HDAC1 interacts with the p50 NF-κB subunit via its nuclear localization sequence to constrain inflammatory gene expression

The NF-κB p50 subunit is an important regulator of inflammation, with recent experimental evidence to support it also having a tumor suppressor role. Classically, p50 functions in heterodimeric form with the RelA (p65) NF-κB subunit to activate inflammatory genes. However, p50 also forms homodimers which actively repress NF-κB-dependent inflammatory gene expression and exert an important brake on the inflammatory process. This repressive activity of p50:p50 is thought to be in part mediated by an interaction with the epigenetic repressor protein Histone Deacetylase 1 (HDAC1). However, neither the interaction of p50 with HDAC1 nor the requirement of HDAC1 for the repressive activities of p50 has been well defined. Here we employed in silico prediction with in vitro assays to map sites of interaction of HDAC1 on the p50 protein. Directed mutagenesis of one such region resulted in almost complete loss of HDAC1 binding to p50. Transfected mutant p50 protein lacking the putative HDAC1 docking motif resulted in enhanced cytokine and chemokine expression when compared with cells expressing a transfected wild type p50. In addition, expression of this mutant p50 was associated with enhanced chemoattraction of neutrophils and acetylation of known inflammatory genes demonstrating the likely importance of the p50:HDAC1 interaction for controlling inflammation. These new insights provide an advance on current knowledge of the mechanisms by which NF-κB-dependent gene transcription are regulated and highlight the potential for manipulation of p50:HDAC1 interactions to bring about experimental modulation of chronic inflammation and pathologies associated with dysregulated neutrophil accumulation and activation

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.This study was funded by a Wellcome Trust Investigator Award (106951/Z/15/Z) and a Royal Society Wolfson Research Merit Award (WM130089) to J.M.G.H., an EPSRC DTP (Biological Informatics) PhD Studentship to M.H., a Barbour Foundation PhD Studentship to J.L.M., and by a J.G.W. Patterson Foundation grant to L.G.Peer reviewe

cRel expression regulates distinct transcriptional and functional profiles driving fibroblast matrix production in systemic sclerosis

Objectives: NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. Methods: Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. Results: cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. Conclusion: cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease

Simultaneous measurement of single-cell mechanics and cell-to-materials adhesion using fluidic force microscopy

The connection between cells and their substrate is essential for biological processes such as cell migration. Atomic force microscopy nanoindentation has often been adopted to measure single-cell mechanics. Very recently, fluidic force microscopy has been developed to enable rapid measurements of cell adhesion. However, simultaneous characterization of the cell-to-material adhesion and viscoelastic properties of the same cell is challenging. In this study, we present a new approach to simultaneously determine these properties for single cells, using fluidic force microscopy. For MCF-7 cells grown on tissue-culture-treated polystyrene surfaces, we found that the adhesive force and adhesion energy were correlated for each cell. Well-spread cells tended to have stronger adhesion, which may be due to the greater area of the contact between cellular adhesion receptors and the surface. By contrast, the viscoelastic properties of MCF-7 cells cultured on the same surface appeared to have little dependence on cell shape. This methodology provides an integrated approach to better understand the biophysics of multiple cell types.</p