Investigating the genomic mechanisms mediating daily timekeeping in the suprachiasmatic nucleus (SCN) in mammals

With my BBSRC Discovery Fellowship, I propose to unravel the gene regulatory mechanisms that governs daily timekeeping in the master pacemaker. Here, I hypothesise environmentally induced circadian change in 3-D chromosomal conformation drives the spatiotemporal gene regulation in the central clock.

The 24-hour (hr) rotation of the earth around its own axis results in daily cycles of light and temperature and directs the internal body clock present in almost all living creatures. This intrinsic circadian (approximately one day) clocks align the molecular, behavioural and physiological processes such as sleep-wake cycles, to changing daily environmental conditions.

Typically, in multicellular organisms such as humans, environmental light travels from the retina to specific region of the brain; suprachiasmatic nuclei (SCN) also known as central pacemaker and triggers a series of rhythmic molecular and biochemical events. The signal is then passed to different regions of the brain and tissues (heart, liver, kidney etc.) to synchronize the local peripheral clocks and generate a coherent physiological response.

Precise and timely regulation of the gene expression in the SCN is crucial for circadian timekeeping and overall fitness. However, the gene-regulatory mechanisms that renders SCN as a powerful master oscillator is still unknown.

Until recently, and because of technical limitations, peripheral tissues and cell lines were used as a "proxy" for the real SCN to study the gene-regulatory processes that underpin daily timekeeping. However, this is patently unsatisfactory as it lacks the critical neuronal cellular dimension intrinsic to the role of the SCN as our central brain pacemaker.

My research is focussed on investigating the gene- regulatory elements and processes that are vital for daily timekeeping mechanisms, and have recently discovered the prevalence of cycling tissue-specific gene enhancer elements in the SCN.

In my fellowship project, I will aim to gain in-depth mechanistic insights into the genomic regulation operative in the central clock to understand the systematic maintenance of daily rhythms. Enhancers are short stretches of DNA that can modulate both proximal and distal gene expression. Almost forty years after their discovery, enhancers are recognised as playing a critical role in the spatiotemporal control of gene expression.

Therefore, I will initially focus on the functional characterization of the putatively mapped SCN enhancers by using massively parallel reporter assay (MPRA). Next, I propose to study the genome-wide binding of the key DNA loop extrusion proteins CTCF (CCCTC-Binding Factor) and cohesin in the context of circadian timekeeping. In addition, I would also like to adopt advanced Capture-C technology to produce the first ever chromosomal contact map of the SCN and explore the missing link between enhancer and downstream target gene expression.

Finally, I plan to carry out high-throughput spatial transcriptomics to visualize and quantify targeted transcribing enhancer and gene expression at sub-cellular level.

Overall, my findings will clearly demonstrate how daily environmental stimuli modulate the epigenomic landscape in order to achieve spatiotemporal gene regulation in the mammalian brain. The proposed research will highlight the tissue-specific enhancer elements and involved processes that facilitates daily timekeeping. It will constitute the starting points to understand how aberrant gene -regulatory features (enhanceropathies) could result in circadian misalignment and lead to the development of various diseases and disorders.

Moreover, this study holds great potential to advance our current understanding on systemic regulation of chromosomal conformation and DNA topology and the biological basis of time keeping.