Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Page 11 of 17 Figure 7 The txnip and trib3 promoters contain potential c-Jun binding sites. A, DNA sequences of functionally important ATF sites known to bind c-Jun and ATF2 in the c-jun, dp5 and mkp1 promoters. The c-jun promoter contains two ATF sites, the jun2 and jun1 TREs, the dp5 promoter contains one site, and the mkp1 promoter contains two sites. Each of these sites has been shown to bind c-Jun and ATF2 in ChIP assays and to be important for promoter induction after NGF withdrawal [24,26,34]. The orientation of the sequences is 5’ to 3’. B, A conserved ATF site was identified in the txnip promoter using CONSITE software (http://asp.ii.uib. no:8090/cgi-bin/CONSITE/consite) to analyse orthologous pairs of genomic sequences. This sequence (5’-TGAGGTAA-3’) is identical to the reverse complement of the jun2 TRE (5’-TTACCTCA-3’) in the c- jun promoter. Numbers refer to position in relation to Exon 1 of the rat txnip gene, as defined in the NCBI entry for rat txnip (http:// www.ncbi.nlm.nih.gov/gene/117514). * indicates nucleotides conserved in all four species. Two non-conserved nucleotides in the mouse ATF site are shown in red. C, A conserved ATF site was identified in the trib3 promoter using CONSITE software to analyse orthologous pairs of genomic sequences. This sequence (5’- TTACATCA-3’) is identical to the reverse complement of the ATF site in the dp5 promoter. Numbers refer to position in relation to Exon 1 of the rat trib3 gene, as defined in the NCBI entry for rat trib3 (http://www.ncbi.nlm.nih.gov/gene/246273). * indicates nucleotides conserved in all four species. One non-conserved nucleotide in the human ATF site is shown in red. withdrawal-induced apoptosis in sympathetic neurons. However, since then only a small number of genes have been shown to be regulated in this system and these were identified either by candidate gene approaches or the differential display technique. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. However, advances in tech- nology have now allowed us to identify the majority of the genes regulated by NGF withdrawal in sympathetic neurons. Using Affymetrix exon arrays and RNA iso- lated from rat sympathetic neurons, we investigated the global pattern of gene expression at 16 hours after NGF withdrawal. This time-point represents the transcrip- tional commitment point for sympathetic neurons undergoing NGF withdrawal-induced apoptosis and induced genes known to be required for NGF withdra- wal-induced death, e.g. c-jun, bim, and egln3, are expressed at a high level at this time. We were able to detect almost all of the genes known to be regulated after NGF withdrawal (Table 1) indicating the reliability of the microarray data. However, one exception was the previously described up-regulated gene puma [19] which is required for NGF withdrawal-induced death [21]. On further investigation, we found that no probe sets matching the puma gene were represented on the rat Affymetrix exon 1.0ST microarray. Nevertheless, micro- array technology remains a reliable tool and represents the best method for obtaining a complete overview of patterns of gene expression in this system. In addition, microarray studies can identify candidate genes for func- tional studies. For example, in the microarray experi- ments described in this paper we identified mkp1 as a gene induced after NGF withdrawal that could be a tar- get of the MLK-JNK-c-Jun pathway. We subsequently showed that mkp1 is a direct transcriptional target of the MLK-JNK-c-Jun pathway in sympathetic neurons and an important regulator of JNK activity and the rate of NGF withdrawal-induced death [24]. Microarrays have previously been used to study gene expression in potassium-deprived cerebellar granule neurons under- going apoptosis [37]. The most highly up-regulated gene in this study, trim17, was subsequently shown to encode a novel E3-ubiquitin ligase that can initiate neuronal apoptosis in several in vitro models of transcription- dependent apoptosis, including cerebellar granule neu- rons and NGF-deprived sympathetic neurons [38]. Approximately 95% of the genes identified in our microarray study have never been shown before to be transcriptionally regulated during NGF withdrawal- induced apoptosis. We have been able to identify poten- tial targets of the MLK-JNK-c-Jun pathway by including CEP-11004 in our experimental design. We selected 16 hours as the optimal time-point for our microarray