Transcriptional and post-translational mechanisms that regulate Drosophila circadian rhythms
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Circadian rhythms are present in most living organisms with 24-hour cylces in behavioral or physiological processes. Circadian rhythms are generated by the endogenous molecular circadian clock, which relies on the negative transcriptional-translational feedback loop (TTFL). TTFL is a conserved clock regulatory mechanism among different species, which gates many aspects of 24-hour molecular changes from cyclic gene expression to rhythmic protein translocation to time-dependent protein-protein /DNA interactions. Drosophila has been a well-established model organism to study the circadian clock. The first clock mutants were identified in a large-scale Drosophila screen in 1971, which, later on, leads to a series of pioneer discoveries of core clock genes in both mammals and flies. In Drosophila, the TTFL consists of a handful of genes, including Clock (Clk), cycle (cyc), period (per), timeless (tim). CLK and CYC form a heterodimer acting as transcriptional activators, and they bind to the E-box region of downstream genes, such as per and tim to promote their transcription. The encoded PER/TIM proteins translocate to nucleus at late night to repress CLK/CYC activity, thus inhibiting their own transcriptional activity. Aside from the CLK/CYC-dependent regulation on per transcription, we found the transcriptional factor, cAMP Response Element-Binding Protein A (CREBA), also binds to per promotor region. The first half of my dissertation focuses on the underlying mechanism of CREBA on circadian clock regulation. Here, we show that downregulation of the transcription factor CREBA causes behavioral and molecular phenotypes reminiscent of those observed when ATX2 or TYF (RNA binding proteins) were depleted. ATX2 and TYF were previously identified to be involved in per translation by binding to polyA sequence of per mRNA. We also found that CREBA genetically and physically interacts with the ATX2/TYF complex. Interestingly, ATX2 also binds to per promotor but in a CREBA-dependent manner. Consistent with this, we observed that both CREBA and ATX2 regulate circadian period via the 5’ end of the per gene. We therefore propose a model in which CREBA deposits the ATX2/TYF complex on per mRNAs during transcription.The second half of my dissertation focuses on that Suppressor of RAS (SUR-8) regulates PER stability. SUR-8 is expressed within circadian neurons and depletion or overexpression of Sur-8 disrupts circadian rhythms. Further behavioral and immunostaining analysis indicates that PER is the primary target of SUR-8. SUR-8 induces phosphorylation changes of PER to further regulate its stability. PER protein phosphorylation levels are significantly increased in Sur-8 knockdown, accounting for the reduced PER levels in Sur-8 RNAi. The downregulation of protein phosphatase 1 catalytic subunit, PP1-87b, phenocopies Sur-8 knockdown and we further identified SUR-8, PP1-87B, and PER are in a protein complex. Moreover, SUR-8 mediates the interaction between PER and PP1-87B, confirming the role of SUR-8 as a scaffold protein. Interestingly, there is more binding of SUR-8 to PER at night than early morning, suggesting a temporal regulation. Together, our findings demonstrate that SUR-8 links PP1-87B with PER to control PER stability and ultimately, to regulate circadian rhythms.