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Abstract:
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Phenobarbital (PB) is the prototype for a class of agents that produce marked induction in the genetic expression of a variety of metabolic enzymes in many species. One of the consequences of this induction is a change in the metabolic fate and disposition of xenobiotics, which in turn influences their pharmacologic efficacy or toxicity. The biological mechanisms effecting these induction processes are not understood. The cytochromes P450 (CYP) 2B1 and CYP2B2 are the major PB-inducible genes in the rat, and have been the most intensively studied. Although PB-induction of these genes occurs via an increase in gene transcription initiation, cis-acting DNA sequences involved have not been identified. This is largely due to the difficulty of maintaining PB-inducibility in cultured cells. The aim of this dissertation research is to identify and characterize cis-acting elements involved in regulating CYP2B1/2B2 expression.In the initial phase of this research transgenic mice carrying rat CYP2B2 gene constructs were analyzed. Transgenes lacking the section of the CYP2B2 gene between $-$20 and $-$.8kb were expressed at high levels regardless of PB exposure. This suggests a cis-acting element 5$\sp\prime$ of $-$.8kb represses CYP2B2 transcription, which is reversed in the presence of PB. Transient transfection studies also provided evidence of repressor elements regulating CYP2B1/2B2 genes. Successive 5$\sp\prime$ deletions of their flanking sequences increased CAT reporter gene expression, indicating that multiple repressor elements reside in the 5$\sp\prime$ sequence. A number of in vitro methods were used to analyze protein-DNA interactions occuring along the CYP2B1/2B2 5$\sp\prime$ sequence. Proteins binding a recognition site at $-$2.2kb were further characterized according to their binding site affinity and immunoreactivity to antibodies raised against known transcription factors and specific phospho-amino acids. This element was identified as a recognition motif for the transcription factor NF-1. The NF-1 binding site is present in CYP2B2 DNA identified by others as required for PB-inducibility, suggesting an important role for this protein in the PB induction response. NF-1 binds in both control and PB-induced states, but has a higher affinity after PB treatment. Increased binding is not correlated with NF1-L transcript levels nor differential reactivity to anti-phospho-amino acid antibodies. |