R, Kirtana (2024) Roles of KDM5A and MLLs in Modulating Cellular Processes with Respect to Reversible H3K4me3 Status. PhD thesis.
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Abstract
Differential expression of genes involved in certain processes is a collaborative outcome of crosstalk between signalling molecules and epigenetic modifiers, which together dictate the regulation of genes in response to environmental stimuli. Epigenetic mechanisms involve changes in gene expression by modulating the chromatin configuration. Epigenetic modifiers that set in a euchromatin led to gene activation and those that participate in heterochromatinization will repress gene expression. These changes in nucleosome architecture are due to addition or removal of small chemical groups which either increase the affinity of DNA and histones or weaken their interactions. Here, in this study, we attempt to understand the dynamics of one such modification – the H3K4me3, which is an activation signal. The expression of its writer – MLL and eraser – KDM5A protein, was varied using siRNA and overexpression constructs. To strengthen our study, we utilized both knockdown and transient overexpression approaches to decode the exact mechanism by which H3K4me3 modification regulates different cellular processes. EMT is a multistage process and many transcriptional factors and co-factors play a role in regulating this process. MLLs and KDM5A are functionally antagonistic proteins as one acts as writer and the other erases the active chromatin mark, i.e., H3K4me3. KDM5A promotes EMT by occupying promoters of both epithelial and mesenchymal markers. Through this work, we show that when bound to E-cadherin promoter, KDM5A acts as a classical repressor by demethylating H3K4me3, but on mesenchymal marker promoters, it acts as a transcriptional activator by inhibiting the activity of HDACs and increasing H3K18ac. Further we report that when enzymatically inactive, KDM5A occupancy on E-cadherin enhances either MLL1 or MLL2 by physically interacting with them and that signalling pathways like ITG-FAK may regulate the enzymatic activity of KDM5A by phosphorylation. We further show that KDM5A is a part of COMPASS complex as it interacts with MLL1, MLL2 and WDR5. Metabolic plasticity is a key for tumor survival in the dynamic microenvironment, and this process involves shifting gene expression from a certain phenotype to one that supports better adaptability. Epigenetic regulation is one way of attaining this transformation. Here, we investigated if glucose metabolic gene regulation is dependent on KDM5A and MLL1, and if targeting the H3K4me3 would help in modulating the resilience of cancer cells. We present that KDM5A modulates most of the metabolic genes in a demethylase dependent manner as assesses by H3K4me3 occupancy on G6PD and catalase promoters. Chemo-preventive active compounds found in medicinal plants were shown to epigenetically alter cancer cells without affecting normal cells, thus we tested if phytochemicals rewire the epigenetic state of cancers cells to regulate metabolic genes. From our investigation, we understood that curcumin could enhance KDM5A and reduce MLL2 expression in both cancer cell lines (HeLa and PC3) leading to a state of silencing on metabolism related genes. In HaCaT cells, curcumin treatment reduced KDM5A and enhanced MLL2 thus setting a state of activation. When HeLa cells were treated with curcumin, KDM5A mediated reduction of TCA, ATP, PPP related genes was noted. Also, KDM5A mediated increase in ROS mediated apoptotic cell death was observed in HeLa, but HaCaT cells remained unaffected. Thus, we could conclude that targeting KDM5A/MLLs expression would indeed transpire cancer cells metabolism and help in sensitizing cancer cells to ROS dependent apoptotic cell death. Cell cycle progression is regulated by many extracellular stimuli and intracellular signaling, all of which converge in the nucleus. Inside the nucleus, interaction between different epigenetic modifiers and transcription factors regulate the expression of proteins involved in cell cycle control. Along with the cyclin-CDK complexes that direct the cell passage through different stages of cell cycle, the chromatin configuration also dictates the progression by monitoring the attainment of correct chromatin compaction. Here, in this study we analyzed how modulation of H3K4me3 by MLL1 and KDM5A affect cell cycle progression. Slow and fast cycling cell lines exhibited diverse mechanisms of regulation, from our in-silico screening, we understood that the expression of the MAPK effector – RAS controls the activity of KDM5A and MLL proteins to balance H3K4me3 throughout cell cycle. In fast cycling cell lines like HeLa and HaCaT, which are characterized by high RAS expression, we understand that KDM5A is active and MLL is inactive, so when KDM5A is overexpressed, it leads to an S-phase arrest due to reduced global H3K4me3 associated heterochromatinization. In contrary to this, slow cycling, low RAS expressing PC3 cell line exhibits inactive KDM5A and active MLLs, thus leading to enhanced H3K4me3 mediated loose chromatin configuration that blocks cell cycle at G2/M phase. To confirm that KDM5A/MLL catalyzed changes in global H3K4me3 was downstream effect of growth factor (GF) signaling mediated regulation of their enzyme activity, we performed KDM5A overexpression in serum deprived HeLa cells. In these conditions, we observed a G2/M arrest instead of S-phase block (as observed in normal cultured cells), proving that absence of GF mediated signaling led to inactive KDM5A and active MLLs. Further, we also tested if transient overexpression of RAS in PC3 cells could activate KDM5A thereby leading to an S phase arrest, so we overexpressed RAS and KDM5A together and noted an S-phase block instead of G2/M arrest as observed in KDM5A overexpressing PC3 cells. Concluding the study, as KDM5A is downregulated in cervical and prostate cancers, mechanisms that enhance its expression would lead to cell cycle arrest and metabolic rewiring which ultimately aid in targeting the hallmarks of cancer – i.e., high metastatic, proliferative, and metabolic adaptability phenotypes.
Item Type: | Thesis (PhD) |
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Uncontrolled Keywords: | Epigenetic signalling; KDM5A; MLLs; H3K4me3; COMPASS; HDACs; FAK signalling; Cell cycle; RAS signalling; Glycolysis; TCA cycle; PPP; Redox system; Apoptosis. |
Subjects: | Life Science > Molecular Meidicine Life Science > Immunology Life Science > Biochemistry |
Divisions: | Sciences > Department of Life Science |
ID Code: | 10616 |
Deposited By: | IR Staff BPCL |
Deposited On: | 30 Jul 2025 19:54 |
Last Modified: | 30 Jul 2025 19:54 |
Supervisor(s): | Patra, Samir Kumar |
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