Periodic Reporting for period 1 - CLOCK (Characterization of the circadian chromatin landscape using a novel CRISPR/Cas9-guided proximity-labelling technique)
Okres sprawozdawczy: 2020-12-01 do 2022-11-30
Circadian clocks are molecular oscillators present in most mammalian cells that drive the circadian rhythms (~24 h) of a wide range of molecular, physiological and behavioral functions. Circadian clocks are essential for health. In humans, their disruption (e.g. caused by shift work, jet lag, etc.) has been associated with the development of multiple pathologies (e.g. cancer, metabolic diseases such as diabetes and obesity, as well as cardiovascular and neurodegenerative diseases). In the European Union (EU) where these diseases are widespread (cardiovascular disease being the leading cause of death with 35% of all deaths, and cancer the second with 26% - according to the OECD study, 2019), understanding the basic mechanisms underlying circadian rhythmicity is therefore crucial for the development of healthcare strategies.
A central aspect of molecular oscillator function is the tight regulation of circadian transcription. Over the years, several cis-regulatory proteins and enhancer elements (i.e. specific sequences located around the promoter region, e.g. E-box, RRE, D-box) have been shown to be essential for circadian transcription. However, mainly due to technical limitations, these studies have focused on a few regulators and have left many gaps in the understanding of the dynamics of circadian transcription. With recent advances in quantitative genomic-local proteomics, we saw an opportunity to overcome these limitations and sought to provide the first comprehensive and unbiased characterization of circadian protein binding to key circadian regulatory regions.
A central aspect of molecular oscillator function is the tight regulation of circadian transcription. Over the years, several cis-regulatory proteins and enhancer elements (i.e. specific sequences located around the promoter region, e.g. E-box, RRE, D-box) have been shown to be essential for circadian transcription. However, mainly due to technical limitations, these studies have focused on a few regulators and have left many gaps in the understanding of the dynamics of circadian transcription. With recent advances in quantitative genomic-local proteomics, we saw an opportunity to overcome these limitations and sought to provide the first comprehensive and unbiased characterization of circadian protein binding to key circadian regulatory regions.
A central part of the project was to transfer the recently developed quantitative genomic-local proteomics technology to the specifics of studying circadian regulatory regions. We adapted the CRISPR/Cas9-APEX2 (CASPEX) tagging method to target the circadian regulatory E-box region of the mouse Dbp gene (a known clock-controlled gene). Briefly summarized, this method utilizes CRISPR-based genome targeting properties, where guide RNAs can be designed to bring a CAS9 protein fused with a peroxidase (APEX2) to a genetic region of interest (in this case the E-box of Dbp); the peroxidase can then be triggered to tag proteins near the E-box. Finally, the tagged proteins are enriched for analysis by quantitative proteomics.
By developing a panel of immunodetection protocols, we have optimized protein labeling and enrichment in our NIH3T3 Dbp-array mouse fibroblast model (chosen for its increased Dbp gene copy number). In collaboration with experts in liquid chromatography mass spectrometry, we performed proteomic analysis of samples where guide RNAs were expressed and bring CASPEX to the E-box. As a control, samples were prepared where CASPEX is expressed but not specifically addressed at a locus. In addition, samples were prepared either during the active or repressive phase of circadian transcription. Statistical analysis for the control samples provided a list of proteins specifically localized to E-boxes and included known clock proteins (i.e. BMAL1, PER1). In parallel with the preparation of samples spanning a complete circadian cycle, we explored the significance of novel candidate proteins for the human circadian oscillator. We used a high-throughput RNAi-based screening system established by the host team to evaluate which newly discovered candidate proteins are required for the maintenance of normal circadian rhythmicity.
At the European Biological Rhythms Society Congress, 2022, the international community showed great interest in the project and its development; and in agreement with the host team, further work is underway to publish a comprehensive description of the circadian chromatin landscape based on the results obtained during this funding period.
By developing a panel of immunodetection protocols, we have optimized protein labeling and enrichment in our NIH3T3 Dbp-array mouse fibroblast model (chosen for its increased Dbp gene copy number). In collaboration with experts in liquid chromatography mass spectrometry, we performed proteomic analysis of samples where guide RNAs were expressed and bring CASPEX to the E-box. As a control, samples were prepared where CASPEX is expressed but not specifically addressed at a locus. In addition, samples were prepared either during the active or repressive phase of circadian transcription. Statistical analysis for the control samples provided a list of proteins specifically localized to E-boxes and included known clock proteins (i.e. BMAL1, PER1). In parallel with the preparation of samples spanning a complete circadian cycle, we explored the significance of novel candidate proteins for the human circadian oscillator. We used a high-throughput RNAi-based screening system established by the host team to evaluate which newly discovered candidate proteins are required for the maintenance of normal circadian rhythmicity.
At the European Biological Rhythms Society Congress, 2022, the international community showed great interest in the project and its development; and in agreement with the host team, further work is underway to publish a comprehensive description of the circadian chromatin landscape based on the results obtained during this funding period.
Through this project, the candidate has successfully adapted for the first time a novel genomic locus proteomic method to the study of the daily change in protein binding. Thus, the method will allow a better understanding of the dynamic changes that regulate gene transcription, and can be used to understand the integration at the transcriptional level of external signals through signaling pathways. Specifically for the understanding of circadian enhancer regions, the project has identified a selection of proteins that are part of the protein complexes regulating circadian transcription on the E-boxes. Data research on these identified proteins showed that several of them are involved in oncogenic or neurodegenerative diseases. During the funding period, the candidate developed strategies to explore the connection of these candidate proteins with circadian oscillation and pathological development. In ongoing work with the host institute, the candidate will continue to characterize the function of these promising protein candidates.