Genetics and Epigenetics of Behaviour (GEB) Laboratory, Istituto Italiano Di Tecnologia, via Morego, 30, 16163, Genova, Italy.
BioMed X Innovation Center, Im Neuenheimer Feld 515, 69120, Heidelberg, Germany.
Epigenetics Chromatin. 2021 Jan 6;14(1):1. doi: 10.1186/s13072-020-00373-5.
DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period.
In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression.
We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3.
The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.
DNA 甲基化已成为大脑过程（包括昼夜节律）的重要表观遗传调节剂。然而，DNA 甲基化如何干预环境信号（如光驯化）与细胞时钟的转录和翻译分子机制之间的联系，目前尚不清楚。在这里，我们研究了 Afh 小鼠，它们的 Fbxl3 基因发生点突变，昼夜周期延长。
在这项研究中，我们使用了体内、体外和体外相结合的方法。我们测量了 Afh 动物的视网膜反应，并在各种脑组织中进行了简化代表性双硫测序（RRBS）、焦磷酸测序和基因表达分析。在体外，我们使用原代神经元培养物结合微电极阵列（MEA）技术和基因表达。
我们观察到突变神经元网络的功能障碍，以及对光依赖刺激的视网膜反应减少。我们检测到感光黑素细胞 opsin（OPN4）表达异常。此外，我们发现视交叉上核纤维末端的 DNA 甲基化途径以及转录因子 Rev-Erbα 和 Fbxl3 之间的联系发生了改变。
这项研究的结果主要代表了对 Afh 模型对外界刺激的电生理和分子表型反应的理解的贡献。此外，由于 DNA 甲基化最近已成为神经元网络的新调节剂，对昼夜行为有重要影响，我们讨论了 Afh 突变对昼夜生物学表观遗传景观的影响。
