Abstract
Renin is the rate-limiting enzyme in the renin-angiotensin system and thus dictates the level of the pressor hormone angiotensin-II. The classical site of renin expression and secretion is the renal juxtaglomerular cell, where its expression is tightly regulated by physiological cues. An evolutionarily conserved transcriptional enhancer located 11 kb upstream of the human RENIN gene has been reported to markedly enhance transcription in renin expressing cells in vitro. However, its importance in vivo remains unclear. We tested whether this enhancer is required for appropriate tissue- and cell-specific expression, or for physiological regulation of the human RENIN gene. To accomplish this, we used a retrofitting technique employing homologous recombination in bacteria to delete the enhancer from a 160-kb P1-artificial chromosome containing human RENIN, two upstream genes and one downstream gene, and then generated two lines of transgenic mice. We previously showed that human renin expression in transgenic mice containing the wild type construct is tightly regulated as is expression of the linked genes. Deletion of the enhancer had no effect on tissue-specific expression of human RENIN, but using the downstream gene as an internal control, found that human RENIN mRNA levels were 3-10-fold decreased compared with constructs containing the enhancer. Despite this decrease in expression, renin protein remained localized to renal juxtaglomerular cells and was appropriately regulated by cues that either increase or decrease expression of renin. Our results suggest that sequences other than the enhancer may be necessary for tissue-specific, cell-specific, and regulated expression of human RENIN.
Highlights
Renin is the first and rate-limiting aspartyl protease in the renin-angiotensin system (RAS)2 cascade that leads to the production of angiotensin-II (Ang-II) by the consecutive cleavage
We previously showed that human renin expression in transgenic mice containing the wild type construct is tightly regulated as is expression of the linked genes
We previously reported that transgenic mice carrying a 160-kb P1 artificial chromosome (PAC) encoding the human RENIN (hREN) gene including 75 kb of upstream and 70 kb of downstream sequences exhibited appropriate tissue-specific, cell-specific, copy number proportional, and exquisitely regulated expression in response to cues that either stimulate or inhibit renin expression [23, 24]
Summary
Generation of the Kidney Enhancer-deficient PAC160 Construct—The PAC160 (Genome Systems control number 4917) was used as the template for deletion of the human kidney enhancer (KE) as previously reported [23, 26]. The flanking sequences were designed to delete the 241-bp KE (GenBankTM accession number AF140238) segment without altering adjacent sequences Another PCR fragment was cloned containing a LoxP511-. The PAC160⌬KEϩCAM vector was isolated and subjected to battery of PCR and Southern blots to ensure faithful recombination This vector was transformed into E. coli strain BS591 (a gift of Brian Sauer), which constitutively expresses cre-recombinase, to excise the CAM gene. The PAC160⌬KE vector was isolated and purified using the large construct DNA isolation kit from Qiagen This PAC was subjected to a number of PCR and Southern blot analysis to ensure fidelity of the KE deletion. After selection for homologous recombinants, the LoxP511 flanked CAM gene was removed by propagation in E. coli constitutively expressing cre-recombinase. A value of p Ͻ 0.05 was considered statistically significant
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