The role of ubiquitin in erythroid terminal differentiation
Ganapathy Subramanian, Balakrishnan
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Ubiquitin is a major protein that has been highly conserved in all eukaryotic organisms and is involved in various cellular processes such as metabolic homeostasis, organelle biosynthesis, cell cycle regulation, DNA repair, apoptosis, antigen processing and gene expression. Ubiquitination refers to the process by which ubiquitin attaches to a protein and marks it for its degradation or modifies the function of the protein. The cell nucleus undergoes a number of changes during the process of erythroid terminal differentiation (ETD), which includes a condensation of chromatin and gene inactivation. It has been hypothesized that ubiquitination of nuclear proteins such as histones H2A and H3 may play an important role in the modification of chromatin structure during ETD, and that the ubiquitin conjugating enzyme HR6B is responsible for ubiquitination of these histones. The overall distribution and intracellular localization of the HR6B enzymes was to be documented using techniques such as western blotting using newly generated polyclonal antibodies. These turned out to be unsuccessful due to the fact that wild type HR6B could not be detected with the newly generated polyclonal antibodies. Studies were also carried out to identify which histones were ubiquitinated during ETD using antibodies specific to histones H2A and H3 and ubiquitin on western blots. The western blots showed that histones H2A get mono-ubiquitinated. However, there was still some ambiguity in terms of which histone, H2A or H3, is di-ubiquitinated when the blots were re-probed with anti-ubiquitin antibodies. Another aim was to identify which other genes not previously studied, if any, involved in the ubiquitin proteasome pathway are up-regulated during the erythroid terminal differentiation using PCR arrays. It was found that the HR6B gene was up-regulated about 19 fold, while another gene E2-20K, previously studied in our lab, was found to be up-regulated about 68 fold. These results correlated with our hypothesis that HR6B was expressed during ETD using independent techniques such as northern blotting which showed about 4 fold up-regulation. This project is significant in that it will lead to a better understanding of the mechanisms involved in ETD.