Figure 4. Identification of MZF1 as a CHIP-regulated transcription factor in breast cancer cells.
(A) DNA-binding activities of 345 transcription factors were analyzed in nuclear extracts of CHIP-lo (MSCV-puro control) vs. CHIP-hi (MSCV-CHIP) BT474 and MDA-MB-231 cell lines. Y-axis, loge-fold binding in CHIP-hi over CHIP-lo cells. The 3-fold increase (upper) or decrease (lower) in binding (dotted lines) was used as cut-off. MZF1 (Open symbol). (B, C) Validation of CHIP-dependent downregulation of protein levels of selected transcription factors identified in A by western blotting of CHIP-lo vs. CHIP-hi BT474 (B) or MDA-MB-231 (C) cell lysates. (D) Real-time qPCR analysis of MZF1 mRNA levels in CHIP-hi vs. CHIP-lo BT474 cells. (E) A biotin-labeled double-stranded oligonucleotide with MZF1-specific DNA sequence was used to probe nuclear extracts of CHIP-hi (MSCV-CHIP, C) vs. CHIP-lo (MSCV-puro, P) BT474 cells. 200-fold excess of the non-labeled oligonucleotide served as a competitor. (F) Immunoblot analysis of MZF1 protein levels in CHIP-hi vs. CHIP-lo BT474 cells. (G) CHIP-dependent ubiquitination and degradation of MZF1. Anti-MZF1 immunoprecipitations from lysates of HEK-293T cells transfected with GFP-MZF1 (1μg) +/- Myc-CHIP (1 or 2 μg) were immunoblotted for ubiquitin or MZF1 (upper panel), and whole cell lysates (lower panel) were blotted for MZF1, CHIP or HSC70 (loading control). (H, I) HEK-293T cells were transfected with MZF1-GFP (250 ng) +/- Myc-CHIP (1 μg). Cells were treated with vehicle (-) or proteasomal inhibitor MG132 (50 μM) or lysosomal inhibitor bafilomycin A1 (100 nM) for 6 or 16 hr., respectively. Lysates were immunoblotted for MZF1 or CHIP. (J, K) Quantitation of the MZF1 levels in CHIP-co-transfected cells in the presence of MG132 or Bafilomycin A1, relative to vehicle controls assigned a value of 1. Data shown represent mean +/- S.D. of n=3; * = p<0.05, ** = p<0.01, *** = p<0.001. (L) The tetratricopeptide repeat (TPR) domain of CHIP is required for binding to MZF1. HEK-293T cells transfected with MZF1-GFP and their lysates used for pulldown with GST or the indicated GST-CHIP fusion proteins. Bound MZF1-GFP was visualized by immunoblotting for GFP (upper panel) or MZF1 (middle panel). Ponceau Red staining of the membrane shows the relative amounts of fusion proteins used in pulldowns (lower panel).