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Peter Groskreutz Regulator


ALWAYS READ AND FOLLOW PESTICIDE LABEL DIRECTIONS. It is a violation of federal and state law to use any pesticide product other than in accordance with its labeling. NOT ALL formulations of dicamba or glyphosate are approved for in-crop use with Roundup Ready 2 Xtend soybeans. NOT ALL formulations of dicamba, glyphosate or glufosinate are approved for in-crop use with products with XtendFlex Technology. ONLY USE FORMULATIONS THAT ARE SPECIFICALLY LABELED FOR SUCH USES AND APPROVED FOR SUCH USE IN THE STATE OF APPLICATION. Contact the U.S. EPA and your state pesticide regulatory agency with any questions about the approval status of dicamba herbicide products for in-crop use with Roundup Ready 2 Xtend soybeans or products with XtendFlex Technology.




Peter Groskreutz Regulator



The CRC risk locus at 4q32.2 identified by Schmit et al. [6] is located between the genes NAF1 and FSTL5 (Fig 1A). The association between the risk locus at 4q32.2 and the increased risk of developing CRC could be caused by the risk locus at 4q32.2 being located in a gene regulatory region. We examined previously published cap analysis of gene expression (CAGE) data (accession number: GSE95437) [14] to analyze whether the risk locus could be found in a gene regulatory region (Fig 1A). We found no transcriptional activity determined by CAGE around the FSTL5 promoter, conversely, there was clearly transcriptional activity around the NAF1 promoter. Furthermore, Fig 1A shows transcriptional activity in the risk locus at 4q32.2. This could indicate that the risk locus at 4q32.2 could be located in a gene regulatory region, as it has been shown that active gene regulatory regions produces unspecific transcription [15].


(a) Location of the risk locus at 4q32.2. CAGE analysis [14] of the region containing the two genes NAF1 and FSTL5 and the risk locus at 4q32.2, shown in yellow, are presented in the top [16]. The bottom presents a zoom in of the risk locus at 4q32.2, that shows the 6 SNPs found in Schmit et al., highlighted in blue. From left: rs17042479 (A/G), rs79783178 (-/AT), rs35509282 (A/T), rs11736440 (A/G), rs9998942 (C/T) and rs57336275 (C/T) (b) Plasmid map of the construct used in the promoter reporter gene assay of promoter activity (c) Promoter reporter assay. Promoter activity of FSTL5 (FSTL5pro) and NAF1 (NAF1pro) in colon cancer cell lines. From left to right: Caco2 cells, DLD-1 cells and SW480 cells. Luciferase activities were corrected for transfection efficiency and normalized to the activity of the pGL4.10 without promoter (pGL4.10). N = 4. Statistical significance was determined using one-way ANOVA. (d) Plasmid map of the construct used in the promoter reporter gene assay of the gene regulatory region (e) Promoter reporter assay. The luciferase activities were corrected for transfection efficiency and normalized according to the expression of the NAF1 promoter (NAF1pro). From left to right: Caco2 cells, DLD-1 cells and SW480 cells. NAF1pro + SNP rs17042479(A) is the construct with the NAF1 promoter and the reference allele of SNP rs17042479. NAF1pro + SNP rs17042479(G) is the construct with the NAF1 promoter and the risk allele of SNP rs17042479. N = 4. Statistical significance was determined using one-way ANOVA.


We analyzed the promoter activity of NAF1 and FSTL5 in the three colon cancer cell lines Caco2, DLD-1 and SW480 in a promoter reporter gene assay (Fig 1C). The results of the promoter reporter assay demonstrated that the FSTL5 promoter, FSTL5pro showed a slightly higher reporter gene activity in the two colon cancer cell lines DLD-1 and SW480 compared to background (the promoter-less pGL4.10 luciferase reporter plasmid). The FSTL5 promoter, FSTL5pro, reporter gene activity was not significantly changed from background (the promoter-less pGL4.10 luciferase reporter plasmid) in Caco2 cells. Additionally, the NAF1 promoter, NAF1pro, was highly active in all three colon cancer cell lines Caco2, DLD-1 and SW480. The promoter activity analysis revealed that the FSTL5 promoter was not nearly as active in the three colon cancer cell lines as the NAF1 promoter. As the CAGE data [14] (Fig 1A) also showed a very low transcriptional activity of the FSTL5 promoter region in vivo, we conclude that any possible gene regulatory activity of the risk locus at 4q32.2 is most likely to take place on the NAF1 promoter, and we therefore focused our further analyses on the NAF1 promoter.


A promoter reporter analysis was performed to determine if the risk locus at 4q32.2 functions as a gene regulatory region on the NAF1 promoter activity, and whether the SNP rs17042479 (the SNP most significantly associated with CRC [6]) modifies such gene regulatory activity. Therefore, two versions of the risk locus were cloned into the NAF1 promoter construct, NAF1pro. One containing the reference SNP rs17042479 (A) and one containing the risk SNP rs17042479(G). Analyzing the NAF1 promoter activity in different constructs yielded similar results in the three colon cancer cell lines: Caco2, DLD-1, and SW480 (Fig 1C). The possible gene regulatory region with the reference allele SNP rs17042479 (A) significantly reduced the promoter activity of NAF1 by 18% and 11% in the two colon cancer cell lines DLD-1 and SW480, respectively. In Caco2 cells, the possible gene regulatory region with the reference allele SNP rs17042479 (A) had no effect on NAF1 promoter activity. The possible gene regulatory region with the risk allele SNP rs17042479(G) reduced the NAF1 promoter activity by 18%, 38% and 34% in the three colon cancer cell lines Caco2, DLD-1 and SW480, respectively. In conclusion, it was demonstrated that the gene regulatory region with the risk SNP rs17042479 (G) has an increased repressor effect on the promoter activity of NAF1 and is significantly different from the gene regulatory region with the reference allele SNP rs17042479 (A) in DLD-1 cells and SW480 cells.


The results from the bioinformatic analysis of the risk locus at 4q32.2 and the promoter reporter analysis indicated an association between the risk locus at 4q32.2 and the increased risk of developing CRC. This could be caused by a gene regulatory region containing SNP rs17042479, located in the risk locus at 4q32.2, which alters the expression of NAF1. To investigate the effect of SNP rs17042479 on cancer characteristics and the NAF1 expression, along with the NAF1 expression impact on cancer characteristics we analyzed a clinical dataset from patients diagnosed with CRC.


If the risk locus at 4q32.2 functions as a gene regulatory region, it has the potential to alter the expression of a gene that influences the development of CRC, which could explain the link between the risk locus at 4q32.2 and the increased risk of developing CRC. We analyzed whether the risk locus changes the activity of the NAF1 promoter through a promoter reporter gene assay. The promoter reporter gene analysis showed that the region in the risk locus at 4q32.2 functions as a repressor on the NAF1 promoter (Fig 1E). Signifying the risk locus at 4q32.2 potential as a gene regulatory region and providing an explanation for why the risk locus at 4q32.2 is linked to CRC. The risk locus was identified in a GWAS [6], where the SNP rs17042479 was identified as the most significant SNP associated to CRC. We therefore investigated if the SNP rs17042479(G) changes the activity of the gene regulatory region and found that it significantly increased the repressor effect of the region in the risk locus at 4q32.2. This indicated that the link between the 4q32.2 risk locus and CRC could be caused by gene regulatory activity altering the activity of the NAF1 promoter. Although promoter reporter gene assays have made significant contributions to the analysis of eukaryotic gene expression and regulation [26], the promoter reporter assay has the limitation that the DNA in the plasmid is not organized in a chromatin structure like the genomic DNA in the cell. As a result, the promoter reporter assay analyzes promoter activity without taking into account the influence of epigenetic mechanisms. Epigenetics is important in the regulation of gene expression, often in a close interplay with transcriptional regulators [27]. Patients with the risk allele of SNP rs17042479 included in this study is heterozygous for the SNP rs17042479, it is possible that the effect would be more evident if the patients included were homozygous for the risk allele of SNP rs17042479.


The association between SNP rs17042479(G) and the increased risk of developing CRC could be explained by a changed expression of NAF1 mediated through the gene regulatory effect of SNP rs17042479(G) affecting cancer characteristics. The patients with the risk SNP rs17042479(G) were associated with lower expression of NAF1 compared to patients with the reference SNP rs17042479(A) in tumor tissue (Fig 3C). The NAF1 expression in healthy tissue was not altered between the two genotype groups. The observation that the risk allele of SNP rs17042479 (G) seems to have different impact in healthy tissue compared to malignant tissue could indicate cancer-related transcriptions factors binds better to the risk allele compared to the reference allele. An example of a transcription factor with increased activity in colon cancer cells is TCF7L2 (TCF4), the main transcription factor activated by Wnt signaling. The Wnt signaling pathway is a key pathway in colorectal cancer pathogenesis and is constitutively active in APC mutated colon cancer cells [31]. A TCF7L2 binding site has previously been described in an enhancer of the MYC gene [32]. A colon cancer-associated single nucleotide variant in this binding site increases the binding of TCF7L2 and the expression of MYC. As most studies of gene regulatory activity is done in cell lines; it could be an overlooked phenomenon that nucleotide variants can have different effects on gene regulatory elements in normal and diseased cells. The NAF1 expression in tumor tissue was lower than the NAF1 expression in healthy tissue (Fig 3A). This suggests that low NAF1 expression in tumor tissue is associated with a poor prognosis for CRC patients. It would have been interesting to investigate a survival analysis of the two genotype groups and high and low NAF1 expression, but due to the size of the dataset this was not possible. Human Protein Atlas ( ) has analyzed if the survival rate of CRC was affected by the NAF1 expression in a Kaplan Meier plot. The 597 patients included in the Kaplan Meier analysis was divided into the two groups high and low NAF1 expression. The survival analysis showed a difference in survival rate between the two groups high NAF1 expression (5-year survival: 73%) and low NAF1 expression (5-year survival: 57%) [33]. 041b061a72


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