A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts.

Area of Science:

• Molecular Biology
• Chronobiology
• Biochemistry

Background:

• Circadian rhythms are regulated by transcription factors like CLOCK and BMAL1.
• Daily oscillations in CLOCK-BMAL1 DNA binding activity are linked to gene expression.
• Existing methods for assessing CLOCK-BMAL1 binding can be time-consuming or require specialized equipment.

Purpose of the Study:

• To develop a sensitive, rapid, and versatile assay for measuring CLOCK-BMAL1 DNA binding activity.
• To characterize the performance of the new assay using tissue extracts and cell cultures.
• To demonstrate the assay's utility in studying factors influencing CLOCK-BMAL1 binding.

Main Methods:

• Development of the Clock Protein-DNA Binding Assay (CPDBA) using crude tissue extracts.
• Assay optimization for sensitivity (detecting <2-fold differences) and speed (results in ≤2 hours).
• Application of CPDBA to assess changes in CLOCK-BMAL1 binding after protein dephosphorylation and casein kinase I inhibition.

Main Results:

• The CPDBA accurately recapitulates native CLOCK-BMAL1 DNA binding rhythms.
• The assay detected significant increases in CLOCK-BMAL1 binding activity (~1.5 to 2-fold) upon protein dephosphorylation or casein kinase I inhibition.
• CPDBA demonstrated sensitivity with minimal sample input (~10 µg) and no need for specialized equipment or probes.
• The assay successfully measured CLOCK-BMAL1 binding to reconstituted chromatin.

Conclusions:

• The Clock Protein-DNA Binding Assay (CPDBA) is a highly sensitive, fast, efficient, and versatile tool for probing clock function.
• CPDBA provides a valuable method for studying the regulation of circadian rhythms at the molecular level.
• This assay can be applied to various biological samples, including crude extracts and reconstituted chromatin, facilitating broader research into circadian biology.