The Roles Of Histone Acetyltransferase MOF In The Regulation Of Cell Stress

When exposed to conditions of environmental stress or internal damage including genetoxic agents,heat shock,hypoxia,bacterial or viral infections, heavy metals and so on,cells will produce a series of defense or adaptive response,that is cellular stress.The cellular stress response is conserved in evolution,including a series of highly ordered events.The protective function of cellular stress is to help the cells cope with lethal conditions and promote cell survival.The major proteins involved in cell protection are stress proteins or heat shock proteins (HSPs). Their expression is increased or induced after exposure of cells to conditions of environmental stress.HSPs are highly conserved.Under normal condition,levels of HSPs protein remains low and are responsible for maintaining homeostasis.Expect for heat shock,many other biological, chemical and physical factors,such as free radicals, reactive oxygen species, inflammatory factors, radiation,can also induce the expression of HSPs.A small family of transcription factors called heat shock factors (HSFs) are the regulators of stress-inducible expression of HSP genes. They bind consensus heat shock elements (HSEs) which are located at various distances upstream of the site of 5’transcription initiation of HSP genes.The HSFs family consists of four members, including HSF1, HSF2, HSF3 and HSF4. Among them,HSF1 is a master regulator of HSPs in mammalian cells.Under non-stressed conditions, HSF1 exists as an monomer in complex with chaperones and other co-chaperones. After exposure of cells to conditions of stress HSF1 is released.After a number of post-translation modifcations the factor convert into an active form,moves toward a nucleus and binds within the promoter regions of HSP genes and induce the expression of them.MOF (also called MYST1 or KAT8) is a MYST family HAT and highly conserved in evolution.MOF is the major enzyme to catalyze acetylation of histone H4 lysine 16 (H4K16ac) and essential for the development of the embryo.MOF plays an important role in cell physiological processes, including cell proliferation, differentiation, tumor occurrence and DNA damage response and repair.Recent studies have shown that MOF directly binds to the genes required for cell cycle progression and maintain their expression. In response to stress,MOF is the regulator of transcriptional programs and an important factor for cellular stress response and adaptation.Therefore, the study of the relationships between MOF,HSF1 and HSP90 during cellular stress will help us understand the mechanism of cellular stress response deeply.ObjectiveIn this study, we subjected MCF7 cells,HCT116 cells and Hela cells to Actin-omycin D, mitomycin C,UV lights and heat stress in vitro to create a model system for investigating the trends in expression and association between MOF, HSF1 and HSP90 under adverse environmental conditions by western blotting and immuno-precipitation.We also detected the location of MOF,HSF1 and HSP90 upon normal condition and cellular stress wtih immunofluorescence staining and separation and extraction of nuclear and cytoplasmic Proteins,so as to determine their roles in cellular stress.Research Methods1.Culture human breast cancer MCF7 cells, colon cancer HCT116 cells,cervical cancer Hela cells and divide them into control group and experiment group. Cells of experiment group were treated to induce stress injury.2.After the cells were subjected to ActD(0.08μM),MMC(0.8μm),UV(40J/CM2) or heat stress at 42℃ for different periods of time,MOF and H4K16ac protein levels were detected by western blotting analysis in the normal and stressed cells.3.Interfer the expression of MOF and induce stress injury and then detect of cell morphological changes upon cell stress of low MOF cells cells compared to normal cells4.Overexpression of Mof and test cell survival rate comparing to the control group upon cellular stress by MTT.5.Separate and extract proteins of nuclear and cytoplasmic to determine the location of MOF in cells and whether it shuttle between the nucleus and the cytoplasm.6.Detect the localization and changes of MOF, HSF1 and HSP90 in nuclear by immunofluorescence staining.7.Detect the the interaction between MOF and HSF1/HSP90 on normal physiological condition or upon stress.8.Interfer the expression of MOF by siRNA,then test the location and accumulation of HSF1 and HSP90 in MOF kockdown cells upon stress.Results1.The MOF expression levels significantly increased in the cells following exposure to stess compared to the levels observed at the beginning and H4K16ac followed a similar trend in expression to MOF.2.MOF overexpression promotes cell survival upon stress while MOF konckdown increased cell death.3.MOF always localized in the nucleus during cellular stress, without shuttling between the nucleus and the cytoplasm.4.MOF gathered in the nucleus into spots which look like stress bodies.HSF1 is the marker of stress bodies.5.Results of immunofluorescence staining indicated that both MOF and HSF1 accumulated in nucleus and colocalized upon cellular stress.6.MOF interact with HSF1 and HSP90 during cellular stress or under normal condition,while HSF1 and HSP90 interact with each other only under physiological state.7.Expression of HSF1 and HSP90 protein was not influenced while accumulation of HSF1 and HSP90 into stress bodies was abolished upon MOF knockdown.ConclusionsMOF is an important factor of transcriptional regulation in stress response.HSF1 transactivate a series of genes involved in cell stress response and a maker of nuclear stress bodies.Our studies reveal that MOF interacts with HSF1 and HSP90 and always located in the nucleus.MOF along with HSF1 and HSP90 form stress bodies in response to cellular stress.Our experiment has a certain value in the further understanding of the mechanism of cellular stress response.