Dominika LETKOVÁ will defend her PhD in Biology, prepared under the supervision of Kiran PADMANABHAN.

Abstract

In mammals, robust circadian clocks coordinate gene-expression programs in a tissue-specific manner. Rhythmic binding of the core-clock transcription factors (TF) BMAL1-CLOCK on chromatin results in a ‘day to night’ dynamics in chromatin states and in turn, ~10-15% of transcriptome in every tissue is cycling. In order to allow BMAL1-CLOCK rhythmic binding at ‘specific time and place’ on the genome, chromatin has to be accessible and such dynamic changes in chromatin states epigenetically encoded. Therefore, how chromatin architecture gets reorganized upon BMAL1-CLOCK binding, remains an active field of research.

Over the past decades, it has been shown that circadian clock machinery is tightly linked with dynamics in chromatin landscapes. Deposition of epigenetic marks, such as acetylation or methylation of histone proteins, nucleosome remodeling and circadian gene expression are closely coordinated. Core-clock factors are directly involved in the recruitment of chromatin-modifying enzymes and deposition of epigenetic signatures, including histone variants, such as H2A variant, H2A.Z. Dynamic incorporation and/or eviction of H2A.Z within nucleosomes near transcription start sites (TSS) is highly concomitant with incorporation of the H3 histone variant, H3.3. In dividing cells, it has been shown that H3.3/H2A.Z histone variants are incorporated within the same nucleosomes at promoters and TSS, generating extremely labile, easily removable dual-variant nucleosomes. Nevertheless, while H2A.Z role in 24hr rhythms and chromatin dynamics is well established across species, the role of H3.3 in clock function remains unknown.

Therefore, we aimed to study the link between the histone variant H3.3 and the molecular machinery of mammalian circadian clocks. In other words, how could H3.3 nucleosomes shape the chromatin architecture to contribute to 24hr transcriptional dynamics in close connection with circadian core-clock regulators in murine tissues, such as liver. Purification of specific H3.3 liver protein complexes over circadian time allowed us to identify dynamic assembly of PBAF/cBAF chromatin remodelers with the core-clock TF BMAL1 on H3.3 variant chromatin. We found that H3.3 deposition is cycling over time, and PBAF/cBAF-BMAL1 assemble on dynamic H3.3 nucleosomes marked with specific epigenetic marks such as H3K115ac and H3K122ac, resulting in their fragility. Furthermore, circadian clock disruption (Per knock-out mouse models) results in a higher loading of H3.3 on chromatin and maintenance of fragile H3.3 ‘day-time-like’ epigenetic landscapes. Moreover, near-total depletion of ARID2 and BRG1 shared ATPase subunits respectively lead to disassembly of PBAF and reorganization of cBAF chromatin remodelers, followed by a ‘switch’ to cBAF/BRM complexes. In addition, the absence of circadian feedback results in increased BMAL1 binding on chromatin due to the higher accessibility of BMAL1-CLOCK binding sites. All of these events highlight a permanently active ‘day-time like’ chromatin state upon circadian disruption, implicating H3.3.

Last, but not least, circadian disruption is implicated in several disorders such as metabolic syndrome and sleep loss, neurodegenerative diseases and cancer, all of which have a strong epigenetic component, including mutations in H3.3 and chromatin remodelers. Better understanding of the physiological link between chromatin dynamics and circadian transcription would allow better understanding of disease development and its progression, as well as an aid in the development of novel therapeutic approaches.