Jin Hong Lan, Yang Liu, Jeong Kwang Won
Gachon Institute of Pharmaceutical Sciences, College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon, 21936, Republic of Korea.
Mol Biol Rep. 2017 Apr;44(2):243-250. doi: 10.1007/s11033-017-4106-3. Epub 2017 Apr 28.
Flightless-I homolog (FLII) is a member of the gelsolin family of proteins, and has been identified as a coactivator of estrogen receptor-mediated transcription. Here, we investigate the role of FLII in the glucocorticoid receptor (GR) signaling pathway. Reporter gene assay and real-time quantitative PCR in A549 were performed to investigate the function of FLII in the expression of GR target genes. Co-immunoprecipitation assay and in vitro binding assay were used to identify binding domain of FLII. Chromatin immunoprecipitation assay were carried out with FLII-depleted A549 cells to determine the role of FLII at GR binding sites. We demonstrate that FLII potentiates GR-mediated reporter gene activity synergistically with CARM1 and p300 to enhance GR transcriptional activity in the presence of dexamethasone (Dex) in A549 cells. Depletion of endogenous FLII inhibited the expression of Dex-regulated GR target genes in A549 cells, indicating that FLII is required for GR-mediated transcription. Further, we observed that FLII binds to GR via its N-terminal leucine-rich repeat (LRR) region, suggesting that the enhancement of GR activation may occur through the interaction of GR and FLII. Moreover, chromatin immunoprecipitation analysis demonstrated that FLII is recruited to the GR binding sites. In addition, depletion of endogenous FLII decreased the recruitment of p300, and subsequently RNA polymerase II, to specific sites of GR target genes. Taken together, these studies reveal a functional involvement of FLII in activating transcription of GR target genes, suggesting a physiological role for FLII in the GR signaling pathway.
无翅 I 同源物（FLII）是凝溶胶蛋白家族的成员，已被鉴定为雌激素受体介导转录的共激活因子。在此，我们研究 FLII 在糖皮质激素受体（GR）信号通路中的作用。在 A549 细胞中进行报告基因检测和实时定量 PCR，以研究 FLII 在 GR 靶基因表达中的功能。采用免疫共沉淀检测和体外结合检测来鉴定 FLII 的结合结构域。对 FLII 缺失的 A549 细胞进行染色质免疫沉淀检测，以确定 FLII 在 GR 结合位点的作用。我们证明，在 A549 细胞中，FLII 与 CARM1 和 p300 协同增强 GR 介导的报告基因活性，从而在存在地塞米松（Dex）的情况下增强 GR 的转录活性。内源性 FLII 的缺失抑制了 A549 细胞中 Dex 调节的 GR 靶基因的表达，表明 FLII 是 GR 介导转录所必需的。此外，我们观察到 FLII 通过其 N 端富含亮氨酸重复序列（LRR）区域与 GR 结合，提示 GR 激活的增强可能通过 GR 与 FLII 的相互作用发生。此外，染色质免疫沉淀分析表明 FLII 被招募到 GR 结合位点。另外，内源性 FLII 的缺失减少了 p300 以及随后 RNA 聚合酶 II 向 GR 靶基因特定位点的募集。综上所述，这些研究揭示了 FLII 在激活 GR 靶基因转录中的功能参与，提示 FLII 在 GR 信号通路中具有生理作用。
