Investigating the molecular role of transcription factor MAFG in circadian rhythm regulation

The molecular mechanisms underlying circadian photoentrainment are complex, comprising several regulatory factors. There is evidence to suggest differential chromatin accessibility in the master clock, SCN, in response to light at different times of the day. Several genes identified close to differential regions of chromatin accessibility are already known to play an important role within the molecular clock and are recognized to be important for entrainment. Previous studies in the lab using ATAC-seq (Assay for Transposase Accessible Chromatin) showed that chromatin near the gene Mafg (MAF BZIP Transcription Factor G) closed following a nocturnal light pulse (LP), indicating repression of the gene. Additionally, the response element to which MAFG binds, which is the AP-1 motif, was highly over-represented in the light regulated accessible chromatin profile, leading us to hypothesise that MAFG, that is part of the AP-1 transcription factor family, regulates photoentrainment through the regulation of AP-1. To investigate this, I conducted in vitro and in vivo analyses to evaluate MAFG function in circadian entrainment. Sections of the Mafg promoter that responded by closing in response to light were cloned into a luciferase reporter plasmid and transfected into U2OS cells. Forskolin (which simulates a LP via the AP-1 pathway) reduced reporter expression, and this was further reduced in cells with siRNA- mediated Mafg knockdown. Furthermore, knockdown of Mafg in BMAL1:Luc U2OS circadian reporter cells showed a reduction in amplitude. In vivo, the baseline circadian phenotype, and photic entrainment phenotype of Mafg knockout mice was studied by housing Mafg+/+ and Mafg-/- mice in strict light/dark cycles. Periodogram analysis showed reduced amplitude of circadian period in knockout mice, suggesting that they have weak & fragmented rhythms. Mafg knockouts also showed slower re- entrainment in a 6-hour phase-advancing jetlag study and reduced phase shifting in response to a nocturnal LP (CT 16), indicating reduced photic responses in Mafg -/- animals. However, qPCR analysis of gene expression in the SCN isolated from CT16 light pulsed Mafg+/+ and Mafg-/- mice shows very little difference in a subset of the light regulated genes assessed between the genotypes. In summary, a novel role for MAFG in regulating circadian photoentrainment was shown, however discrepancies in the mechanism of action remain to be addressed

The Mycoplasma hyorhinis genome displays differential chromatin accessibility

Whilst the regulation of chromatin accessibility and its effect on gene expression have been well studied in eukaryotic species, the role of chromatin dynamics and 3D organisation in genome reduced bacteria remains poorly understood [1,2]. In this study we profiled the accessibility of the Mycoplasma hyorhinis genome, these data were collected fortuitously as part of an experiment where ATAC-Seq was conducted on mycoplasma, contaminated mammalian cells. We found a differential and highly reproducible chromatin accessibility landscape, with regions of increased accessibility corresponding to genes important for the bacteria's life cycle and infectivity. Furthermore, accessibility in general correlated with transcriptionally active genes as profiled by RNA-Seq, but peaks of high accessibility were also seen in non-coding and intergenic regions, which could contribute to the topological organisation of the genome. However, changes in transcription induced by starvation or application of the RNA polymerase inhibitor rifampicin did not themselves change the accessibility profile, which confirms that the differential accessibility is inherently a property of the genome, and not a consequence of its function. These results together show that differential chromatin accessibility is a key feature of the regulation of gene expression in bacteria

The Mycoplasma hyorhinis genome displays differential chromatin accessibility

Whilst the regulation of chromatin accessibility and its effect on gene expression have been well studied in eukaryotic species, the role of chromatin dynamics and 3D organisation in genome reduced bacteria remains poorly understood [1,2]. In this study we profiled the accessibility of the Mycoplasma hyorhinis genome, these data were collected fortuitously as part of an experiment where ATAC-Seq was conducted on mycoplasma, contaminated mammalian cells. We found a differential and highly reproducible chromatin accessibility landscape, with regions of increased accessibility corresponding to genes important for the bacteria's life cycle and infectivity. Furthermore, accessibility in general correlated with transcriptionally active genes as profiled by RNA-Seq, but peaks of high accessibility were also seen in non-coding and intergenic regions, which could contribute to the topological organisation of the genome. However, changes in transcription induced by starvation or application of the RNA polymerase inhibitor rifampicin did not themselves change the accessibility profile, which confirms that the differential accessibility is inherently a property of the genome, and not a consequence of its function. These results together show that differential chromatin accessibility is a key feature of the regulation of gene expression in bacteria