The homeodomain protein Cux1 interacts with Grg4 to repress p27 kip1 expression during kidney development

Abstract

The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.

Figure 1. In vivo and in vitro interactions between Cux1 and Grg4

A: Kidney lysates from 3-day-old wild type (WT) and Cux1 transgenic (TG) mice were immunoprecipitated with rabbit polyclonal anti-Cux1 (top panel) or rabbit polyclonal anti-Grg4 (bottom panel) as indicated. The immunoprecipitates were then blotted for Grg4 (top panel) or Cux1 (bottom panel) as indicated. Grg4 can be seen in WT and TG kidneys, after immunoprecipitation with Cux1 (right lanes, top panel). Cux1 can be seen in WT and TG kidneys, after immunoprecipitation with Grg4 (right lanes, bottom panel). No bands are observed when protein G beads alone are precipitated (no Ab). B: Bacterially expressed Cux1 protein was incubated with GST- Grg4 fusion protein immobilized on Glutathione agarose beads. Following washing, GST beads alone with Cux1, wash #3, and GST-Grg4 immobilized beads with Cux1 were transferred to a membrane and blotted with antibody directed against Cux1 protein. Only GST-Grg4 with Cux1 protein and the Cux1 input showed a positive band, indicating a direct physical interaction between Cux1 and Grg4.Lane 1 shows 50% of the total purified recombinant Cux1 protein used for the assay.

Figure 2. Repression of p27^kip1 promoter activity by Cux1 and Grg4

293T cells were transiently transfected with 1 µg of reporter construct containing p27 upstream sequences from −1609 to +178 fused to the luciferase reporter gene (+), together with different concentrations of the Cux1 and Grg4 expression vectors (amounts shown are in µg) or with empty pcDNA3.1 expression vector (amounts shown are in µg). In the presence of Grg4, significantly less Cux1 is required to repress p27 promoter activity (3^rd and 4^th bar). Promoter activity was plotted as fold change, normalized to the expression of a co-transfected renilla expression construct. Activity is expressed as the mean of three separate experiments performed in triplicate. Error bars indicate standard deviation. ANOVA showed a significant reduction in luciferase activity following cotransfection with Grg4 in a dose dependent manner (P< 0.002).

Figure 3. Co-localization and interaction of Cux1, Grg4, and HDACs

A: HDAC1 was expressed at highest levels in the nephrogenic zone, where it was localized to mesenchyme, ureteric buds (UB), and early nephric structures, including S-shaped bodies (S), but down regulated in capillary loop staged glomeruli (C). B: HDAC3 was also expressed in the nephrogenic zone, including the ureteric bud (UB) and S shaped bodies (S). In capillary loop stage glomeruli (C), HDAC3 expression was observed in the presumptive podocytes (P). C: Grg4 was expressed at low levels in the nephrogenic zone, including S-shaped bodies (arrowhead), and upregulated in the presumptive podocytes of capillary loop staged glomeruli (arrows). D: HDAC3 was similarly expressed in the nephrogenic zone, with highest levels in the presumptive podocytes (arrows) and in S-shaped bodies (arrowheads). E, F: Co-localization of HDAC3 and Grg4 without (E) or with (F) DAPI in capillary loop staged glomeruli (arrows). G: Newborn kidney lysates were immunoprecipitated (IP) with protein G beads alone (beads only), rabbit IgG (IgG), rabbit polyclonal anti-HDAC1 (left panel), or rabbit polyclonal anti-HDAC3 (right panel) as indicated. The immunoprecipitates were then blotted for Cux1 (left panel) or Grg4 (right panel) as indicated. Cux1 can be seen in newborn kidneys after immunoprecipitation with HDAC1 (left lane, left panel), and Grg4 can be seen in newborn kidneys after immunoprecipitation with HDAC3 (left lane, right panel).

Figure 4. p27^Kip1 is a direct Cux1 target in the developing kidney

Complexes isolated from Notch^ic cells (A–C) or from newborn kidneys (D) were immunoprecipitated with anti-Cux1, anti-Grg4, anti-HDAC1, anti-HDAC3, or anti-RNA polymerase II antibodies followed by reverse cross-linking of protein and DNA, and PCR amplification of a 249 bp fragment of the p27kip1 promoter spanning −1609 to −1360 (A), or a 191 bp fragment of the p27kip1 promoter spanning −687 to −496 (C, D), relative to the transcription start site. A: Inverse image of ethidium-stained agarose gel showing PCR amplification of a 249 bp p27kip1 product from input DNA (lane 3), following ChIP with antibody directed against Cux1 (lane 5), Grg4 (lane 6), HDAC1 (lane 7), and HDAC3 (lane 8), indicating that Cux1, Grg4, HDAC1, and HDAC3 interact with a site within the 5’ region of the p27kip1 promoter in vivo. B: To control for non-specific chromatin immunoprecipitation, a 317 bp product from −1272 to −955 of the p27kip1 promoter was amplified from input DNA (lane 2), following ChIP with antibody directed against Cux1 (lane 4), Grg4 (lane 5), HDAC1 (lane 6), and HDAC3 (lane 7). C: PCR amplification of a 191 bp p27kip1 product from input DNA (lane 2), following ChIP with antibody directed against Cux1 (lane 4), Grg4 (lane 5), HDAC1 (lane 6), and HDAC3 (lane 7), indicating that Cux1, Grg4, HDAC1, and HDAC3 interact with a site within the 3’ region of the p27kip1 promoter in vivo. D: PCR amplification of a 191 bp p27kip1 product from input DNA (lane 5), following ChIP with antibody directed against Cux1 (lane 3), and RNA polymerase II (lane 6), indicating that Cux1 interact with a site within the 3’ region of the p27kip1 promoter in newborn kidneys in vivo. In A controls were water (lane 2) and no antibody (lane 4). In both B and C, controls were water (lane 1) and no antibody (lane 3). In D controls were water (lane 2) and normal rabbit IgG (lane 4).

Figure 5. Two separate Cux1 binding sites on the p27^kip1 promoter

DNAse I footprint analysis of the p27^kip1 promoter regions bound by Cux1 in vivo. −687 to −496 (panel B) and −1609 to −1360 (panel C) PCR amplification products from ChIP assays were radiolabeled and incubated with purified in vitro synthesized Cux1 protein (amounts shown are in µl) and then digested with DNase1. Regions of DNA protected from DNase1 digestion are indicated by boxed regions. The corresponding sequence for each protected region is shown, with the previously identified Cux1 target sequences underlined. Panel A shows radiolabeled SV40 DNA following incubation with AP2 protein and DNase1 digestion as a positive control. Boxed region shows region of DNA protected from DNase1 digestion.