Requirement for chromatin-remodeling complex in novel tumor suppressor HIC1-mediated transcriptional repression and growth control

Abstract

HIC1 is a newly discovered tumor suppressor and transcriptional repressor that is frequently silenced in human tumors. HIC1 protein expression has been linked to better outcomes in breast cancers. The molecular mechanism underlying HIC1-mediated transcriptional and growth suppression, and the relevant targets of HIC1-mediated transcriptional modulation, is currently unclear. We have identified an HIC1 DNA-binding site in E2F-responsive gene promoters and demonstrate that HIC1 targets E2F-responsive genes for transcriptional regulation and growth suppression. We and others have recently discovered that Brg1, a central component of the SWI/SNF chromatin-remodeling family, is required for the transcriptional regulation of multiple cell cycle control-related genes, including E2F-responsive promoters. We studied HIC1 interactions with, and dependence upon, Brg1 activity, and found that HIC1 can recruit Brg1 to E2F-responsive promoters and that its transcriptional repression of these genes is dependent upon Brg1. These data indicate that HIC1 is a central molecule in a novel mechanism controlling cell growth and that the disruption of this HIC1-mediated pathway may lead to abnormal cell proliferation and, ultimately, cancer.

Figure 1

HIC1 interacts with Brg1. (a) Extracts of HSF8 cells were analysed by immunoprecipitation (IP), PAGE and immunoblotting (IB) using the indicated antibodies. CE represents cell extract before IP. (b) Extracts from MCF7 cells (control) or MCF7 cells transfected with Flag-tagged HIC1 (Flag-HIC1) were analysed by PAGE and IB, using the indicated antibodies. (c) Cell extracts of MCF7 cells transfected with Flag-HIC1 shown in panel b were immunoprecipitated by the indicated antibodies, followed by PAGE and IB using the antibodies shown. (d) The ³⁵S-labeled proteins indicated were incubated with GST or the indicated fusion proteins, followed by pull-down analysis using standard protocols. RL represents the ³⁵S-labeled proteins without binding. GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

Figure 2

HIC1 represses E2F-mediated transcription of endogenous genes. (a) HSF8 cells were transfected with empty vector (control) or the indicated expression vector plasmids. The cells were collected 72 h after transfection and the transcriptional activity of endogenous Sirt1, E2F1 and cFos (negative control) genes was analysed by quantitative RT–PCR, using standard protocols of the supplier. The experiments were repeated four times, yielding comparable results, and error bars shown represent the s.e.m. from all four assays. β-Actin expression was examined in each experiment as an internal control and used for normalization. The relative expression in the control non-transfected cells was assigned a value of 10. (b) The occupancy of HIC1 at the E2F1 and cFos promoters in unsynchronized actively growing HSF8 cells was analysed by ChIP assay with quantitative PCR, using an anti-HIC antibody (or anti-tubulin antibody as a specificity control). Relative quantitation of input DNA (not immunoprecipitated) is shown as ‘total.’ All the experiments were repeated four times and the averages are shown, with error bars representing s.e.m. ChIP, chromatin immunoprecipitation; RT–PCR, reverse transcriptase PCR.

Figure 3

Brg1 is required for HIC1-mediated transcriptional repression. (a) Brg1-negative SW13 cells were transfected with an empty vector (control) or the indicated expression vector plasmids. The cells were collected 72 h after transfection and the expression of the Sirt1, E2F1 and cFos (negative control) genes was analysed by quantitative RT–PCR. The relative expression of these genes was calculated and normalized to the expression of β-actin. Error bars represent the s.e.m. from four independent assays. (b) Promoter association of Brg1 and HIC1 was examined by quantitative ChIP and double-ChIP assays, using the indicated antibodies. Relative association, as reflected by the quantitative PCR values, was calculated and normalized to the input DNA results. Error bars represent s.e.m. from four independent assays. ChIP, chromatin immunoprecipitation; RT–PCR, reverse transcriptase PCR.

Figure 4

Knockdown of Brg1 abrogates HIC1-mediated transcriptional repression. (a) HSF8 cells were transfected with the empty control vector, HIC1, a dominant-negative Brg1 vector or a vector-based Brg1 siRNA, followed by quantitation of transcripts to measure the activity of the endogenous HIC1-responsive promoters, and the non-responsive control promoter (cFos), as indicated. (b) The cells were analysed by immunoblot using the indicated antibodies, which confirmed the depletion of Brg1 by the transfected siRNA. (c) Quantitative RT–PCR was performed for the genes indicated, which further confirmed the depletion of Brg1 by the siRNA. As a control, the tubulin gene was not affected by the Brg1 siRNA, verifying the specificity. A second Brg1-specific siRNA, purchased from Santa Cruz, produced similar results (data not shown). RT–PCR, reverse transcriptase PCR; siRNA, small interfering RNA.

Figure 5

HIC induces the recruitment of Brg1 to responsive genes. (a) HSF8 cells were transfected with empty vector (control), an HIC1 expression vector and/or a vector-based Brg1 siRNA. The cells were collected 72 h after transfection and analysed by quantitative ChIP assay of the Sirt1, E2F1 or cFos (control) gene, using the indicated antibodies. (b) As a positive control, SP1 association with the cFos promoter in the same cells explained above was analysed using quantitative ChIP assay. ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA.

Figure 6

HIC1 is required for the recruitment of Brg1 to responsive genes. (a) HSF8 cells were transfected with empty vector (control), vector-based HIC1 siRNA or control siRNA. The cells were collected 72 h after transfection followed by immunoblot analysis using the indicated antibodies (tubulin as control), which confirmed the depletion of HIC1 by siRNA. (b) The cells were analysed by quantitative RT–PCR for the indicated genes (tubulin as control), which further verified the depletion of HIC1 by siRNA. (c) Quantitative ChIP assay using the indicated antibodies and quantitative RT–PCR were performed for the E2F1 gene. ChIP, chromatin immunoprecipitation; RT–PCR, reverse transcriptase PCR; siRNA, small interfering RNA.

Figure 7

HIC1 is required for serum deprivation-induced growth arrest. HSF8 cells were stably transfected with an empty vector, or control siRNA, or HIC1 siRNA. The cells were serum starved for 72 h and their cell cycle profiles were analysed by propidium iodide staining and flow cytometry. The experiments were independently repeated four times, yielding similar results. Profiles from one representative experiment are shown, with the percentages of cells in each phase of the cell cycle derived from the four independent experiments indicated. siRNA, small interfering RNA.

Figure 8

Cell cycle dependency of HIC1 versus E2F promoter occupancy. (a) HSF8 cells were serum starved for 72 h (G₀), and one portion was then treated with serum for 12 h (late G₁). The E2F1 and HIC1 genes in these HSF8 cells were analysed for transcription factor occupancy by quantitative ChIP or double-ChIP assays, using the indicated antibodies. (b) The E2F1 and HIC1 genes in these HSF8 cells were analysed for activity by quantitation of transcripts. The experiments were repeated four times, which yielded comparable results, and the error bars shown represent the s.e.m. from all four assays. ChIP, chromatin immunoprecipitation.