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MOLECULAR MECHANISMS INVOLVED IN
THE REGULATION OF 25-HYDROXYVITAMIN D3 1£-HYDROXYLASE BY CALCITONIN AND 25-HYDROXYVITAMIN D3 24-HYDROXYLASE BY 1,25(OH)2VITAMIN D3

by
Yan Zhong
Biochemistry and Molecular Biology

BMD, 2000 Nanjing Medical University, P.R. China

Thesis Advisor: Sylvia Christakos, Ph.D.
Professor
Biochemistry and Molecular Biology

MSB E609b

Tuesday, April 7, 2009
1:00 p.m.


Abstract

Vitamin D is a principal factor required for maintaining normal calcium homeostasis (1,2). The active form of Vitamin D, 1,25(OH)2D3, is mainly catalyzed by the 25-hydroxvitamin D3-1a-hydroxylase (1a(OH)ase) in kidney.
Although parathyroid hormone (PTH) induces 1a(OH)ase under hypocalcemic conditions, previous studies showed that calcitonin, not PTH, has an important role in the maintenance of serum 1,25(OH)2D3 under normocalcemic conditions. In the first part of my thesis work, I studied the calcitonin-induced 1a(OH)ase expression in kidney cells and the mechanisms that underlie this regulation. C/EBP£] is up-regulated by calcitonin in kidney cells and results in a significant enhancement of calcitonin induction of 1a(OH)ase transcription and protein expression. The SWI/SNF chromatin remodeling complex was found to cooperate with calcitonin in the regulation of 1a(OH)ase. ChIP analysis showed that calcitonin recruits C/EBP£] ƒnƒnto the 1a(OH)ase promoter and ReChIP analysis showed that C/EBP£] and BRG1, an ATPase that is a component of the SWI/SNF complex, bind simultaneously to the 1a(OH)ase promoter. These findings are the first to address the dynamics between calcitonin, C/EBP£] and SWI/SNF in the regulation of 1a(OH)ase and provide a mechanism, for the first time, for calcitonin induction of 1a(OH)ase.
The second part of my thesis investigated the mechanisms involved in the regulation of 25-hydroxyvitamin D3 24-hydroxylase (24(OH)ase), an enzyme responsible for 1,25(OH)2D3 degradation.
We find that H3 specific lysine 9 methyltransferase G9a, that has been reported to function as a co-repressor of transcription, functions as a transcriptional activator in the 1,25(OH)2D3 regulation of 24(OH)ase. Transient transfection studies indicate that G9a in combination with GRIP1 (a p160 co-activator) and the H3 specific arginine methyltransferase CARM1/PRMT4 results in a significantly synergistic enhancement of 1,25(OH)2D3 induced 24(OH)ase transcription. Studies using mutants of CARM1 or G9a suggest that the methyltransferase activity of CARM1 or G9a contributes to the synergistic coactivator function. In addition studies using the GRIP1 mutant which lacks the binding site for CARM1 indicate a critical role of GRIP1 in recruiting secondary coactivators for VDR mediated transcriptional activation. Results of ChIP assays indicate that G9a is associated with the 24(OH)ase VDRE. These findings suggest that the histone modification induced by GRIP1 can result in G9a acting as a transcriptional activator and that cooperativity between histone methyltransferases and p160 co-activators may play a fundamental role in VDR mediated transcriptional activation.


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