Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas, and Universidad Autónoma de Madrid-CSIC-UAM, Madrid, Spain.
PLoS One. 2013 May 13;8(5):e62849. doi: 10.1371/journal.pone.0062849. Print 2013.
FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility.
METHODOLOGY/PRINCIPAL FINDING: In order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors.
CONCLUSIONS/SIGNIFICANCE: In search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells.
FoxE1 是甲状腺特异性叉头转录因子，对甲状腺发育以及成年期甲状腺分化状态的维持至关重要。FoxE1 识别并结合甲状腺球蛋白（Tg）和甲状腺过氧化物酶（Tpo）启动子中的短 DNA 序列，但 FoxE1 与 Tg 和 Tpo 启动子以外的调节区域的结合几乎尚未被探索。深入了解 FoxE1 的调节功能对于阐明其在内分泌综合征和癌症易感性中的作用是必要的。
方法/主要发现：为了进一步研究 FoxE1 的下游靶标，我们在敲低 FoxE1 后进行了全基因组表达筛选，从而深入了解甲状腺滤泡细胞中 FoxE1 的转录网络。经过验证，我们确认 Adamts9、Cdh1、Duox2 和 S100a4 为上调基因，而 Casp4、Creld2、Dusp5、Etv5、Hsp5a、Nr4a2 和 Tm4sf1 为下调基因。在假定的 FoxE1 调节基因的启动子区域以及经典甲状腺基因 Nis、Pax8 和 Titf1 的启动子中，我们按照先前针对其他叉头因子的描述，在紧邻 NF1/CTF 结合序列的位置进行了 FoxE1 结合基序的计算机搜索。使用染色质免疫沉淀，我们检测到 FoxE1 在两个相关甲状腺基因 Nis 和 Duox2 中的新型调节区域的特异性体内结合。此外，我们证明了 FoxE1 和 NF1/CTF 同时结合到 Nis 上游增强子区域，并且这两个转录因子都能明确地激活 Nis 启动子。
结论/意义：在寻找 FoxE1 在甲状腺细胞中功能的潜在下游介质时，我们鉴定了两个新的直接 FoxE1 靶标基因。据我们所知，这是首次证明 Nis 和 Duox2 参与执行 FoxE1 触发的转录程序。此外，这项研究指出了 FoxE1 在调节甲状腺细胞中大量基因方面的重要作用。
