Abstract
NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.
Highlights
Nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) is a transcription factor that regulates the oxidative stress response
Identification of NF-E2-related Factors in Zebrafish—We sought initially to assess the diversity of the NF-E2-related CNC-basic-leucine zipper (bZIP) family in zebrafish as compared with the four members found in humans: NF-E2, NRF1 (NFE2L1), NF-E2-related factor 2 (NRF2) (NFE2L2), and NRF3 (NFE2L3)
The conserved synteny suggests that the nrf1 and nrf2 duplicates, like the hox clusters to which they are linked, arose as part of the fish-specific whole-genome duplication that occurred after divergence of fish and mammalian lineages [9, 18]
Summary
NRF2 is a transcription factor that regulates the oxidative stress response. Results: Zebrafish have duplicate nrf genes, nrf2a and nrf2b, with distinct functions during embryonic development. Nuclear factor erythroid 2 (NF-E2)/p45-related factor 2 (NRF2; called NFE2L2), a member of the cap’n’collar (CNC)-basic-leucine zipper (bZIP) protein family, plays an important role in the regulation of antioxidant genes and Phase II metabolism in vertebrates This transcription factor, which activates gene transcription through its interactions with antioxidant/electrophile response elements (ARE/EpRE), is a key regulator of the oxidative stress response, influencing numerous biological processes, such as aerobic respiration, embryonic development, inflammation, and carcinogenesis [1, 2]. An advantage of the zebrafish model is that it often contains duplicate copies of genes that are present as only single copies in mammals, allowing for additional insight into the multiple functions of the human counterpart [9] With this in mind, we sought to characterize the oxidative stress response in zebrafish embryos and the role of zebrafish homologs of genes in the NRF2 gene family. We demonstrate that nrf2a and nrf2b5 have undergone subfunction partitioning and provide evidence that the Nrf2b protein functions as a repressor to regulate constitutive gene expression during embryonic development
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