Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1
Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1
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DOI：
10.1210/en.2005-0100
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发表时间：
2005-06-01
期刊：
影响因子：
4.8
通讯作者：
Boockfor, FR
中科院分区：
文献类型：
--
作者：
Leclerc, GM;Boockfor, FR
最近的研究结果表明，从我们的实验室和其他人的催乳素基因表达（PRL-GE）振荡在单一的生活mammotropes，但很少有信息是可用的分子过程，有助于这种现象。为了阐明这种活性的来源，我们产生了一系列的结构，其含有与荧光素酶报告基因融合的长度递减的PRL启动子。将这些构建体注射到单细胞中并测定光子活性。我们发现脉冲活性与所有质粒测试，甚至与最小的启动子片段的331 bp。该片段的序列分析鉴定了两个潜在的E盒（已知结合CLOCK和BMAL 1昼夜节律蛋白的元件）。此外，PRL细胞（垂体，MMQ和GH（3））的RT-PCR显示了clock和bmal 1以及其他五个时钟基因（per 1，per 2，cry 1，cry 2和tim）的表达，表明昼夜节律系统可能在PRL细胞中起作用。接下来，我们突变了2.5-kb PRL启动子内的两个E-box的核心序列，发现只有E-box 133的突变完全消除了PRL-GE脉冲。EMSA显示，CLOCK和BMAL 1能够在体外结合到E-box 133位点。我们的结果表明，PRL-GE脉冲是依赖于一个特定的E盒结合位点的PRL启动子。此外，CLOCK/BMAL 1可以结合到该位点的迹象表明，这些昼夜节律蛋白，单独或与其他因素结合，可以调节间歇性PRL启动子活性的mammotropes，也许作为一个临时开关的PRL的开/关表达。
Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes ( elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells ( pituitary, MMQ, and GH(3)) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E- box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.