Scottish Centre for Regenerative Medicine

Acetylation of lysine residues in the tail domain of histone H3 is well characterised, but lysine residues in the histone globular domain are also acetylated. Histone modifications in the globular domain have regulatory potential because of their impact on nucleosome stability but remain poorly characterised. In this study, we report the genome-wide distribution of acetylated H3 lysine 115 (H3K115ac), a residue on the lateral surface at the nucleosome dyad, using chromatin immunoprecipitation. In mouse embryonic stem cells, we find that detectable H3K115ac is enriched at the transcription start site of active CpG island promoters, but also at polycomb-repressed promoters prior to their subsequent activation during differentiation. By contrast, at enhancers, H3K115ac enrichment is dynamic, changing in line with gene activation and chromatin accessibility during differentiation. Most strikingly, we show that H3K115ac is detected as enriched on ‘fragile’ nucleosomes within nucleosome-depleted regions at promoters and active enhancers, where it coincides with transcription factor binding, and at CTCF-bound sites. These unique features suggest that H3K115ac correlates with, and could contribute to, nucleosome destabilisation and that it might be a valuable marker for identifying functionally important regulatory elements in mammalian genomes.

Background:Autologous buccal mucosa cell transplantation has emerged as a promising treatment strategy for urethral stricture disease. However, ambiguity has persisted regarding the optimal cell type and culture conditions that aid successful urethral repair. Clinical study of our previously reported cell-based endoscopic approach, the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS), demonstrated durable epithelial regeneration and long-term urethral patency. The present work provides mechanistic insights supporting the BEES-HAUS approach of combining two-dimensional (2D) monolayer-cultured fibroblast-like cells and three-dimensional (3D) thermo-responsive gelation polymer (Festigel)-cultured cells. Methods:Human buccal tissues (n=22) were cultured in two methods; one portion using the monolayer method (2D), and the other in 3D using Festigel. Flow cytometry for phenotype markers and ELISA for IGF-1 were carried out. Results:3D Festigel-cultured cells acquired an epithelial phenotype, with AE1/AE3 expression up to day 21, while 2D cultures yielded fibroblast-like CD140b-positive/AE1-AE3-negative cells. IGF-1 secretion was significantly higher in 2D cultures than 3D Festigel (p < 0.05), indicating a supportive paracrine role. These findings explain the complementary contribution of epithelial integration and IGF-1-mediated support observed as successful clinical outcome of the BEES-HAUS procedure. Conclusion:This study, a first of its kind, clarifies the rationale and advantages of combining 3D Festigel-expanded epithelial cells with the paracrine effect of IGF-1-secreting 2D fibroblast-like cells in a single transplantation strategy, thereby explaining the successful clinical outcomes reported in BEES-HAUS. Further research on this hybrid cell combination is recommended to expand this approach for regenerating and repairing other tissues and organs.

Genomic instability and inflammation are distinct hallmarks of aging, but the connection between them is poorly understood. Here we report a mechanism directly linking genomic instability and inflammation in senescent cells through a mitochondria-regulated molecular circuit involving p53 and cytoplasmic chromatin fragments (CCF) that are enriched for DNA damage signaling marker γH2A.X. We show that p53 suppresses CCF accumulation and its downstream inflammatory phenotype. p53 activation suppresses CCF formation linked to enhanced DNA repair and genome integrity. Activation of p53 in aged mice by pharmacological inhibition of MDM2 reverses transcriptomic signatures of aging and age-associated accumulation of monocytes and macrophages in liver. Mitochondrial ablation in senescent cells suppresses CCF formation and activates p53 in an ATM-dependent manner, suggesting that mitochondria-dependent formation of γH2A.X + CCF dampens nuclear DNA damage signaling and p53 activity. These data provide evidence for a mitochondria-regulated p53 signaling circuit in senescent cells that controls DNA repair, genome integrity, and senescence- and age-associated inflammation, with relevance to therapeutic targeting of age-associated disease.

Duchenne muscular dystrophy (DMD): is a rare, life-limiting genetic disorder for which no curative treatment currently exists. While various gene therapy approaches, some approved and others still under clinical investigation have been explored, they have not consistently produced the desired outcome and, in some cases, have been associated with serious adverse effects, including mortality. A critical factor we wish to highlight is the hostile inflammatory environment inherent to skeletal muscles’ pathology in DMD, which may be further aggravated by gene therapy, either due to the viral vector used or the gene component itself. Therefore, a comparative and detailed evaluation of inflammatory biomarkers between control and treatment arms in such clinical trials is essential to determine whether therapeutic benefits are being compromised by inflammation. Based on the implications of such hostile environment on the therapeutic outcome, adding a safer and efficacious management strategy to mitigate the inflammation during gene therapies is considered indispensable. Therefore, we recommend further research on adjuvant anti-inflammatory approaches to ensure safety and improvement of the therapeutic outcome of gene therapies for DMD.