# Mechanisms of Circadian Repression

> **NIH NIH R01** · TEXAS A&M UNIVERSITY · 2024 · $361,503

## Abstract

Daily rhythms in animal behavior, physiology and metabolism are driven by cell-autonomous circadian clocks
that are synchronized by environmental cycles but maintain ~24h rhythms even in their absence. These clocks
keep circadian time and control overt rhythms via transcriptional feedback loops (TFLs). Because clock
dysfunction negatively impacts human health, characterizing mechanisms that drive TFLs is of critical
importance. The goal of this proposal is to understand how feedback repression, a key event controlling
rhythmic transcription, is achieved using two complementary model systems; the monarch butterfly and the
fruit fly Drosophila melanogaster. Animals possess two TFL paradigms with orthologous components: A
Drosophila-like (dl) paradigm in which CLOCK (CLK) activates and PERIOD (PER) represses transcription,
and a mammal-like paradigm (ml) in which CLK-BMAL1 activates and PER-CRYPTOCHROME (PER-CRY)
complexes repress transcription. Monarch butterflies have an ml clock, but unlike mammals, monarchs carry
single copies of clock activator and repressor genes, thus making it an attractive model to dissect clock
mechanisms relevant to mammals. Common features of dl and ml clocks are that PER complexes containing
CASEIN KINASE 1 (CK1) initiate transcriptional repression `on-DNA' by binding CLK complexes on E-box
elements, followed by CK1-dependent PER and CLK phosphorylation, removal of PER-CLK complexes from
E-boxes to initiate `off-DNA' repression, and ultimately PER degradation. How PER orchestrates transcriptional
repression is poorly understood. We recently identified a region in CLK that acts as a conserved molecular hub
to coordinate transcription activation and repression. The TRITHORAX (TRX) histone methyltransferase, which
activates transcription by binding this hub, is also essential for repressing transcription by permitting CLK-PER
binding. TRX mediates repression by directly or indirectly methylating the chaperonin HSP68, which is required
for CLK-PER binding and repression. We also discovered that CLOCK-Interacting Protein Circadian (CIPC)
also binds the CLK hub to repress transcription across animals. CIPC and TRX binding to the CLK hub
suggests that CIPC inhibits transcription by displacing TRX, altering TRX substrate specificity to permit HSP68
R45 methylation, promoting PER-CLK binding, removing CLK-CYC from DNA and/or promoting co-repressor
binding. These hypothetical CIPC functions will be tested in Aim 1. Our discovery that TRX methylation of
HSP68 is required for PER-CLK binding and repression suggests that HSP68 acts in concert with progressive
phosphorylation of unstructured PER and CLK regions to efficiently drive sequential structural changes that
control DNA binding and protein interactions needed to maintain a ~24h circadian cycle. This hypothesis will
be tested in Aim 2. Successful completion of these aims will provide mechanistic insight into how circadian
repression determines the phase, period and amplitude o...

## Key facts

- **NIH application ID:** 10811734
- **Project number:** 5R01GM124617-06
- **Recipient organization:** TEXAS A&M UNIVERSITY
- **Principal Investigator:** PAUL E HARDIN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $361,503
- **Award type:** 5
- **Project period:** 2017-08-11 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10811734

## Citation

> US National Institutes of Health, RePORTER application 10811734, Mechanisms of Circadian Repression (5R01GM124617-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10811734. Licensed CC0.

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