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    • Cux is a cell cycle dependent

    2021-09-23

    Cux1 is a JZL184 dependent transcription repressor that is aberrantly expressed in the Pkd1 null, Pkd1CD, and cpk mouse models of PKD, as well as in human ADPKD cells (Vanden Heuvel et al., 1996; Sharma et al., 2005; Paul et al., 2011; Alcalay et al., 2008). In Pkd1 null mice and in human ADPKD cells there is a reduction in the levels of a nuclear isoform of the cysteine endopeptidase cathepsin-l (Alcalay et al., 2008). This enzyme proteolytically processes Cux1 in S phase of the cell cycle, thus the reduction of cathepsin-l results in the accumulation of full length Cux1 protein in PKD (Alcalay et al., 2008; Goulet et al., 2004). Deletion of the cathepsin-L processing site in Cux1 in Cys1cpk mice similarly resulted in the accumulation of Cux1 (Alcalay et al., 2008). The increased expression of Cux1 in PKD is associated with increased cell proliferation and disease progression resulting from the down regulation of the cyclin kinase inhibitor p27 (Alcalay et al., 2008; Porath et al., 2017). However, forced expression of Cux1 in transgenic mice results in multiorgan hyperplasia, but does not result in cystic kidney disease (Ledford et al., 2002). Moreover, sustained overexpression of Cux1 failed to induce rapid cyst formation after the disruption of cilia in the kidneys of adult mice (Sharma et al., 2013). To determine whether Cux1 is required for PKD progression, we crossed mice carrying a targeted deletion of Cux1 with Pkd1CD mice, which have Pkd1 deleted in the collecting ducts (Porath et al., 2017). Mice that were homozygous mutant for both Cux1 and Pkd1 showed no cystic disease, and mice that were heterozygous for Cux1 and homozygous for Pkd1 showed significantly reduced cystic disease (Porath et al., 2017). These studies demonstrated that Cux1, while not sufficient to cause polycystic kidney disease when overexpressed, is required for cystic disease progression. These results suggest that targeting Cux1 regulation of the cell cycle may be an effective treatment for ADPKD. We have previously shown that Cux1 interacts with the co-repressor Grg4 and HDAC1 and HDAC3 in the context of the p27 promoter, and that this complex binds to two regions of the p27 promoter in its native configuration. Analysis of the two sites bound by Cux1 showed sequences that are recognized by different DNA binding domains in Cux1, suggesting that Cux1 could bind both sites simultaneously forming a loop in the p27 regulatory region. Our results support this model showing that a loop is formed between an AT rich region located a1–1.3 kb from the transcription start site and a GC rich region and a CCAAT box located at −0.35 kb from the transcription start site. Interaction with the GC rich region and CCAAT sequences located at −0.35 kb are associated with passive repression and involve the first two cut repeats of Cux1, which compete with transcriptional activators for binding (Mailly et al., 1996). Interaction with the AT rich sequences located at −1.3 are associated with active repression and involves the third cut repeat and the homeodomain (Mailly et al., 1996). Binding of this domain to target sequences is thought to involve the recruitment of HDACs. We have previously shown that HDAC1 and HDAC3 can be immunoprecipitated, along with Cux1 and Grg4, at both of these sites in vivo (Sharma et al., 2009). Moreover, the present results demonstrate that HDAC activity is necessary for Cux1 repression of p27. Recently, a number of studies have determined that treatment of Pkd1 and Pkd2 animal models of polycystic kidney disease with HDAC inhibitors reduces polycystic kidney disease (Cao et al., 2009; Xia et al., 2010; Fan et al., 2012). Similarly, our results show that treatment of Pkd1CD mouse embryos with TSA reduced cyst growth. Moreover, p27 expression is increased in the TSA treated mice. Thus, the mechanism of cyst reduction appears to be increased expression of p27 reducing cell proliferation. Interestingly, Cux1 expression was not changed in the TSA treated embryonic kidneys compared to the control kidneys, further supporting the requirement of HDAC activity for Cux1 repression of p27. Our results provide further support for the use of HDAC inhibitors as therapeutic treatments for PKD, and provide a potential mechanism of action for their effectiveness in treating PKD.