Cut proteins have five evolutionarily conserved domains Thes
Cut proteins have five evolutionarily conserved domains. These include a homeodomain, three Cut repeats, and a coiled coil structure. The cut repeats, called CR1, CR2, and CR3, are composed of 70 amino acids, and, along with the homeodomain, are each capable of binding DNA. Mammalian cut proteins function as bms my trials dependent transcription factors that can function as activators or repressors (Lievens et al., 1995; Nirodi et al., 2001; Nishio, 2004; Zhu et al., 2004; Truscott et al., 2003; Moon et al., 2001; Cadieux et al., 2008; Truscott et al., 2008; Mailly et al., 1996). Cux1 can repress target genes by 1) competing for CCAAT or Sp1 binding site occupancy, preventing the binding of the corresponding transcriptional activators, or 2) active repression involving the recruitment of histone deacetylases (HDACs) (Li et al., 1999; Coqueret et al., 1998). Cux1 represses the cyclin kinase inhibitors p21 and p27 and promotes proliferation of nephron progenitor cells (Coqueret et al., 1998; Ledford et al., 2002; Sharma et al., 2009). Transgenic mice constitutively expressing Cux1 develop multiorgan hyperplasia, including renal hyperplasia, phenocopying p27 knockout mice (Ledford et al., 2002; Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). In contrast, p21 knockout mice do not exhibit renal hyperplasia (Deng et al., 1995).
Cux1 is highly and ectopically expressed in several mouse models of polycystic kidney disease (PKD), including cpk, Pkd1 null, and Pkd1CD mice, which carry a collecting duct specific deletion of Pkd1 (Vanden Heuvel et al., 1996; Sharma et al., 2005; Paul et al., 2011), and is upregulated in human kidney cells isolated from the cysts of ADPKD patients (Alcalay et al., 2008). However, Cux1 transgenic mice do not develop cystic kidneys, indicating that overexpression of Cux1 alone is insufficient to develop PKD in mice (Ledford et al., 2002). However, sustained expression of a shorter isoform of Cux1 was shown to induce cysts in transgenic mice after 12 months (Cadieux et al., 2008). This is similar to the protracted cyst development that occurs when the Pkd1 gene is deleted in mice after a developmental switch (Piontek et al., 2007). A number of studies have shown that renal injury induces rapid cyst growth in mice when Pkd1 or cilia genes are disrupted after a developmental switch (Piontek et al., 2007; Davenport et al., 2007; Lantinga-van Leeuwen et al., 2007; Patel et al., 2008). However, when cilia were disrupted in adult Cux1 transgenic mice, rapid cyst development did not occur, even though cell proliferation remained high (Sharma et al., 2013). Rather, mice carrying both a collecting duct deletion of Pkd1 and a targeted deletion of Cux1 showed that Cux1 is required for cyst growth (Porath et al., 2017). Reduced Cux1 expression in PKD mice suppresses cyst growth, and tubules in which both Cux1 and Pkd1 were deleted showed increased expression of p27 and diminished cell proliferation. Cux1 interacts with the co-repressor Grg4 to repress p27 (Sharma et al., 2009), and along with the histone deacetylases HDAC1 and HDAC3, all four proteins bind to two different sites on the p27 promoter (Sharma et al., 2009). Thus, to determine whether inhibition of Cux1 repression of p27 could be a therapeutic approach for the treatment of PKD, we characterized the role of HDACs in p27 repression and evaluated Cux1 and p27 expression following treatment of Pkd1CD mice with the HDAC inhibitor trichostain A (TSA).
Materials and methods
Results We previously showed that Cux1 represses p27 promoter activity in a concentration dependent manner and that repression is significantly enhanced when Cux1 is associated with the co-repressor Grg4 (Sharma et al., 2009). Further studies showed that, in addition to Grg4, Cux1 interacts with HDAC1 and HDAC3 and that all four proteins interact with two separate sites on the p27 promoter in RKE cells, a kidney epithelial cell line (Sharma et al., 2009). DNAse footprinting showed that these sites correspond to an AT rich sequence located approximately −1.3 kb from the transcription start site, and a CCAAT box and two GC rich sequences approximately −0.35 kb from the transcription start site. Both the AT rich sequence and the CCAAT and GC rich sequences have been previously described as binding sites for Cux1 (Mailly et al., 1996). The AT rich sequence is recognized by the homeodomain of Cux1, while the CCAAT box and GC rich sequences are recognized by cut repeats 1 and 2.