Laboratorio de Genómica Funcional, Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5090000, Chile.
ANID-Millennium Science Initiative-Millennium Institute for Integrative Biology (iBIO), Santiago 8330025, Chile.
Int J Mol Sci. 2023 Jul 25;24(15):11874. doi: 10.3390/ijms241511874.
The BcWCL1 protein is a blue-light photoreceptor from the fungus . This protein has a central role in circadian regulation and is an ortholog to WC-1 from . The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1). Here, we demonstrated that BcWCL1 is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1 enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1 AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1 protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.
BcWCL1 蛋白是一种来自真菌的蓝光光受体。该蛋白在生物钟调节中起着核心作用，是来自的 WC-1 的同源物。BcWCL1 和 WC-1 蛋白具有相似的蛋白结构域，包括用于光感应的 LOV（光氧电压）结构域、用于蛋白-蛋白相互作用的两个 PAS（Per Arnt Sim）结构域，以及来自 GATA 家族的 DNA 结合结构域。最近，在没有 PAS 结构域的版本（BcWCL1）中证明了 BcWCL1 的蓝光反应。在这里，我们证明了 BcWCL1 在蓝光刺激下能够通过其 N 端区域进行自二聚化。有趣的是，我们观察到 BcWCL1 作为单个组件在酵母中能够实现转录激活。通过使用嵌合转录因子和荧光素酶报告基因，我们评估了 BcWCL1 的 N 端和 C 端区域的不同片段的转录活性，确定了 N 端区域的一个功能性转录激活结构域（AD），属于 9aaTAD 家族。最后，我们确定 BcWCL1 AD 的转录激活水平可与真核细胞中常用的 AD（Gal4 和 p65）相媲美。总之，BcWCL1 蛋白以蓝光依赖的方式自二聚化并激活转录，为该光受体在酵母光遗传学中的未来应用开辟了道路。
