UC Santa Cruz

Nearly all walks of life, from single cell cyanobacteria to humans, have evolved an intimate connection to the light dark cycle and coordinate physiology and behavior to the solar day. This phenomenon is known as circadian rhythms and is an adaptation that organisms use to anticipate daily environmental changes. In mammals, disruption of circadian rhythms through environmental stimulus or genetic means leads to the onset of many diseases such as: diabetes, cardiovascular disease, premature aging and cancer. Nearly every cell in the human body has an endogenous molecular clock that controls integrated biochemical processes on a ~24-hour period. At the core of this molecular clock is the heterodimeric basic helix-loop-helix Per:Arnt:Sim (bHLH-PAS) transcription factor CLOCK:BMAL1 that together with its repressors, Period (PER) and Cryptochrome (CRY), forms an autoregulatory feedback loop that results in the rhythmic transcription of nearly 40% of the genome including essential genes in metabolism, hormone secretion and the cell cycle. While this model for transcription-translation feedback loop has been accepted for over two decades, it is still not well understood how CLOCK:BMAL1 activity is directly regulated to generate intrinsic 24-hour timing in mammals. Using a combination of biochemistry, structural and cell biology we have elucidated a mechanism by which CRY1 directly interacts with CLOCK:BMAL1 to form a critical repressive circadian complex in circadian rhythms. We have also discovered an uncharacterized PAS domain containing protein in humans, PAS Domain containing protein 1 (PASD1), that inhibits CLOCK:BMAL1 activity and suppresses circadian cycling in cancer cells, providing a molecular link from oncogenesis to circadian disruption.

Chapter 2 describes our work to elucidate how CRY1 directly inhibits CLOCK:BMAL1 activity. CRYs close the tightly regulated transcriptional feedback loop to control circadian rhythms, however, the mechanistic underpinnings of how CRYs interact with and repress CLOCK:BMAL1 have remained elusive. Previous studies in our lab showed that tuning affinity of CRY1 for the transactivation domain (TAD) of BMAL1 controls circadian period by competing with the coactivator CBP/p300. CRY1 also binds to CLOCK, although it was not yet understood how multivalent interactions with CLOCK:BMAL1 contribute to CRY1 function. I have shown that CRY1 directly binds the CLOCK:BMAL1 PAS-AB core and this interaction is driven by a single domain in this multi-domain protein complex, CLOCK PAS-B. Furthermore, I have worked with experts in small angle x-ray scattering analysis to generate a model of the CRY1:CLOCK:BMAL1 complex that situates CRY1 atop the CLOCK:BMAL bHLH PAS-AB domains in the solution envelope providing the first low resolution description of the CRY1:CLOCK:BMAL1 complex. These studies have paved the way for circadian-directed therapeutics and have provided a basis for comparative analysis between the CRY1 and CRY2 proteins as described in Chapter 5. Further studies on the assembly of circadian repressive complexes, including CRY and PERIOD proteins, is described in Chapter 6.

In Chapter 4 we report the discovery of a previously uncharacterized repressor of circadian rhythms, PAS domain containing protein 1 (PASD1). Upon joining Dr. Partch’s lab I chose to work on the “long-shot project” – an interesting but undeveloped project that was based entirely off Dr. Partch’s initial discovery of an uncharacterized gene that bears significant similarity to a clock protein, potentially possessing the ability to modulate the circadian clock. This CLOCK-like protein, PASD1, is not expressed in healthy somatic tissues, but is instead limited to gametogenic tissues where there are no functional clocks. Using transcriptional reporter assays I confirmed initial results that PASD1 inhibits transactivation of genes by the core circadian transcription factor, CLOCK:BMAL1. Through series of truncation experiments I found that the C-terminus of PASD1 is sufficient to repress CLOCK:BMAL1 in the nucleus. Furthermore, deletion of one region that is highly conserved with CLOCK alleviates repression by PASD1 to suggest it utilizes molecular mimicry to interfere with CLOCK:BMAL1 function. Furthermore, knockdown of PASD1 in both colon and lung cancer cell lines improves the amplitude of cycling, indicative of a more robust oscillator. These data together provide a tool to rescue the clock in PASD1+ tumors.

In summary, I have used biochemistry and biophysics to describe how the essential circadian protein, CRY1, serves as a potent repressor of CLOCK:BMAL1 activity to establish circadian rhythms. This work provides molecular details of the CRY1-CLOCK:BMAL1 interaction that are being actively used to create circadian-based therapeutics and study the structure of circadian repressive complexes. I have also used cell biology to identify a novel repressor of CLOCK:BMAL1 that is highly up-regulated in many forms of cancer and suppresses circadian cycling.