Research On Expression And Mechanisms Of Regulation Of Circadian Clock-related Genes In Mammals During Development

A variety of mammalian life activities (such as body temperature, blood pressure and changes in hormone secretion, sleep and wake states, and alternation behavior) have shown the phenomenon of autonomous circadian rhythm。In mammals, the circadian timing system is organized as a hierarchy of oscillators located in most tissues of the body (peripheral oscillators), synchronized by a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Lesions of SCN disrupt circadian rhythms of wheel running, drinking, hormonal secretion and sleep patterns. Over the past few years, the molecular models of the circadian clock have evolved as additional clock genes have been identified. The molecular clockwork is composed of a network of transcriptional-translational feedback loops that drive rhythmic, about 24-hour expression patterns of core clock components. The basic character of the circadian system is the prevalence of the oscillation at the levels of genes reflects at cells, tissues, and system levels.Prokineticin 2 (PK2) as a novel peptide expressed in the SCN could influence clock output. The role of PK2 as a clock output factor fulfil the following criteria:its prominent circadian express in the SCN while its receptor express in the SCN projection targets; it is a secreted molecule; the production and/or release of PK2 is regulated by core clock genes and respond to light entrainment; administration of PK2's could result in inhibitory effect on locomotor activity after i.c.v. administration. There is not enough information about the PK2 system in the Syrian hamster, the widely used species in circadian rhythm research. So we cloned the cDNA sequences of shPK2 and its receptor shPKR2 in the Syrian hamster through a homologous cloning approach in this study. We further characterized those genes' temporal expression within the adult animals'SCN by using in situ hybridization and Realtime PCR, demonstrating they are expressed in the central circadian rhythm. Furthermore, we characterized the developmental expression of those genes during the perinatal period. Recent studies indicate that the clock functions during development undergo complex changes at the molecular level, with gradual maturation of its molecular oscillating mechanisms. In this study, we found no obvious circadian expression of PK2 during the prenatal period, while a clear rhythm detectable only on postnatal day 2. This result is consistent with previous studies of developmental expression of clock genes by using the highly robust PK2 as a marker to probe the functional state of the central clock during development.Expression of clock genes undergo dramatic changes within the SCN during development, while postnatal upregulation of several genes (including mPerl and mCryl) accompanying the maturation of clockwork operation. Recently, more and more studies have shown that epigenetics also involve in regulating the transcription of circadian rhythm, accompaning by histone H3 acetylation and chromosomal remodeling.In this study, we performed DNA methylation analyses for mPerl, mPer2 and mCryl promtoers during the perinatal period in the suprachiasmatic nucleus, adjacent aera of suprachiasmatic nucleus, liver and sperm. These genes are part of the transcription/translation feedback loop underlying circadian rhythm generation. We detected significant demethylation for the mPerl promoter around birth in the SCN. By using luciferase reporter assays, mPerl promoter methylation has inhibitory effect on gene expresion. So we conclude that epigenetic mechanisms are potentially involved in regulating mPerl expression during development.