Biology subjects

Kitchen, G. B.

Publications and source records attributed to Kitchen, G. B..

3 recordsLinked to original sources

bioRxiv2021

Circadian clock function does not require the histone methyltransferase MLL3

AO_SCPLOWBSTRACTC_SCPLOWThe circadian clock controls the physiological function of tissues through the regulation of thousands of genes in a cell-type specific manner. The core cellular circadian clock is a transcription-translation negative feedback loop, which can recruit epigenetic regulators to facilitate temporal control of gene expression. Histone methyltransferase, mixed lineage leukemia gene 3 (MLL3) was reported to be required for maintenance of circadian oscillations in cultured cells. Here, we test the role of MLL3 in circadian organisation in whole animals. Using mice expressing catalytically inactive MLL3, we show that MLL3 methyltransferase activity is in fact not required for circadian oscillations in vitro in a range of tissues, nor for maintenance of circadian behavioural rhythms in vivo. In contrast to a previous report, loss of MLL3-dependent methylation did not affect global levels of H3K4 methylation in liver, indicating substantial compensation from other methyltransferases. Further, we found little evidence of genomic repositioning of H3K4me3 marks. We did, however, observe repositioning of H3K4me1 from intronic regions to intergenic regions and gene promoters, however there were no changes in H3K4me1 mark abundance around core circadian clock genes. Output functions of the circadian clock, such as control of inflammation, were largely intact in MLL3-methyltransferase deficient mice, although some gene specific changes were observed, with sexually dimorphic loss of circadian regulation of specific cytokines. Taken together, these observations call for a major reassessment of the inter-relationship between the circadian clock and MLL3-directed histone methylation, and a deeper examination of other epigenetic mechanisms which may facilitate circadian clock function. SO_SCPLOWIGNIFICANCEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSTATEMENTC_SCPLOWA highly cited paper published in PNAS previously reported an essential role for the histone methyltransferase MLL3 in maintaining circadian oscillations in cultured cells. We tested the role of MLL3 in vivo and in primary tissues showing that MLL3 in fact plays no role in organising the core circadian clock, and has no functional impact on whole animal circadian behaviour. However, in further analysis, we newly discover a role for MLL3 in conferring circadian control to components of the inflammatory response, doing so in a sexually dimorphic manner. As the MLL family of histone methyltransferases are being targeted by pharmaceuticals for cancer, it is important to understand how methyltransferases may be driving circadian rhythms in gene expression.

molecular biology↗

bioRxiv2020

Ezh2 restrains macrophage inflammatory responses, and is critical for neutrophil migration in response to pulmonary infection.

Mucosal immunity is critical to survival, with huge attention at present due to the Coronovirus pandemic. Epigenetic factors are increasingly recognized as important determinants of immune responses, and EZH2 closest to application due to the availability of highly-specific and efficacious antagonists. However, very little is known about the role of EZH2 in the myeloid lineage, with some conflicting reports. Here we show EZH2 acts in macrophages to limit inflammatory responses to activation, and selective genetic deletion results in a remarkable gain in protection from infection with the prevalent lung pathogen, pneumococcus. In contrast, EZH2 is required for neutrophil chemotaxis, and animals lacking neutrophil EZH2 show increased susceptibility to pneumococcus. In summary, EZH2 shows complex, and divergent roles in different myeloid cells, likely contributing to the earlier conflicting reports. Compounds targeting EZH2 are likely to impair mucosal immunity, however, may prove useful for conditions driven by pulmonary neutrophil influx, such as adult respiratory distress syndrome (ARDS). DigestEpigenetic control of mucosal immunity is important, and has translational relevance with the advent of inhibitor drugs now in the clinic for cancer indications. Here we show divergent role for EZH2 in macrophages and neutrophils. Loss of EZH2 in macrophages results in a gain of inflammatory and immune function, and protection from pneumonia. However, EZH2 is required for neutrophil chemotaxis, resulting in impaired anti-bacterial defence. We show that inhibition, or loss of EZH2 in macrophages results in a gain of immune function, with increased responses to infectious mimics such as LPS. However, the impact was far more dramatic in-vivo, with striking protection from the consequences of infection with pneumococcal bacteria. Loss of EZH2 resulted in a gain in activity of a number of inflammatory signaling cascades, including NFkB, PPARg, and IRFs1, and 7. This widespread macrophage re-programming varied between macrophages sites of origin, with the greatest impact seen in peritoneal macrophages which resulted in emergence of a new population of MerTK low cells. In contrast, in the neutrophils loss of EZH2 greatly impairs motility, and chemotaxis. This results in dramatic impairment of immune responses to the same pneumococcal infection. Extension of these studies to the mucosal epithelium revealed that EZH2 in bronchoalveolar epithelial cells had no impact on responses to infection with influenza. Taken together EZH2 plays diverse roles in the myeloid lineage, with profound impacts on inflammatory responses. The most striking observation was the difference seen between macrophages and neutrophils. EZH2 inhibition is likely to greatly impair mucosal immunity. Impact StatementHere we show a striking, but highly cell-type specific impact of the EZH2 methyltransferase on inflammatory, and anti-infective circuits; inhibition of EZH2 in macrophages augments macrophage cytokine production, but by impairing neutrophil migration impairs anti-bacterial responses.

cell biology↗

bioRxiv2019

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however the importance of circadian mechanisms in fibrosis is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERB in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERB in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERB revealed regulation of the poorly described transcription factor TBPL1. Both REVERB and TBPL1 altered integrin{beta}1 focal adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of two human cohorts. In the UK Biobank circadian strain markers (sleep length, chronotype and shift work) are associated with pulmonary fibrosis making them novel risk factors. In a separate cohort REVERB expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERB inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, suggesting targeting REVERB could be a viable therapeutic approach.\n\nSignificanceThe circadian clock plays an essential role in energy metabolism, and inflammation. In contrast the importance of the clock in the pathogenesis of fibrosis remains poorly explored. This study describes a striking alteration in circadian biology during pulmonary fibrosis where the relatively arrhythmic alveolar structures gain circadian but desynchronous rhythmicity due to infiltration by fibroblasts. Disruption of the clock in these cells, which are not widely implicated in circadian pathophysiology, results in a pro-fibrotic phenotype. Translation of these findings in humans revealed previously unrecognised important circadian risk factors for pulmonary fibrosis (sleep length, chronotype and shift work). In addition, targeting REVERB repressed collagen secretion from human fibrotic lung tissue making this protein a promising therapeutic target.

cell biology↗