by Kumar Kotlo, Douglas E. Hughes, Victoria L. M

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Functional Polymorphism of the Anpep Gene Increases Promoter Activity in the Dahl Salt-Resistant Rat by Kumar Kotlo, Douglas E. Hughes, Victoria L.M. Herrera, Nelson Ruiz-Opazo, Robert H. Costa, R. Brooks Robey, and Robert S. Danziger Hypertension Volume 49(3):467-472 March 1, 2007 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Polymorphisms in Anpep 5′ flanking region of Anpep gene; 4 Figure 1. Polymorphisms in Anpep 5′ flanking region of Anpep gene; 4.4-kb 5′ flanking regions of SS/Jr and SR/Jr were sequenced; 11 SNPs, 4 of which were within or adjacent to cis-elements (colors), were identified. Figure 1. Polymorphisms in Anpep 5′ flanking region of Anpep gene; 4.4-kb 5′ flanking regions of SS/Jr and SR/Jr were sequenced; 11 SNPs, 4 of which were within or adjacent to cis-elements (colors), were identified. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. EMSA analysis reveals interaction of a protein with fragment 9 in SR/Jr (C-2272) but not SS/Jr (T-2272) rat Anpep. Figure 2. EMSA analysis reveals interaction of a protein with fragment 9 in SR/Jr (C-2272) but not SS/Jr (T-2272) rat Anpep. Each SNP was incorporated into a separate PCR product (fragments 1 to 11). No differential binding of the probes between rat strains was detected except for fragment 9, which shows an additional DNA–protein complex in SR/Jr but not SS/Jr strains (arrow). (Fragment 9 differs between strains only in the T/C2272 SNP.) The other bands present both in SS/Jr and SR/Jr strains could be nonspecific or because of binding of other transcription factors. See text for details. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Chromatin immunoprecipitation assay–C/EBPα transcription factor binds to the endogenous kidney Anpep promoter region of SR/Jr rats. Figure 3. Chromatin immunoprecipitation assay–C/EBPα transcription factor binds to the endogenous kidney Anpep promoter region of SR/Jr rats. Chromatin fragments derived from SS/Jr and SR/Jr rat kidneys were cross-linked and immunoprecipitated with polyclonal antibody against C/EBPα. Immunoprecipitates from each sample were analyzed by real-time PCR using primers spanning fragment 9 and control primers as detailed in the Methods section. The number on the y axis indicates the ΔCT (See Methods for details). Numbers on the top of bars of graph indicate the relative expression derived by 2ΔCT. n=5; **P<0.01 vs SS/Jr. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. EMSA analysis of C/EBPα binding in fragment 9 Figure 4. EMSA analysis of C/EBPα binding in fragment 9. a, C/EBPα associates with canonical binding site in fragment 9. Figure 4. EMSA analysis of C/EBPα binding in fragment 9. a, C/EBPα associates with canonical binding site in fragment 9. Nuclear extracts were prepared from HK-2 cells, and EMSA was performed using C/EBPα binding site with wild type (WT), and mutant (M) C/EBPα binding site with 4 nucleotide changes as described in the Methods section. Nuclear extracts were incubated with competitor oligonucleotides (WT and M) and C/EBPα antibody as indicated 10 minutes before adding the labeled probe. The position of the super shifted band is indicated by the arrowhead. b, C/EBPα stimulates Anpep activity of SR/Jr rat in HK-2 cells. pGL3 control vector, SR/Jr Anpep promoter, and SR/Jr Anpep promoter harboring mutant C/EBP binding site (SR/cEBPmutant) were cotransfected with β-galactosidase expression plasmid (internal control), and luciferase assay was carried out as described in the Methods section. n=6; *P<0.05 vs SR C/EBP mutant; **P<0.01 vs pGL3. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. C/EBPα increases promoter activity in SR/Jr but not in the SS/Jr strain. Figure 5. C/EBPα increases promoter activity in SR/Jr but not in the SS/Jr strain. Luciferase reporter activity assay in HK-2 cells for 4.4-kb Anpep regulatory regions of SS/Jr and SR/Jr and control pGL3 vectors cotransfected with C/EBPα (+C/EBPα) and dominant-negative C/EBPα expression vectors (+C/EBPαDN) (see Methods). The luciferase activity was normalized with β-galactosidase activity of each sample; n=6; *P<0.05, **P<0.01 vs SS/Jr and pGL3. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Insertion of fragment 9 from SR/Jr rat into 4 Figure 6. Insertion of fragment 9 from SR/Jr rat into 4.4-kb 5′ flanking region of SS/Jr (SS/SR) increases the promoter activity. Figure 6. Insertion of fragment 9 from SR/Jr rat into 4.4-kb 5′ flanking region of SS/Jr (SS/SR) increases the promoter activity. Control pGL3 and the 4.4-kb 5′ flanking regions of SS/Jr, SR/Jr rats, and SS/Jr with insertion of fragment 9 from SR/Jr rat (SS/SR) cloned in front of luciferase reporter gene in pGL3 vector were transiently cotransfected with cytomegalovirus β-galactosidase internal control vector into HK-2 cells. The luciferase activity was normalized with β-galactosidase activity of each sample. n=6; *P<0.05 vs SS and pGL3. Kumar Kotlo et al. Hypertension. 2007;49:467-472 Copyright © American Heart Association, Inc. All rights reserved.