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

Q: What is the clinical evidence from this study?

Population: MAS gene promoter regulation in the renin-angiotensin system. Intervention: Peroxynitrite-induced tyrosine nitration of ZNF274/KAP-1 complex and SOX/SRY transcription factor expression vs. Un-nitrated ZNF274/KAP-1 protein complex and control vectors/lysates. Primary outcome: MAS proximal promoter transcriptional repression and ZNF274/KAP-1 protein-protein complex binding dissociation.

Q: What does this research mean for the field?

The MAS proximal promoter is repressed by SOX family proteins, while tyrosine nitration of the KRAB/KAP-1 complex disrupts KAP-1 binding to relieve repression, establishing a nitric oxide-mediated transcriptional feedback loop. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

Jeremy W. Prokop; Frank J. Rauscher; Hongzhuang Peng; Yuanjie Liu; Fabiano Conde Araujo; Ingrid Kazue Mizuno Watanabe; Fernando M. Reis; Amy Milsted

doi:10.1042/cs20130385

October 16, 2013Clinical ScienceOpen Access

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

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Key result

SOX proteins repress the MAS promoter, while tyrosine nitration triggers KAP-1 complex dissociation.

Key Points

To characterize the promoter architecture of the MAS receptor gene and determine the transcriptional and post-translational feedback mechanisms governing its expression.
Analyzed evolutionary conserved regions and ENCODE database tracks to identify transcription factor binding sites across the human MAS promoter.
Evaluated transcriptional repression by cloning the MAS proximal promoter (+4 to −2200) into luciferase reporter vectors alongside expression constructs for SRY and SOX family members.
Conducted glutathione pull-down assays and peroxynitrite exposure experiments to examine binding interactions among GST-tagged ZNF274, KAP-1, and wild-type or mutated SRY.
The proximal MAS promoter was repressed by SRY as well as its closest SOX family homologs, SOX2, SOX3, and SOX14, with direct SRY binding mapped to position −98 from the ATG start codon.
Peroxynitrite treatment induced tyrosine nitration of KAP-1, which completely abolished its ability to bind the KRAB domain of ZNF274.
Mutational analysis of the SRY bridge domain showed that K136E and K140E mutations eliminated binding to the minimal KRAB domain (KRAB-O).

Structured PICO

P

Population

In vitro biochemical assays using purified recombinant human proteins (SRY, ZNF274, KAP-1), mammalian reporter constructs, and comparative genomic bioinformatics (ENCODE and ECR Browser).

I

Intervention

Expression of SOX family proteins (SRY, SOX2, SOX3, SOX14), site-directed mutagenesis of SRY (K136E and K140E), and peroxynitrite treatment (2 μL added to 10 μg purified KAP-1 protein or preformed ZNF274/KAP-1 complex) to induce tyrosine nitration.

C

Comparator

Un-nitrated KAP-1 protein, wild-type SRY protein, empty vector (pET28), and control EBNA lysate or Philips DNA positive control.

O

Outcome

Transcriptional activity/repression of the MAS proximal promoter pGL3 luciferase construct, direct DNA binding of SRY to the MAS promoter (-98 bases from ATG) via EMSA, and protein-protein interaction/dissociation between ZNF274 (KRAB domain) and KAP-1 or SRY via GST pull-down and Western blot.surrogate

Identifies a potential nitric oxide-driven epigenetic feedback mechanism regulating MAS receptor transcription via tyrosine nitration and dissociation of the repressive ZNF274/KAP-1 complex, alongside transcriptional repression by SRY and related SOX proteins.

Limitations

In vitro biochemical study; the degree to which ZNF274 and KAP-1 proteins are nitrated in vivo has not been determined
Additional targets of tyrosine nitration due to MAS activation require further investigation
Although ZNF274 and KAP-1 proteins were shown to be nitrated in vitro, the degree to which these proteins can be nitrated in vivo has not been determined.
Additional studies must be performed to address in vivo nitration and look at more targets of tyrosine nitration due to MAS activation.

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Implication

Animal findings on MAS regulation are hypothesis-generating; leaves open translation to human cardiovascular therapies.

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.

Discussion

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Cite This Study

Prokop et al. (2013) studied MAS gene promoter regulation in the renin-angiotensin system. Peroxynitrite-induced tyrosine nitration of ZNF274/KAP-1 complex and SOX/SRY transcription factor expression vs. Un-nitrated ZNF274/KAP-1 protein complex and control vectors/lysates was evaluated on MAS proximal promoter transcriptional repression and ZNF274/KAP-1 protein-protein complex binding dissociation. SRY, SOX2, SOX3, and SOX14 repressed the MAS proximal promoter, while tyrosine nitration of the ZNF274/KAP-1 complex caused KAP-1 dissociation, suggesting a nitric oxide-mediated feedback mechanism.

synapsesocial.com/papers/6a8318f8708dc38473582317 https://doi.org/10.1042/cs20130385

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

Key Points

Structured PICO

Limitations

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Implication

Abstract

Discussion

Cite This Study

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

Key Points

Structured PICO

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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