| Abstract |
The MHCII family of genes is of central importance to the development and function of the adaptive immune system. Transcription of all members of the family is controlled by the Class II Transactivator, CIITA, and a set of DNA-binding factors, among which RFX5, the main subunit of the RFX trimer, is the most important. Previous work in our lab had shown that RFX5 is subject to arginine methylation by the PRMT family of methyltransferaces. The aim of this work was to map the arginine residues-targets of methylation and to investigate the biological significance of these methylations.
The initial phase of this work consisted of methylation mapping, and was done in two stages: First, the regions that contained the arginines-targets of methylation were determined, by subsequent rounds of cloning, expressing and methylating gradually smaller regions of RFX5. The exact arginine residues-targets were then determined by single aminoacid mutagenesis of all the candidate arginine residues. This mapping revealed that methylation targets are located in specific regions of RFX5. Their importance was assessed during the second phase of the project, using both experimental and bioinformatic approaches, the latter revealing the existence of an AT-hook, an auxiliary DNA-binding domain, on RFX5. The existence and function of this domain were previously unknown. On the contrary, AT-hooks are known targets of arginine methylation by PRMT6. The last part of this work focused in investigating the newly identified AT-hook’s contribution to MHCII transcription, and the role of PRMT6, if any, in its’ regulation. To this end, we created and analysed stable cell lines using either the wild type RFX5, or a version bearing an AT-hook deletion (RFX5-Δhook). PRMT6 effect was then studied using in vivo overexpression experiments.
Our results show that the AT-hook function varies depending on the MHCII isotype examined. PRMT6 effect, which is mediated by the AT-hook, also differs
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depending on the isotype examined. PRMT6 effect thus constitutes the first discovered mechanism by which differential regulation of the MHCII genes can be achieved under normal conditions, a discovery that apart from its’ mechanistic interest has also immunological implications and potential clinical applications.
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Διαφορική ρύθμιση των γονιδίων του μείζονος συμπλόκου ιστοσυμβατότητας τάξης ΙΙ από τη μεθυλοτρανσφεράση PRMT6, μέσω μιας ΑΤ-hook, μιας νέας πρωτεϊνικής επικράτειας του RFX5
