Chan, E. C. | bioRxiv Science

bioRxiv2024

Transcriptomic analysis of TGFβ-mediated fibrosis in primary human Tenon's fibroblasts

Glaucoma filtration surgery (GFS) is performed to slow down disease progression in glaucoma, a leading cause of irreversible blindness worldwide. Following surgery, pathological wound healing may lead to conjunctival fibrosis and filtering failure. Myofibroblasts are the key cells responsible for postoperative conjunctival scarring. This study aims to further understand the molecular mechanisms of conjunctival fibrosis following GFS. We utilised RNA-sequencing (RNA-seq) to delineate the TGF{beta}1 induced changes in the transcriptome of human Tenons fibroblasts (HTFs). RNA sequencing was performed on HTFs after 5 days of TGF{beta}1 treatment. Following quality control, 3,362 differentially expressed genes were identified, of which 1,532 were upregulated and 1,820 were downregulated. We identified signaling pathways associated with the pathogenesis of conjunctival fibrosis. The DEGs (differentially expressed genes) were enriched in pathways including myofibroblast differentiation, TGF{beta}-signaling, collagen and extracellular matrix organization, epithelial to mesenchymal transition, and cell cycle regulation. The results of this study identified the transition from HTF to myofibroblast is characterised by the upregulation of key genes including LDLRAD4, CDKN2B, FZD8, MYOZ1, and the downregulation of SOD3, LTBP4 and RCAN2. This unprecedented insight into the transcriptional landscape of HTFs and myofibroblast differentiation is essential to understand the pathophysiology of conjunctival scarring and develop new therapeutic agents.

bioinformatics↗

bioRxiv2022

CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters

Circadian clock and chromatin remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. This conclusion is further supported by elevated BRM binding to the per promoter in flies subjected to constant light. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin remodeling complex. Author SummaryCircadian clocks are endogenous time-keeping mechanisms that allow organisms to anticipate and adapt to daily changes in their external environment. These clocks are driven by a molecular oscillator that generates rhythms in the expression of many genes, termed clock-controlled genes. The genomic DNA containing these clock-controlled genes are also modified in a rhythmic manner throughout the day. DNA are more tightly packaged with histone proteins when transcription of clock-controlled genes is repressed while the interaction between DNA and histone proteins are more relaxed during transcriptional activation. We found that two key clock proteins, CLOCK and TIMELESS, regulate daily rhythmicity in the binding of BRAHMA, a chromatin remodeler, to DNA spanning clock-controlled genes to facilitate their rhythmic gene expression cycles. Moreover, because TIMELESS is sensitive to light, our study provides new insights into how light can affect DNA structure and gene expression.

molecular biology↗