Role of Methylation in <i>Period2</i> (<i>PER2</i>) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical—Studies on the Pig Model

• Main Authors: Przemysław Gilun, Krzysztof Flisikowski, Tatiana Flisikowska, Joanna Kwiatkowska, Barbara Wąsowska, Magdalena Koziorowska-Gilun
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: International Journal of Molecular Sciences
• Subjects: gene expression; E-box; methylation; <i>PER2</i>; carbon monoxide; oscillation
• Online Access: https://www.mdpi.com/1422-0067/22/15/7796

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of <i>Period2</i> gene (<i>PER2</i>), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the <i>PER2</i> promoter was performed. qPCR showed a differential pattern of <i>PER2</i> mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine <i>PER2</i> in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of <i>PER2</i> expression.