Attenuation of Forkhead signaling by the retinal determination factor DACH1

Abstract

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant ellipse, encodes a key component of the retinal determination gene network that governs cell fate. Herein, cyclic amplification and selection of targets identified a DACH1 DNA-binding sequence that resembles the FOX (Forkhead box-containing protein) binding site. Genome-wide in silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP coupled with high-throughput sequencing mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the cyclic amplification and selection of targets-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis.

Fig. 1.

Identification of DACH1-response element. (A) The ³²P-labeled oligonucleotide of DACH1 binding sequence (DACH1 probe) was used in EMSA. The probe migration was retarded by DACH1 binding (lane 1). The protein/DNA complex was competed by 100-fold cognate cold competitor (lane 2). The addition of anti-FLAG antibody delayed mobility of the DNA–protein complex (lane 4). The DACH1 DBD deletion (ΔDBD) failed to bind the DNA probe (lanes 5–7). (B) Immunopurified DACH1 was incubated with DACH1 probe. The DNA–protein complex was competed by cold probe (lane 2) and the mobility of the complex was delayed by anti-FLAG antibody (lane 4 vs. 3). (C) In vitro–translated DACH1 or ΔDBD were incubated with DACH1 probe. No addition of DNA (lane 1) or empty vector DNA (lane 2) was included as a negative control. The DACH1/DNA complex (lane 3) was supershifted by anti-FLAG antibody (lane 6) compared with IgG control (lane 5). Deletion of DBD abolished DNA binding (lane 7).

Fig. 2.

Genome-wide in silico promoter analysis. (A) Sequence logo depicts sequence conservation of the template sequence for all single-mismatch hits. (B) The clustered gene expression data from MDA-MB-231 breast cancer cells for the exact and single-mismatch genes with minimal expression in cyan and high expression in red. The yellow bars indicate the cutoffs between minimal, low, medium, and high expression subsets. (C) Transcription factors that are enriched for the medium low, medium high, and high expression subsets of the genes in MDA-MB-231 cells with exact match or single-mismatch DACH1 binding site templates. Homology refers to the percentage of genes within the subset that possess the element transcription factor binding site. The P values were calculated using the hypergeometric test (P < 0.05).

Fig. 3.

DACH1 inhibits expression of FOXM1 targeted genes. (A) Western blot analysis of DACH1 protein abundance in breast cancer cell lines. (B) ChIP assay was conducted with Ponasterone A–inducible DACH1 MDA-MB-231 cells with either the antibody against FLAG epitope (FLAG tag in the aminoterminus of DACH1) or control IgG. DACH1 occupies the FOXM1-binding sites of G₂/M regulatory proteins included in ChIP assays. (C) DACH1 inhibits the transcriptional activity of G₂/M regulatory genes. The promoter sequences of the Cdc25B-950, Skp2, and E-cadherin genes were linked to the luciferase reporter. The reporter was transfected together with vectors expressing WT DACH1 and DBD-deletion mutant (ΔDBD). The data are shown as mean ± SD.

Fig. 4.

DACH1 competes with FOXM1 for DNA binding and inhibits FOXM1-mediated contact-independent growth. (A) The Dox-inducible U2OS/FOXM1 cells were transduced with a DACH1 retroviral vector. Cells were treated with either Dox or vehicle control. The transcript abundance of G₂/M regulatory genes including Skp2 and CENPB were measured with RT-PCR. β-Actin served as an internal loading control. (B) A Ponasterone A–inducible MDA-MB-231 cell line was induced to express DACH1. ChIP analysis was conducted for the G₂/M regulatory genes promoters using antibody to FOXM1 or DACH1 (anti-FLAG) as indicated. FOXM1 occupancy at the promoters of G₂/M regulatory genes was reduced following DACH1 recruitment. (C) HEK 293T cells were cotransfected with the indicated luciferase reporter genes together with expression vectors FOXM1, DACH1, or control vector. DACH1 repressed FOXM1-induced Cdc25B promoter activity. (D) U2OS cells stably expressing FOXM1 were transduced with MSCV-DACH1-IRES-GFP and vector control. Colony growth was measured by size and number. DACH1 expression reduced the FOXM1-induced colony formation.

Fig. 5.

Genome-wide identification of DACH1 DNA binding by ChIP-Seq. (A) Distribution of DACH1 binding sites in the resulting nonredundant and non-overlapping classes of regions by ChIP-Seq analysis within a promoter of 2 kb upstream of transcription start site. (B) Overlap between in silico and ChIP-Seq analysis of target genes. (C) DACH1-dependent tag density at selected gene promoter that contains DACH1 binding sites defined by the ChIP-Seq. Arrow indicates the start and direction of transcription. (D) Validation of a subset of genes whose promoters contain DACH1 binding site(s).