MAS promoter regulation: a role for Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism

Abstract

The ACE2 (angiotensin-converting enzyme 2)/Ang-(1-7) [angiotensin-(1-7)]/MAS axis of the RAS (renin-angiotensin system) has emerged as a pathway of interest in treating both cardiovascular disorders and cancer. The MAS protein is known to bind to and be activated by Ang-(1-7); however, the mechanisms of this activation are just starting to be understood. Although there are strong biochemical data regarding the regulation and activation of the AT1R (angiotensin II type 1 receptor) and the AT2R (angiotensin II type 2 receptor), with models of how AngII (angiotensin II) binds each receptor, fewer studies have characterized MAS. In the present study, we characterize the MAS promoter and provide a potential feedback mechanism that could compensate for MAS degradation following activation by Ang-(1-7). Analysis of ENCODE data for the MAS promoter revealed potential epigenetic control by KRAB (Krüppel-associated box)/KAP-1 (KRAB-associated protein-1). A proximal promoter construct for the MAS gene was repressed by the SOX [SRY (sex-determining region on the Y chromosome) box] proteins SRY, SOX2, SOX3 and SOX14, of which SRY is known to interact with the KRAB domain. The KRAB-KAP-1 complex can be tyrosine-nitrated, causing the dissociation of the KAP-1 protein and thus a potential loss of epigenetic control. Activation of MAS can lead to an increase in nitric oxide, suggesting a feedback mechanism for MAS on its own promoter. The results of the present study provide a more complete view of MAS regulation and, for the first time, suggest biochemical outcomes for nitration of the KRAB domain.

Figure 1. Regulation of MAS promoter

Conservation of the MAS promoter in multiple mammalian species. Red represents regions of high conservation, green repetitive elements, blue coding segments, and yellow the UTR sequence as determined with the ECR browser. Known SNPs are shown, with each black line representing an SNP. Three promoter sites (A, B, and C) were identified as highly conserved. Site C contains no known SNPs and also several transcription factor binding sites as identified in ENCODE. The proximal promoter for MAS (orange) was cloned into a pGL3 luciferase reporter vector. The proposed transcriptional start site (TSS) for humans based on primate UTR sequence analysis has a TATA box located 24 bases upstream the TSS.

Figure 2

A) Using the MAS promoter construct, members of the SOX genes were tested for ability to regulate the MAS promoter regulation of luciferase. Error bars represent the standard error of the mean with an n of 3. The bars in red (hSRY, SOX2, SOX3, and SOX14) were statistically different from the control while those in gray were not, based on student’s t-test with p≤0.05. B) Gel shift assays of various DNA elements (EBNA=control, Philips=known Sry element, MAS=−98 to −62 of the MAS promoter sequence, and * indicated when the DNA probe had a biotin tag bound) with different protein constructs. C) Varying the concentration of Sry lysate increased the amount of shifted DNA (top) relative to the free DNA probe (bottom) that was outcompeted with DNA that did not contain a biotin tag (sample 8). D) Densitometry of the free and shifted bands confirms the concentration dependent shift.

Figure 3. Nitration of KAP-1 and ZNF274 suggests possible feedback mechanism in MAS activation by Ang-(1-7)

A) Coomassie stain of glutathione sepharose pull down experiments of ZNF274 and KAP-1 with nitration of the protein induced with peroxynitrite. Conditions for each pull down are shown below the gel. B) Western blot with the polyclonal anti-nitrotyrosine antibody of the samples in A. C) Proposed mechanism for maintaining MAS receptor on the cell surface with stimulation by Ang-(1-7). Activation of the receptor results in increased nitric oxide and thus nitration of the KAP-1 ZNF274 proteins preventing their inhibition of the MAS promoter activity.

Figure 4

A) GST pull down experiments of various SRY constructs using the GST-KRAB-O protein. The GST tag alone did not pull SRY out of solution. Only a band for the hSRY WT can be seen which was further confirmed through western blots against the His tag of SRY (B). C) Models suggesting the critical amino acids for the binding of SRY (gray) to the KRAB domain (yellow) through to polar basic (blue) and polar acidic (red) amino acids while SRY in bound to DNA (cyan).

Figure 5

A) Proposed mechanism for the fold back of element C of the MAS promoter to regulate the transcriptional control of MAS through interaction of ZNF274 and SRY recruiting KAP1. Nitration in the KRAB domain may lead to the differential regulation of the MAS promoter following Ang-(1-7) activation and possible nitration of transcription factors. The hypertensive SHR animal contains an additional copy of Sry which may facilitate additional binding to the promoter sequence thus recruiting additional KRAB domains when compared to the normotensive WKY animal. B) Proposed nuclear mechanism involving nitric oxide repression of the KRAB/KAP1 complex on promoters of multiple RAS genes as a result of activation of MAS and AT2 by Ang peptides.