Single Molecule Resolution Study of Circadian Chromatin Dynamics Regulated by the Pioneer Transcription Factor CLOCK:BMAL1

The mammalian circadian clock is initiated by the heterodimeric transcription factor CLOCK: BMAL1, which binds to the DNA during the day and activates the transcription of Period (Per1, 2, 3) and Cryptochrome(Cry1, 2) that form a repressive complex and inhibit CLOCK:BMAL1 transcription activity at night. However, CLOCK:BMAL1 alone is not sufficient to generate active enhancers. Instead, circadian transcriptional regulation depends on the interplay between CLOCK:BMAL1 and other transcription factors (TFs) at cis-regulatory elements (CREs). Al-though CLOCK:BMAL1 has been proposed to act as a pioneering-like TF that promotes rhythmic chromatin opening, the molecular mechanism of how it cooperates with other TFs to regulate circadian transcription remains poorly understood. To address this, we adapted single-molecule footprinting (SMF), a technique that simultaneously captures nucleosome occupancy and TF protection on individual DNA molecules, to study the physiologically relevant chromatin regulation in the mouse liver. We established an experimental protocol including nuclei purification strategies for six mouse tissues, optimized library enrichment to reduce duplication artifacts, and developed a computational pipeline (BMDsmf) that enables the characterization of chromatin states, nucleosome positioning, and quantification of nucleosome and TF binding at CREs. Applying this framework to mouse livers, we identified that CREs are organized in multiple stereotypical chromatin states that are conserved across circadian time points, tissues, and genotypes. These states reflect dynamic competition between nucleosomes and TFs rather than a binary open/closed model, which may reflect cell heterogeneity in transcriptional bursting. SMF revealed that CLOCK:BMAL1 binding facilitates nucleosome eviction with E-boxes located at its entry-exit sites, which promotes the formation of accessible chromatin states and cooperation with other TF to activate transcription. Furthermore, we uncovered CRE-specific E-box protection, with some sites exclusively bound by CLOCK:BMAL1, and some displayed competition with basic helix-loop-helix (bHLH) TFs. Importantly, we discovered a cooperative CLOCK:BMAL1 binding across multiple E-boxes separated by more than 250 bp, which structurally altered the CLOCK:BMAL1-DNA interface. This work establishes a robust methodology for applying SMF in tissues and provides a powerful framework to study how environmental challenges, such as cancer, high-fat diets, and aging, reprogram circadian gene expression programs through altered chromatin regulation at CREs.

Name thesis:degree_name

Doctor of Philosophy

Discipline thesis:degree_discipline

Biology

Grantor

Texas A&M University

Year dc:date.issued

2025

Author dc:creator

• Nie, Xinyu 11/08/1996-

Advisor dc:contributor.advisor

• Menet, Jerome

Committee members dc:contributor.committeemember

• Sing-Hoi Sze
• Christine Merlin
• Deborah Bell-Pedersen

• Biology, Molecular

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/1969.1/1600712

OAI record oai:identifier

https://oaktrust.library.tamu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aoaktrust.library.tamu.edu%3A1969.1%2F1600712