Reactive oxygen species (ROS) cause cellular damage and affect pathways involved in the aging process. Metallothioneins (MT) are conserved, cysteine-rich metal-binding proteins that function in metal homeostasis and detoxification, as well as the scavenging of ROS. To better understand its transcriptional regulation, genetic screens were used to identify regulatory factors and pathways that control the expression of the C. elegans MT gene,
mtl-1. A mutagenesis screen identified a strain, JF99 that harbored a mutation affecting
atf-7, a negative transcription factor involved in the JNK/p38 pathway. In both JF99 and an
atf-7 deletion mutant, steady-state
mtl-1 mRNA levels were identical but significantly greater than levels in wild type nematodes in the absence of metal. In addition to
atf-7, mutations in
pmk-1 caused a decrease in
mtl-1 expression. PMK-1 translocated to the nucleus in response to cadmium. This data suggested that PMK-1 was also involved in
mtl-1 regulation. A candidate gene screen identified three insulin signaling pathway genes (PDK-1 and the AKT-1/AKT-2 complex) that functioned independently of this pathway. Further genetic analysis confirmed that these genes acted upstream of PMK-1 and ATF-7 to regulate
mtl-1 transcription. Based on the genetic analyses and previous work, we propose that ATF-7 resides on the promoter of
mtl-1 to inhibit the constitutively active transcription factor ELT-2, which is important for intestinal cell-specific transcription. In the presence of cadmium, upstream factors signal PDK-1 and the AKT-1/2 complex to release PMK-1 causing it to translocate to the nucleus and phosphorylate ATF-7, allowing ELT-2 to initiate transcription. The activation of this pathway results in an increase in MT, which can scavenge cadmium and free radicals. Other genes were identified that affected
mtl-1 transcription: MAPK pathway members
mek-1 and
mek-2; a transcription factor
fos-1; a transcriptional regulator
zfp-1; a channel
tax-4; and a PMK-1 pathway member,
tir-1. Further analysis is underway to determine their involvement in
mtl-1 regulation. This new emerging pathway for
mtl-1 transcriptional regulation suggests a more direct role for MT in the response to ROS. It also provides a possible mechanistic link between activation of the insulin signaling pathway, free radical scavenging and longevity.