Biochemical studies of the clock proteins BMAL1 and CRYs in Xenopus : regulation of the CRYs subcellular localization and implications on their role in the modulation of the circadian loop

The photoreceptor layer in the retina of Xenopus laevis harbors a circadian clock. This model system displays a wide array of rhythms, including melatonin release, electroretinograms rhythms and retinomotor movements, suggesting that the ocular clock is important for proper retinal function. Many molecular components known to drive the molecular circadian clock in other organisms have been identified in Xenopus, such as xClock, xPers, xCryl and xCry2b, and, as described in this dissertation, xbmall, demonstrating phylogenetic conservation. The development of the transgenic technique in Xenopus, which leads to the fast generation of transgenic tadpoles, allows precise and reliable molecular perturbations, since it is possible to follow rhythms in eyecups obtained from adults or tadpoles. Because of these reasons, I have investigated the function of two key clock components, xBMALl and xCRYl and xCRY2b, in the Xenopus retina. These components are part of the activation (BMAL1) and repression (CRYs) branch of the mammalian molecular circadian loop. In mammals, CLOCK and BMAL1 heterodimerize and activate transcription of clock genes and are then repressed by the CRYs; it is not known how the two CRYs contribute differently to repression, but this contribution is known not to be redundant. I have found that xBMALl is a transcription factor and that it binds to xCLOCK, suggesting its function is conserved between mammals and Xenopus. In addition, xBMAL 1 's functional domains (the basic, helix- loop-helix and PAS domains) are required for transcriptional activation, xbmall mRNA is highly expressed in the photoreceptor layer and strongly rhythmic, strengthening the hypothesis that xBMALl protein is a key component of the retinal clock. The xCRYs have previously been shown to repress xCLOCK/xBMALl-mediated transcriptional activation in tissue culture. Here, I describe the characterization of nuclear localization signals present in the C-termini of the proteins. By generating several C-terminal mutants, I have shown that the NLSs are differentially regulated between xCRYl and xCRY2b. xCRY2b contains a canonical NLS, which is regulated by phosphorylation, while xCRYl's NLS does not resemble any known sequence. These results offer a possible explanation of how these two proteins contribute differently to the molecular loop. Xenopus offers an excellent system to study the in vivo effects on the circadian system of xBMALl and xCRYs mutants.

Digitization of this thesis was made possible by a generous grant from the Jefferson Trust, 2015.

Thesis originally deposited on 2016-02-18 in version 1.28 of Libra. This thesis was migrated to Libra2 on 2017-03-23 16:34:11.