Transcriptional regulation of the Menkes Copper Atpase (Atp7a) gene by iron.

Abstract

Copper and iron homeostasis‐related genes, Atp7a, metallothionein 1a (Mt1a) and divalent metal transporter 1 (Dmt1) are strongly upregulated in the intestine of iron‐deficient rats. A strong downregulation of hepatic hepcidin expression (>1500‐fold) was also noted. Atp7a and Dmt1 are induced throughout the length of the gut. We have modeled this observation in vitro in IEC‐6 cells, where both genes are induced by iron chelation. Utilizing Atp7a‐specific antibodies, we demonstrate that protein expression is also increased by iron deprivation, and that the protein seems to redistribute to different cellular compartments (i.e. from the trans‐golgi network to vesicular structures and the plasma membrane). Interestingly, actinomycin D inhibited the induction of Atp7a mRNA expression, indicating transcriptional regulation. We thus sought to determine the molecular mechanism of Atp7a gene induction by iron. We first mapped the 5' end of the transcript and found alternative splicing; all splice variants contained exon 1, but their 5' termini varied within a span of 50 bps. Promoter fragments were amplified by PCR off of a BAC genomic clone and cloned into a luciferase‐containing reporter gene vector. Preliminary studies demonstrated that the promoter responded to iron chelation, demonstrating that we have developed an appropriate model with which to determine the precise mechanism by which the Atp7a gene is regulated by iron deprivation.