|M.Sc Student||Zoabi Muhammad|
|Subject||Proteins of the PRC2 Complex are Degraded by the Ubiquitin-|
|Department||Department of Medicine||Supervisor||? 18? Aaron Ciechanover|
|Full Thesis text|
Polycomb repressive complex 2 (PRC2), a histone methyltransferase, is capable of methylating lysine 27 on histone H3 leading to transcriptional repression and silencing of its target genes. PRC2 is composed of the three polycomb proteins EZH2, EED, and SUZ12. Knockdown of any of the three PRC2 members by siRNA results in a dramatic decrease in the levels of the other two members. Inhibition of the proteasome was shown to prevent the loss of these proteins following siRNA treatment, suggesting that they are rapidly degraded by the ubiquitin-proteasome system (UPS) when they are not in the context of the PRC2 complex. These findings brought us to test the ability of the PRC2 proteins to be ubiquitinated and degraded by the proteasome, in vitro and in cells. Interestingly, it was recently shown that chemical inhibition of the methyltransferase component EZH2 by DZNep (3-Deazaneplanocin A) resulted in the loss of all the three PRC2 components. In this study we show that treatment of MCF-7 cells with DZNep causes a depletion of the PRC2 proteins that depends on proteasomal activity. Gene array analysis that followed the disruption of the activity of PRC2 complex by DZNep suggests that among the PRC2 repressed genes there are also the ubiquitin ligases that target this complex components for degradation. Such a negative feedback loop is known for transcriptional activators like p53 that induces the expression of its ubiquitin ligase, Mdm2, but no such loop is known for transcriptional repressors. We have identified several proteins that contain RING finger domains among the PRC2 repressed genes. These genes are putative PRC2 ubiquitin ligases. Here we show that silencing of the E3 ligase PRAJA1, but not, of the other putative E3 ligases TRIM47 and KIAA1542, inhibits EZH2 depletion after DZNep treatment. In addition, we demonstrate that PRAJA1 mediates its autoubiquitination, besides mediating PRC2 components ubiquitination and degradation. We show the ability of PRAJA1 to mediate ubiquitination and degradation of PRC2 proteins in vitro, in addition to its ability to decrease EZH2 protein in cells. We still need to test the ability of PRAJA1 to affect the ubiquitination of EZH2 in cells.