HDAC4 protein expression in normal intestinal epithelium and colon cancer cell lines. (A) Total protein was extracted from various tissues from a wild-type C57BL/6 mouse and levels of HDAC4 quantified by Western blot. Blots were reprobed for actin to ensure equal loading. Immunohistochemical detection of HDAC4 and Ki67 expression in a low-power (10×) longitudinal section of normal human small intestine (B) and HDAC4 in the distal colon of a C57BL/6 mouse (C). (D) Epithelial cells were sequentially isolated along the mouse crypt–villus axis as 10 individual fractions (F), with F1 and F10 representing cells isolated from the villus tip and crypt base, respectively, and HDAC4 and PCNA protein levels determined. (E) Caco-2 cells were allowed to spontaneously differentiate over 21 d, and levels of HDAC4 and PCNA were determined at confluence (day 0) or 7 and 21 d after confluence. (F) LS174T-N2 cells, which are stably transfected with doxycycline-inducible dominant-negative TCF4, were cultured with and without doxycycline for 6–96 h. Doxycycline was replaced every 24 h in fresh medium. Levels of HDAC4 and PCNA protein were quantified by Western blot. (G) Comparison of steady-state levels of p21 mRNA in Weiser F1 and F10 fractions, control and doxycycline-treated LS174T cells (96 h) and day 0 and day 21 Caco-2 cells. Expression of p21 mRNA was calculated relative to an actin mRNA internal standard by QPCR.

Effect of HDAC4 down-regulation on survival of colon cancer cells in vitro. (A) The effect of NT and siHDAC4 (both 100 nM) on subdiploid (apoptotic) cell population assayed by flow cytometry. Values shown are mean + SEM of three independent experiments; p < 0.05 relative to NT, Student's t test. (B) Effect of HDAC4 down-regulation on cleavage of PARP. (C) Cells were treated with NT or siHDAC4 (100 nM) for 72 h, and cytochrome c release was assayed by immunofluorescence. Values were obtained from counts of 200 cells and are expressed as the mean + SEM of three independent experiments; *p < 0.05 relative to NT, Student's t test. (D) Representative fields of NT and siHDAC4-treated cells. 4,6-Diamidino-2-phenylindole (DAPI)-stained nuclei and cytochrome c (cyto C) were detected, and an overlay was generated. The white arrowhead shows a cell releasing cytochrome c into the cytosol, as indicated by a diffuse staining pattern. (E) Clonogenic assay showing the effect of a 72-h incubation with siHDAC4 (100 nM) on formation of cell colonies compared with NT-treated cells. (F) Quantification of colony number from E. Results shown are mean + SEM of three independent experiments; *p < 0.05 relative to NT, Student's t test.

Effect of cell proliferation on subcellular localization of HDAC4. (A) HCT116 cells were transfected with HDAC4-GFP, and serum-starved for 24 h. Cells were then either maintained in 0% serum, or they were serum-pulsed (10%) for 16 h. Representative histograms after flow cytometric analysis are shown. (B) Representative fields of HDAC4-GFP 1-1084-transfected cells (green) after serum starvation, or after serum pulse. Nuclei are stained with DAPI (blue). (C) The distribution of HDAC4-GFP after serum starvation, or serum pulse was analyzed by counting 200 GFP-positive cells per treatment. The distribution was categorized as either nuclear or exclusively cytoplasmic. Values shown are mean + SEM of three independent experiments; *p < 0.05 relative to no serum, Student's t test. (D) Endogenous HDAC4, HDAC1, HDAC6, and PCNA protein levels in nuclear and cytosolic fractions were determined by Western blot after subcellular fractionation of serum-starved and serum-pulsed cells.

HDAC4 regulation of p21 promoter activity in colon cancer cells. (A) Schematic representation of the HDAC4 protein, showing the NLS, NES, and HDAC catalytic domain. (B) Expression of the full-length and deletion HDAC4 GFP-tagged constructs (all 1 μg) in HCT116 cells was determined by anti-GFP Western blot. Also shown are immunofluorescence photomicrographs of the localization of the various HDAC4-GFP deletion mutants used. (C) Effect of HDAC4 overexpression on basal or 2 mM butyrate-induced p21 promoter activity after 24 h. HCT116 cells were cotransfected with pWP-133 (0.25 μg), TK-Renilla (0.1 μg), and increasing concentrations of HDAC4-GFP (1-1084) or the empty vector control (0–1 μg). Values shown are mean + SEM, and they are expressed as a percentage of pWP-133 activity relative to appropriate empty vector controls (empty bars) for basal p21 activity, and as -fold stimulation of p21 promoter activity for butyrate induction. (D) Effect of the HDAC4-GFP deletion constructs on basal or 2 mM butyrate-induced (24-h) p21 promoter activity. HCT116 cells were cotransfected with pWP-133 (0.25 μg), TK-Renilla (0.1 μg), and 1 μg of the HDAC4-GFP full-length and deletion constructs, or the empty vector control (1 μg). Values shown are mean + SEM, and they are expressed as a percentage of pWP-133 activity relative to corresponding empty vector controls (empty bars) for basal p21 activity, and as -fold stimulation of p21 promoter activity for butyrate induction.

Role of Sp1 in HDAC4-mediated repression of p21. (A) Effect of the Sp1 inhibitor mithramycin on siHDAC4 and butyrate-mediated p21 induction. p21 protein levels in HCT116 cells treated with mithramycin (1 μg/ml) for 24 h in combination with either a 72-h exposure to NT siRNA or siHDAC4 (100 μM), or a 24-h exposure to 2 mM butyrate. The effect of down-regulation of Sp1 on induction of p21 by siHDAC4 or butyrate is shown in B and C, respectively. p21, HDAC4, and Sp1 protein levels in HCT116 cells were determined after 72-h treatment with NT siRNA or siHDAC4 with or without concomitant treatment with siRNA targeting Sp1. All cells were treated with an equal final concentration of siRNA (100 nM). (D) Effect of Sp1 down-regulation on p21 promoter activity stimulated by siHDAC4 or butyrate, as determined by luciferase assay. HCT116 cells were transfected with pWP-133 (0.25 μg) and either NT siRNA siHDAC4 or siSp1 (total siRNA transfected was 100 nM), and cultured for 72h. In separate experiments, cells were transfected with pWP-133 (0.25 μg) and either NT siRNA or siSp1 (both 100 nM), and they were cultured with and without 2 mM butyrate for 24 h. TK-Renilla (0.1 μg) was cotransfected in all treatment groups to control for transfection efficiency. Values shown are mean + SEM of three independent experiments; *p < 0.05, Student's t test.

HDAC4 and Sp1 co-occupancy of the proximal p21 promoter. Sequential ChIP analysis in HCT116 cells (see Materials and Methods) after initial immunoprecipitation with anti-HDAC4, followed by a second immunoprecipitation with IgG, no antibody, or anti-Sp1 (A), or initial immunoprecipitation with anti-Sp1 followed by a second immunoprecipitation with IgG, no antibody, or anti-HDAC4 (B). Whether HDAC4 and Sp1 were simultaneously present at the proximal p21 promoter (p21-1), at a region 4-kb upstream from the transcriptional start site of the p21 promoter (p21-up) or at the actin promoter was tested by QPCR. All values were initially expressed relative to relevant Input DNA content.

Effect of HDAC4 down-regulation on growth of colon cancer cells in vitro. (A) Selective down-regulation of HDAC4 expression in HCT116 cells treated for 72 h with the nontargeting siRNA duplex (NT) or a pool of two siRNAs targeting HDAC4 (siHDAC4). Both NT and siHDAC4 were added to a final concentration of 100 nM. Protein levels of HDAC1, HDAC2, HDAC3, HDAC4, and HDAC6 were determined by Western blot. (B) QPCR analysis of the effect of siHDAC4 on mRNA expression of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC10. N/D denotes no mRNA expression for HDAC8 and HDAC9 detected. Results are expressed relative to actin mRNA and are expressed as the percentage of mRNA expression in control (NT) transfected samples. (C) Time course analysis of the effects of NT and siHDAC4 (both 100 nM) on the percentage of cells in G0/G1, S and G2/M phase, determined by flow cytometry. (D) Time course analysis of the effects of NT and siHDAC4 (both 100 nM) on adherent cell number in HCT116 cells. (E) Time course analysis of the effects of NT and siHDAC4 (both 100 nM) on MTT incorporation in HCT116 cells. C–E show the results of three experiments, expressed as mean + SEM; *p < 0.05 relative to relevant control, Student's t test.

Effect of HDAC4 down-regulation on growth of colon cancer cells in vivo. (A) Representative tumors obtained at sacrifice after 7 d of growth of HCT116 cell xenografts in 4-wk-old male SCID mice (bar, 1 cm). HCT116 (5 × 10⁶) cells were transfected with NT siRNA or siHDAC4 (both 100 nM) and injected into the right flank with an equal volume of Matrigel. (B) Tumor volume was quantified from measurements of the longest and smallest diameter of the tumor. Values shown are mean + SEM, n = 8; *p < 0.05 relative to NT, Student's t test. (C) HDAC4 protein levels in HCT116 cells transfected with NT or siHDAC4 and cultured in parallel in vitro for up to 7 d.

HDAC4 regulation of p21 expression in colon cancer cells in vitro. (A) The effect of two independent siHDAC4 duplexes #1 and #2 and a pool of #1 and #2 (designated siHDAC4 and used throughout the study) on HDAC4 and p21 protein expression, compared with the negative controls, NT, siNEG, and siGFP (all 100 nM for 72 h). Additionally, the effect of an independent pool of three siRNA duplexes targeting HDAC4 (siHDAC4 sc) on HDAC4 and p21 protein expression was compared with the negative controls, NT A and NT C (all 100 nM for 72 h). See Materials and Methods for a detailed description of these siRNAs. Protein levels of p21 and HDAC4 were determined by Western blot. (B) The steady-state mRNA levels of HDAC4 and p21 in HCT116 cells treated for 36 h with NT or siHDAC4 (both 100 nM), determined by QPCR. Values are mean + SEM of three replicates, and they are expressed relative to β-actin. *p < 0.05 relative to NT1, Student's t test. (C) Effect of HDAC4 down-regulation on p21 promoter activity, as determined by luciferase assay. HCT116 cells were transfected with the p21 luciferase reporter plasmid, pWP-133 (0.25 μg), and either NT or siHDAC4 (100 nM) for 72 h. TK-Renilla (0.1 μg) was cotransfected in all treatment groups to control for transfection efficiency. Values shown are mean + SEM of three independent experiments; *p < 0.05 relative to NT1, Student's t test. (D) The steady-state mRNA levels of p21 in HCT116 cells treated for 24 h with NT or siHDAC4 (both 100 nM), with and without concomitant addition of 5 μg/ml cycloheximide. mRNA expression was determined by QPCR. Values are mean of a representative experiment, and are expressed relative to β-actin. *p < 0.05 relative to NT1, Student's t test. (E) The effect of protein synthesis inhibition on p21 protein induction mediated by HDAC4 down-regulation. HCT116 cells were treated for 48 h with NT or siHDAC4 (both 100 nM), with and without concomitant addition of 5 μg/ml cycloheximide for a subsequent 24 h.

Role of p21 in HDAC4 siRNA-induced growth arrest and apoptosis induction. (A) Analysis of the effects of NT siRNA and siHDAC4 (both 100 nM) on adherent cell number and the percentage of cells in S phase determined by flow cytometry, in p21-null and wild-type HCT116 cells. Results are expressed as percentage of inhibition mediated by siHDAC4 72 h after transfection, and they are mean + SEM of three experiments. *p < 0.05 relative to effect in p21 wild-type cells, Student's t test. (B) Protein levels of HDAC4, PCNA, and p21 in p21 wild-type and null cells transfected with either NT siRNA or siHDAC4 (100 nM). (C) The role of p21 in apoptosis induction mediated by HDAC4 down-regulation for 72 h. Apoptosis was measured as subdiploid HCT116 cell content by flow cytometry. Values are mean + SEM of three experiments.

HDAC4 binds to the p21 promoter. (A) ChIP analysis of HDAC4 binding to the pWP101 p21 luciferase reporter plasmid template. HCT116 cells were transiently transfected with pWP101 and harvested after 48 h. Primer sets were designed to interrogate the 101 base pairs immediately proximal to the transcription start site of the p21 promoter, which contains four of the six Sp1 binding sites (pWP101-p21) and a separate region of the vector backbone ∼2 kb away (pWP101-up). (B) ChIP DNA was analyzed by PCR amplification and 1% agarose gel electrophoresis. NTC, no template control. (C) ChIP analysis of HDAC4 binding to the endogenous p21 promoter. Primer sets probing the proximal region of the p21 promoter, adjacent to the six Sp1 binding sites, were used (p21-1 and p21-2), as were primers probing a region of the p21 promoter 4Kb upstream from the transcriptional start site (p21-up), or the actin promoter. DNA content after immunoprecipitation with HDAC4 or Sp1 antibodies, or no antibody or nonspecific antibody (IgG) controls, were determined by real-time RT-PCR. All values were expressed relative to Input DNA content. (D) ChIP analysis of HDAC4 and Sp1 binding to the p21-1 promoter locus following transfection of cells with NT siRNA, siHDAC4 or siSp1 (all 100 nM) for 72 h. All values were expressed relative to Input DNA content. (E) ChIP analysis of histone H3 acetylation at the loci described above after transfection of cells with NT siRNA, siHDAC4, or siSMRT (all 100 nM) for 72 h. All values were expressed relative to Input DNA content. (F) Selective down-regulation of SMRT protein and parallel induction of p21 protein expression in HCT116 cells. HCT116 cells were transfected with 100 nM NT siRNA of siSMRT for 72 h. (G) Effect of SMRT down-regulation on p21 promoter activity, as determined by luciferase assay. HCT116 cells were transfected with pWP-133 (0.25 μg) and 100 nM of either NT siRNA or siSMRT and cultured for 72 h. TK-Renilla (0.1 μg) was cotransfected in all treatment groups to control for transfection efficiency. Values shown are mean + SEM of three independent experiments; *p < 0.05, Student's t test. (H) ChIP analysis of the binding of HDAC3 and HDAC4 to the p21-1 promoter locus after transfection of cells with 100 nM NT siRNA, siHDAC4, or siSMRT for 72 h. All values were expressed relative to Input DNA content.

HDAC4 associates with and represses Sp1 transactivity. (A) Overlap of HDAC4-GFP and endogenous Sp1 in HCT116 cells as determined by immunofluorescence analysis utilizing confocal microscopy. (B) HCT116 cells were transfected with HDAC4-GFP (1-1084) for 4 8h, and 300 μg of nuclear extract was immunoprecipitated with anti-GFP or IgG control antibodies. Immunoprecipitates were probed for Sp1 and GFP content by Western blot. Equal amounts of input protein were demonstrated by GFP Western blot. (C) HCT116 cells in the growing phase were harvested, and 300 μg of nuclear extract immunoprecipitated with anti-Sp1 or IgG control antibodies. Immunoprecipitates were probed for HDAC4 content by Western blot. Equal amounts of input protein were demonstrated by Sp1 Western blot. (D) Effect of HDAC4 down-regulation or HDAC4 overexpression on activity of Sp1/Sp3 reporter constructs containing either three consensus Sp1/Sp3 binding sites (Sp1-luc) or three mutant binding sites (mtSp1-luc). HCT116 cells were cotransfected with Sp1-luc or mtSp1-luc (0.1 μg) in combination with TK-Renilla (0.1 μg), NT siRNA or siHDAC4 (100 nM) for 72 h. In separate experiments, HCT116 cells were cotransfected with Sp1-luc or mtSp1-luc (0.1 μg) in combination with HDAC4-GFP 1-1084 or empty vector control (1 μg), and TK-Renilla (0.1 μg) for 24 h. Values shown are the mean + SEM of a representative experiment. (E) A one-hybrid system was also used in which active Sp1 (GAL4-Sp1) or inactive (GAL4-DNSp1) was fused to GAL4, and transactivation was measured using a luciferase reporter linked to five consensus GAL4 DNA binding sites. HCT116 cells were cotransfected with GAL4-Sp1 or GAL4-DNSp1 (0.1 μg) in combination with TK-Renilla (0.1 μg), NT or siHDAC4 (100 μM) for 72 h. In separate experiments, HCT116 cells were cotransfected with GAL4-Sp1 or GAL4-DNSp1 (0.1 μg) in combination with HDAC4-GFP 1-1084 or empty vector control (1 μg), and TK-Renilla (0.1 μg) for 24 h. Values shown are mean + SEM of a representative experiment.