(beta) transcription factors were cloned by recognition site screening with an 85-bp region (-170/-85) of the rabbit uteroglobin gene. Deletion analysis showed this region was essential to prolactin (PRL) action, but conclusions were limited by the complexity of the large deletion. Cyclic amplification and selection of targets (CASTing) was used to identify the RUSH-binding site (-126/-121). Endometrial nuclear proteins were incubated with a pool of degenerate oligonucleotides and immunoprecipitated with RUSH-1alpha(beta) antibodies. Bound DNA was amplified by PCR. The consensus motif (MCWTDK) was identified after five rounds of CASTing, authenticated by CASTing with RUSH-la-specific antibodies and recombinant protein, and refined with EMSA. Dissociation rate constants (K-d = 0.1-1.0 nm; r = 0.99) revealed high-affinity binding. Chromatin immunoprecipitation confirmed in vivo binding of RUSH to the transcriptionally active uteroglobin promoter. CASTing also revealed RUSH-GATA transcription factor interactions. Endometrial GATA-4 expression is progesterone dependent (Northern analysis) and preferentially localized in the epithelium (in situ hybridization). Although physically affiliated with RUSH, uterine forms of GATA-4 were not required for RUSH-DNA binding. Site-directed mutagenesis and transient transfection assays showed the RUSH motif mediates the ability of PRL to augment progesterone-dependent uteroglobin transcription. RUSH is central to the mechanism whereby PRL augments progesterone-dependent gene transcription.