Circadian Expression Of Pineal Biological Clock Genes And Screening Of Peripheral Clock-controlled Genes In Lymphocytes Of Rat

Circadian rhythms can be found at levels of physiological function,biochemical metabolism and behavior change of living organism.Disorder of the rhythms may result in various diseases,such as jet lag caused by flight over different geographic time zones,dyssomnia induced by day and night shift-work,metabolic alteration and aging originated from hormonal parasecretion,mental ailment triggered by special rhythm impairment and so on.It is an important theoretical and practical sense to reveal the regulatory mechanism of circadian rhythm in terms of better understanding physiological,biochemical and behavioral activities of organism,and to explain pathologic process resulted from disturbance in circadian rhythm.Structures of biological clock include nucleus clock organization,i.e., suprachiasmatic nucleus(SCN),pineal gland(PG) and related peripheral tissues manipulating circadian rhythms.The molecular regulatory mechanisms of clock oscillation consist of clock signal input,several clock genes,clock-associated genes, clock-controlled genes and their protein,through the interconnection of intracellular transcription-translation-posttranslation event,to organize a fundamental molecular framework named autoregulatory feedback loop of the clock oscillator for accurate clock signal output.Studies on the oscillatory mechanisms of biological clock have been greatly developed so far,however,some problems remain.Firstly,most research on one of the important clock genes,Clock,as a main component of circadian molecular oscillatory loop in mammal,was focused for SCN rather than for PG.The Daily expression of the Clock gene and clock-associated gene,arylalkylamine N-acetyltransferase gene(NAT,a key gene used to regulate release of chronobiological messenger named melatonin) with their correlation and photoresponses in the PG have not been studied yet.Secondly,the oscillatory evidence of clock in PG independent of SCN,circadian transcriptional discrepancy and light responses difference of the Clock expression between the SCN and PG have not been reported so far.In addition,the concept of clock-controlled gene proposed recently needs concrete experimental proofs.Moreover,if the peripheral lymphocytes contain the clock-controlled gene of Clock,and then the temporal profile of the circadian gene expression and its histological distribution should be further investigated.The present study is aimed to deal with the above-mentioned issues.Objective:By application of animal and lymphocyte models of circadian rhythm, to investigate the circadian rhythmic expressions and photoresponses of clock gene, Clock,and clock-associated gene,NAT in the rat PG and SCN,and to screen and identify the clock-controlled genes of Clock in the peripheral lymphocytes with histological distribution,in order to better illuminate the molecular basis of central circadian rhythm and the mechanisms of the peripheral clock operation.Methods:(1) with free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness(DD) for 8 weeks(n=42) or 12 h-light(5:00-17:00):12 h-dark(17:00-5:00)cycle(LD)for 4 weeks(n=42),respectively.Then,the SCN and PG were dissected out every 4 h in a circadian day,7 rats each time(n=7).All animal treatments and sampling during the dark phases were conducted under aid of red dim light(＜0.1 lux).The total RNA was extracted from each sample and the semi-quantitative reverse transcription PCR (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock or NAT genes in the SCN and PG at different circadian times(CT) or zeitgeber times (ZT)under different light conditions.The grayness ratios of Clock/H3.3 bands or NAT/H3.3 bands were served as the relative estimation of the Clock or NAT gene expressions in the PG and SCN.The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 phases and to simulate the circadian rhythmic curves which were then examined for statistical difference by the amplitude F test.(2) Under the LD(12 h:12 h) regime,peripheral blood of the same rat was sampled twice from heart at 2 hours after illumination and after darkness,respectively.Lymphocytes were dissociated from blood and the total RNA was extracted from the cells.Genes on the peripheral blood lymphocytes which displayed Circadian differential expressions were screened by using various techniques,such as differential display reverse transcription-PCR(dd RT-PCR), subcloning and sequencing of the differential cDNA fragment,reverse Northern dot-blot,quantitative real-time PCR,homologous comparison and analysis of the differential cDNA fragment and so on.(3) After dissociation and culture of peripheral blood lymphocytes,the cDNA fragments showing circadian differential expressions obtained from the lymphocytes were identified with RNA interference(RNAi) and quantitative real-time PCR to determine whether those cDNAs fragments belong to the clock-controlled genes located at the downstream of the Clock gene.(4) In the LD condition,the rhythmic temporal profiles of the clock-controlled genes expressions identified in lymphocytes at different circadian time points were examined with the quantitative real-time PCR.Rats were sampled from different parts of the body in order to determine the histological distribution and expressive abundance of the clock-controlled genes identified in this study.Results:(1) In DD or LD condition,both of Clock and NAT mRNA levels in the PG showed robust circadian oscillation(amplitude F test,P＜0.05) with the peak at the subjective night or at nighttime.(2) In comparison with DD regime,the rhythmic amplitudes and the mRNA levels at peaks of Clock and NAT expressions in LD in the PG were significantly reduced(P＜0.05),but no rhythmic phaseshifts were obtained (P＞0.05).(3) In DD or LD condition,the circadian expressions of NAT were similar in pattern to those of Clock in the PG(P＞0.05).(4) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation(amplitude F test, P＜0.05).The endogenous rhythmic profiles of Clock transcription in the PG were similar to those in the SCN(P＞0.05) throughout the day with the peak at the subjective night(CT15 in the SCN or CT18 in the PG,P＞0.05) and the trough during the subjective day(CT3 in the SCN or CT6 in the PG,P＞0.05).(5) Clock transcription in the SCN in LD cycle also showed the circadian oscillation(amplitude F test,P＜0.05),with an anti-phase to that in DD condition(P＜0.05).The amplitudes and the mRNA levels at peaks of Clock transcriptions in the SCN in LD,were significantly increased(P＜0.05),in comparison with in DD,while the value of corresponding rhythmic parameters in the PG in LD were remarkably decreased (P＜0.05).(6) Under LD cycle,the circadian profiles of Clock transcription induced by light in the PG were quite different from those in the SCN(P＜0.05).Their Clock transcription rhythms were anti-phasic,i.e.,showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG.(7) Ten circadian differential cDNA fragments were identified to be mediated by the Clock in the peripheral blood lymphocytes,including three known genes(catalase,myelin proteolipid protein,and histone acetyltransferase),four known expressed sequence tags(ESTs),and three novel ESTs.Experiment of the RNAi revealed that these ESTs were down-regulated by the inhibition of Clock gene and three of them were identified as clock-controlled genes.The circadian expressions of the genes in LD cycle showed the peaks at dark phases ZT19,ZT19 and ZT20,respectively.Of three genes,the amplitude of catalase circadian expression was largest(P＜0.05).(8) The three clock-controlled genes were found to distribute not only in peripheral blood lymphocytes,but in heart,lung,liver, kidney,thymus,PG,SCN and brain stem as well,with diverse transcriptional levels at different tissues.Conclusion:The findings of the present study indicate:(1) The expressions of clock gene,Clock,and clock-associated gene,NAT in the PG not only show the remarkably synchronous endogenous circadian rhythmic changes,but also response to the ambient light signal in a reduced manner.Light signal does not induce rhythmic phaseshifts of the two gene expressions in the PG.(2) The Clock gene circadian transcriptions in the SCN and PG characterize a synchronous endogenous rhythmic nature and manifest different roles of light entrainment in modulating the circadian transcriptions of Clock in these two central nuclei.(3) The several clock-controlled genes exist at the downstream of the Clock in peripheral blood lymphocytes,with a circadian oscillatory feature.The genes of catalase,myelin proteolipid protein and histone acetyltransferase express daily oscillation in LD cycle with peaks at dark phases and troughs during light phases.(4) These clock-controlled genes expressed in different tissues display different transcriptional levels.