|Institution:||Case Western Reserve University|
|Keywords:||Biochemistry; Molecular Biology; Physiology; Cellular Biology; PRMT5; arginine methylation; SDMA; NF-kappaB; p76; post-translational modification; endothelial cell; inflammation; CXCL10; IP-10; CXCL11; I-TAC; TNF-a; IFNg; transcription|
|Full text PDF:||http://rave.ohiolink.edu/etdc/view?acc_num=case1449579234|
Inflammatory agonists differentially activate gene induction of chemokinesin endothelial cells (EC). The molecular mechanisms that produce distinctchemokine induction profiles following EC activation are incompletelyunderstood. The chemokines CXCL10/IP-10 and CXCL11/I-TAC both facilitateTh1-type leukocytes recruitment to inflammatory lesions. CXCL10 and CXCL11are strongly induced by IFN-gamma (IFNg). However, CXCL11 expression is not triggered byTNF-a (TNF) exposure, whereas TNF potently induces the CXCL10 promoter.Both genes display strong synergistic expression upon simultaneous exposure toTNF and IFNg. In these studies we show that the arginine methyltransferasePRMT5 is critical in the expression of CXCL10 by TNF and CXCL11 in responseto co-stimulation with TNF and IFNg, but that PRMT5 operates throughdistinctive mechanisms to achieve activation of these promoters. Chromatinimmunoprecipitation experiments revealed that proteins containing thesymmetrical dimethylarginine (SDMA) modification catalyzed by PRMT5 arefound on each promoter following stimulation with pro-inflammatory agents.Using immunoblotting and mass spectrometry approaches we found that NF-kappaBp65 (p65), a key transcription factor critical for both CXCL10 and CXCL11induction, contains PRMT5-catalyzed dimethylarginine. PRMT5-mediatedmethylation is required for p65 association with each promoter.A series of experiments revealed that methylation of different arginineresidues on p65 is required for induction of CXCL10 versus CXCL11. CXCL10induction requires methylation of Arg30 and Arg35 in response to TNF. In contrast,methylation of p65 at Arg174 is necessary for CXCL11 induction in EC costimulatedwith TNF and IFNg. Arg30, Arg35, and Arg174 are located in the relhomology domain of p65 but are present in regions associated with differentbiochemical functions. Arg30 and Arg35 are part of the DNA-binding domain.Methylation of these residues probably enhances p65 DNA binding affinity. Arg174is found in a domain involved in protein-protein interactions. Therefore,methylation of Arg174 is likely to promote an association between p65 and acoactivating component of the CXCL11 transcription complex.In conclusion, I show that PRMT5 is a central player in chemokine geneexpression by catalyzing the methylation of multiple arginine residues in p65.Methylation of p65 at specific sites comprises a code that enables uniquechemokine gene expression profiles in EC. Advisors/Committee Members: Nosek, Thomas (Committee Chair), DiCorleto, Paul (Advisor).