Cux1 regulation of the cyclin kinase inhibitor p27kip1 in polycystic kidney disease is attenuated by HDAC inhibitors.

Abstract

Cux1 is a homeodomain protein involved in cell cycle regulation and kidney development. Cux1 represses the cyclin kinase inhibitor p27 during early kidney development, promoting cell proliferation in the nephrogenic zone. Promoter reporter analysis of p27 revealed that Cux1 represses p27 in a concentration dependent manner, and immunoprecipitation showed that Cux1 interacts with the co-repressor Grg4 and the histone deacetylases HDAC1 and HDAC3. Chromatin immunoprecipitation (ChIP) identified the interaction of Cux1, Grg4, HDAC1, and HDAC3 at two different sites in the p27 promoter. To determine whether there was an interaction between these two loci in the developing kidney, we performed chromatin conformation capture (3C) assay. Analysis of newborn kidney tissue with 3C and ChIP-loop showed that the p27 promoter forms a loop intersecting at these two loci and that Cux1 bridges these two sites. To determine whether HDACs are required for Cux1 repression of p27 we analyzed p27 promoter activity in the presence of the HDAC inhibitor trichostatin A (TSA). TSA treatment completely relieved the repression of p27 by Cux1 and Grg4, demonstrating that Cux1 represses p27 in an HDAC dependent manner. To begin to test whether HDAC inhibitors could be used to target Cux1 repression of p27 for the treatment of PKD, we treated Pkd1 targeted pregnant mice with TSA or vehicle beginning at embryonic day 10.5 until embryonic day 18.5. Newborn Pkd1 mutant mice that received vehicle exhibited extensive collecting duct cysts, while newborn Pkd1 mutant mice that received TSA showed a significant reduction in cysts. Moreover, p27 expression was upregulated in TSA treated Pkd1 mice. Taken together, these results suggest that HDACs are required for cyst growth, and further support studies indicating that HDAC inhibitors may be an effective treatment for PKD.