Abstract
Sulforaphane is a dietary isothiocyanate that has been shown to have anticancer, antidiabetic and antimicrobial properties. Found in cruciferous vegetables, sulforaphane acts primarily as a phase II enzyme inducer through the pathway mediated by nuclear factor (erythroid‐derived 2)‐like 2 (NRF2). NRF2 is the key transcriptional activator in this pathway, binding as a heterodimer with small Maf proteins to cis‐acting antioxidant response elements (AREs) found in the promoter regions of downstream oxidative response genes. In Nrf2 knockout mice, oxidative stress exposure causes increased damage to lung, liver and neurological tissue compared to wild type mice. Conversely, an aggressive cancer phenotype has been linked to permanently activated NRF2; indicating NRF2 may directly regulate other pathways unrelated to oxidant defense. In order to investigate NRF2 binding and gene regulation on a genome wide scale, we analyzed chromatin immunoprecipitated (ChIP) NRF2‐bound DNA using ChIP‐on‐chip and ChIP‐Seq technologies. Specifically, a human lymphoblastoid cell line (HapMap GM11994) was exposed in triplicate to 10uM sulforaphane, crosslinked, sonicated and immunoprecipitated with NRF2 antibody. NRF2‐bound DNA was amplified and hybridized to the Agilent Human Promoter chip, which provides −5.5 to +2.5 kilobase coverage around the majority of gene transcriptional start sites (ChIP‐on‐chip). Additionally, ChIP NRF2‐bound DNA was sequenced using the Solexa GAII second‐generation sequencing platform (ChIP‐seq). ChIP‐on‐chip intensity data was input to Agilent's DNA analytics software and 1056 probes in promoter regions of 277 genes displayed sulforaphane ‐ induced NRF2 binding, 20 of which had been previously identified as NRF2‐regulated genes. Ingenuity pathway analysis found these 277 genes were involved in oxidative stress, cellular growth and proliferation, cellular development, small molecule biochemistry and other signaling pathways. We cross referenced the ChIP‐on‐chip data with results from gene expression (induction>1.2‐fold/suppression <0.8‐fold compared to control, p<2.5X10−6) in 60 sulforaphane‐exposed HapMap lymphoblastoid cells lines as determined by Illumina microarray. We identified 32 differentially expressed genes with NRF2 binding, 17 that had not previously been described as regulated by NRF2. Preliminary analysis of the ChIP‐seq data has mapped 2.1 million uniquely aligned sequencing reads resulting in 945 bound regions (high quality peaks). Of these, 5% are located near known NRF2‐binding regions, and approximately 20% of the regions overlap with the ChIP‐on‐chip platform. Using both the ChIP‐on‐chip and ChIP‐seq platform in this study has allowed us to investigate NRF2 on a genome‐wide scale and identify novel sulforaphane‐activated NRF2 gene targets.
Citation Information: Cancer Prev Res 2010;3(1 Suppl):B51.
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