Post-translational Modification Regulation Of Transcription Factors P53 And Sox2

For a eukaryotic gene, its transcription need an active transcription factor and an "open" chromatin confirmation in its regulatory region. Nucleosomes composed of core histones octamer and surrounded DNA are the fundamental repeating units of the eukaryotic "beads-on-a string" chromatin fibre. Chromatin fibre can be compacted into more complex spatial organizations. Modification of core histones play a key role in structure regulation of chromatin. Different modifications integratively affect the interactions among nuceosomes and affinity of core histones to DNA. Compacted chromatin exhibits reduced accessibility for transcription factors. Post-translational modifications of transcription factors also modulate transcription. Poly-ubiquitination leads to transcription factors degradation; phosphorylation, acetylation and methylation sometimes affect transcription factors subcellular distribution, affinity to DNA elements or other macromolecules. Different modifications may regulate transcription factor synergistically or exclusively, all kinds of upstream signals are integrated into modulated transcription activities.We investigated covalent modifications mediated p53 activity regulation, we also explored the roles of post-tranlational modifications in pluripotency maintenance and retinoic acid induced neural differentiation of P19 cells.Part I post-translational modifications and p53 regulation.By performing [3H-methyl] labeling in vivo, methylation of p53 core domain was detected; with a specific antibody, we found arginine methylation was involved in modifications of p53 core domain. P53 R110,209,213 had been reported to be methylated in vivo by mass spectrometry analysis. In the dual-luciferase reporter assays, transcription activity of p53 with tri-valent mutation of R110,209,213 to lysines was severely reduced.Peptidyl-deimination by PAD family is another post-translational modification of arginine residues. PAD4, the only PAD resides in nucleus, deiminates histones and is implicated in chromatin regulation. We sought to figure out whether PAD4 deiminates p53 and regulates its activity. It had been reported that p53 interacts with PAD4 and recruits PAD4 to target gene promoters to modify histones, as a result target gene expressions are restrained. At first we detected whether p53 is a substrate for PAD4.We found that PAD4 deiminates p53 in vitro and in vivo, causes changed mobility of p53 in SDS-PAGE.PAD4 deimination reduces p53 acetylation in vitro.PAD4 represses p53 transcription activity in H1299 cells, a cell line doesn't express endogenous p53.PAD4 induces endogenous p53 expression in HEK293A cells.Taken together, arginine methylation takes part in p53 regulation. PAD4 regulates p53 in three possible ways:direct deimination of p53; deimination of histones in p53 target promoters; induction of endogenous p53 expression.Part II To get a insight into the contribution of protein methylation to pluripotency maintenance and neural differentiation, we treated P19 cells with a methyltransferase inhibitor AdOx. Results are listed below.1. AdOx reduces P19 neural differentiation.2. AdOx impaires P19 cells pluripotency maintenance.3. AdOx causes P19 cells morphological change and blocked cell cycle.4. AdOx differentially inhibits histone methylations.5. AdOx upregulates transcription of pluripotency related genes(Utfl, Fgf4, Sox2, Nanog).6. AdOx reduces Sox2 transcription activity, alters Sox2 subcellular distribution, inhibits the dynamic association of Sox2 with chromatin.7. Protein arginine methyltransferase Carm1 interacts and methylates Sox2 in vitro and in vivo.8. Carml methylates Sox2 preferentially at R113.9. Carml enhances Sox2 transcription activity. R113,115,116K tri-valent mutation reduces the inhibition of Sox2 activity by AdOx. 10. p300 interacts and acetylates Sox2, stimulations of Sox2 activity by Carml and p300 are compatible; Akt negatively regulates Sox2 activity; Carml can prevent Sox2 from site-specific proteolytic cleavage.Taken together:protein methylation is essential for P19 cells pluripotency maintenance and neural differentiation; arginine methylations modulate Sox2 transcription activity; different post-translational modifications jointly regulate Sox2.