The overall goal of this laboratory is to understand the regulation of polycyclic hydrocarbon (PAH)- inducible expression of the cytochrome P4501A1 gene (CYP1A1). This regulation appears to be mediated by cis elements associated with the gene and trans-acting proteins. PAHs such as benzo(a)pyrene (B[a]P) can interact with high affinity and in saturable manner with a rat liver cytosolic 4S protein that has previously been shown to be identifical with glycine N-methy1transferase (GNMT). Our preliminary results indicated that B[a]P can induce the expression of CYP1A1 in the Ah receptor (AhR)-null mouse and that this PAH (as well as several others) induce CYP1A1 in CHO cells that have been stably transfected with the GNMT gene (TCDD) is ineffective in this system). The parent CHO cells have no GNMT, AhR, or Arnt expression while the transfectants elaborate only GNMT. Building on these observations, the specific aims of the current grant are to determine: a) the effects of B[a]P and 3-methylcholanathrene upon expression of CYP1A1 and upon the rate of its transcription in the livers of the Ah receptor- minus knockout mouse and the level of liver GNMT, b) the effects of introducing the GNMT coding sequence into B[a]P-nonresponsive CHO cells by measuring the steady-state CYP1A1 mRNA level and its rate of transcription before and agter B[a]P treatment and by measuring the stability of the GNMT and its mRNA, c) the site of phosphorylation of GNMT, the distribution of unphosphorylated and phosphorylated forms in cytosol and nucleus before/afterB[a]P, the effects of phosphorylation upon function of GNMT and the nature of its oligomeric form, d) the dimer-tetramer transition of GNMT by sedimentation analysis and by chemical crosslinking, and e) nature of the interaction of phosphorylated (and unphosphorylated) 4S GNMT with cis elements of CYP1A1, through the use of in vitro nuclear transcription techniques with truncated templates. We have already demonstrated that the 4S PAH-binding GNMT is a phosphorylated protein with the probable sites of phosphorylation represented by serine and/threonine. Upon definition of the specific amino acids, site-specific mutagenesis of these amino acids will be accomplished (by mutation to alanines). The hypothesis under test is that phosphorylation of the 4S protein leads to enhanced function as a PAH-binder, to increased translocation into the nucleus and to increased transcriptional activation.
