Distinct and Separable Roles for Endogenous CRY1 and CRY2 within the Circadian Molecular Clockwork of the Suprachiasmatic Nucleus, as Revealed by the Fbxl3Afh Mutation
Distinct and Separable Roles for Endogenous CRY1 and CRY2 within the Circadian Molecular Clockwork of the Suprachiasmatic Nucleus, as Revealed by the Fbxl3Afh Mutation
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DOI：
10.1523/jneurosci.4950-12.2013
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发表时间：
2013-04-24
影响因子：
5.3
通讯作者：
Nolan, Patrick M.
中科院分区：
文献类型：
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作者：
Anand, Sneha N.;Maywood, Elizabeth S.;Nolan, Patrick M.
视交叉上核(SCN)的生物钟驱动着日常的行为节律。隐花色素(Crys)是位于SCN发条中心的分子负反馈环中的一种强大的转录抑制因子，在那里它们周期性地抑制自己和时钟控制基因的表达。为了确定CRY1和CRY2在体内昼夜节律时间内的差异贡献，我们利用N-乙基-N-亚硝脲诱导的下班后突变体Fbx13(AFH)来稳定内源CRY。重要的是，这是在CRY2和CRY1缺陷小鼠身上进行的，以隔离测试每一种哭声。在两种哭喊缺乏的背景下，轮子跑和SCN生物发光的昼夜节律都随着Fbx13(AFH)剂量的增加而增加。虽然两种CRY蛋白都使时钟变慢，但CRY1的作用明显强于CRY2，并且在SCN切片中，CRY1而不是CRY2延长了转录抑制的间隔。选择性转录稳定表明，这两个CRY都是时钟控制基因的内源转录抑制因子，但CRY1是最突出的。最后，虽然Cry1(-/-)；Cry2(-/-)小鼠表现为行为心律失常，但它们的SCN表现出短周期(类似于18h)的节律，并具有可变的稳定性。Fbx13(AFH/AFH)对这些不依赖哭声的节律没有影响，证实其昼夜节律作用完全是通过Crys介导的。因此，CRY1和CRY2的稳定对于解释Fbx13(AFH/AFH)的昼夜节律延长是必要的，也是充分的。这两种CRY蛋白都剂量依赖性地延长固有的高频SCN节律，而CRY2也减弱了CRY1更有效的延长周期的作用。因此，加入CRY介导的转录反馈可以稳定固有的SCN振荡，建立由CRY1和CRY2的选择性贡献决定的18-29小时之间的周期。
The circadian clock of the suprachiasmatic nucleus (SCN) drives daily rhythms of behavior. Cryptochromes (CRYs) are powerful transcriptional repressors within the molecular negative feedback loops at the heart of the SCN clockwork, where they periodically suppress their own expression and that of clock-controlled genes. To determine the differential contributions of CRY1 and CRY2 within circadian timing in vivo, we exploited the N-ethyl-N-nitrosourea-induced afterhours mutant Fbxl3(Afh) to stabilize endogenous CRY. Importantly, this was conducted in CRY2- and CRY1-deficient mice to test each CRY in isolation. In both CRY-deficient backgrounds, circadian rhythms of wheel-running and SCN bioluminescence showed increased period length with increased Fbxl3(Afh) dosage. Although both CRY proteins slowed the clock, CRY1 was significantly more potent than CRY2, and in SCN slices, CRY1 but not CRY2 prolonged the interval of transcriptional suppression. Selective CRY-stabilization demonstrated that both CRYs are endogenous transcriptional repressors of clock-controlled genes, but again CRY1 was preeminent. Finally, although Cry1(-/-); Cry2(-/-) mice were behaviorally arrhythmic, their SCN expressed short period (similar to 18 h) rhythms with variable stability. Fbxl3(Afh/Afh) had no effect on these CRY-independent rhythms, confirming its circadian action is mediated exclusively via CRYs. Thus, stabilization of both CRY1 and CRY2 are necessary and sufficient to explain circadian period lengthening by Fbxl3(Afh/Afh). Both CRY proteins dose-dependently lengthen the intrinsic, high-frequency SCN rhythm, and CRY2 also attenuates the more potent period-lengthening effects of CRY1. Incorporation of CRY-mediated transcriptional feedback thus confers stability to intrinsic SCN oscillations, establishing periods between 18 and 29 h, as determined by selective contributions of CRY1 and CRY2.