Transcription Factor MIZ-1 Is Regulated via Microtubule Association Joseph Ziegelbauer, Bei Shan, Deborah Yager, Carolyn Larabell, Björn Hoffmann, Robert Tjian  Molecular Cell  Volume 8, Issue 2, Pages 339-349 (August 2001) DOI: 10.1016/S1097-2765(01)00313-6

Figure 1 T1 Effect on LDLR Expression (A) Structure of T1. (B) Endogenous LDLR gene expression was determined by Northern and Western assays after 2.5 μM T1 treatment Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 2 MIZ-1 Binds the LDLR Promoter (A) Footprinting experiments were conducted with increasing amounts of purified recombinant MIZ-1, incubated with 32P end-labeled LDLR promoter fragments, and treated with DNaseI. Lane 3 contains probe digested with DNaseI without MIZ-1. Lanes 1 and 2 are size markers. A diagram of the LDLR promoter shows the MIZ-1 activated start site and the binding sites of MIZ-1, Sp1, and SREBP. M1 and M2 refer to the mutations discussed in Figure 3. (B) Band shift experiments: purified recombinant MIZ-1 (even lanes) or buffer (odd lanes) was incubated with 32P-labeled oligonucleotides containing different LDLR promoter sequences. The control sequence (ctrl) contains the SREBP and Sp1 binding sites of the LDLR promoter. M-A and M-B are sequences containing the MIZ-1 binding sites (MBS-A and MBS-B) identified in Figure 2A Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 3 MIZ-1 Activation of LDLR (A) In vitro transcription of the LDLR-CAT reporter was assayed in the absence (lane 1) or presence of purified recombinant MIZ-1 (lane 2). (B) Purified recombinant MIZ-1 (lane 2) was added to in vitro transcription reactions containing an LDLR promoter-reporter construct and purified basal transcription factors. (C and D) Purified recombinant MIZ-1 (even lanes) was added to transcription reactions containing either the MBS-B sequence or mutants fused to a reporter construct. Basal transcription factors were supplied from crude HepG2 nuclear extracts (C) or purified basal factors (D). Transient transfection experiments assaying MIZ-1 and T1 effect. (E) Transient transfection experiments used HepG2 cells transfected with increasing amounts of a MIZ-1 expression vector. Either the MBS-B site (BSB) or mutant reporter construct (BM2) was fused into a luciferase reporter vector. (F) HepG2 cells were transiently transfected with a LDLR reporter and a MIZ-1 expression construct. Cells were treated with T1 at 0.2 μg/ml for 14 hr and harvested, and luciferase values of lysates were determined. Fold activation is relative to cells transfected only with reporter constructs without T1 treatment Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 4 MIZ-1 Binds the α2 Integrin Promoter and Activates Transcription (A) Footprinting experiments were conducted with increasing amounts of purified recombinant MIZ-1 (lane 4), incubated with 32P end-labeled α2 integrin promoter fragments, and treated with DNaseI. Lane 2 contained probe digested with DNaseI without MIZ-1. Lane 1 contains the same probe chemically cleaved at guanine and adenosine residues. A diagram of the α2 integrin promoter shows the MIZ-1-activated transcription start site and the binding site of MIZ-1. (B) In vitro transcription of the α2 integrin CAT reporter was assayed in the absence (lane 1) or presence (lanes 2–4) of purified recombinant MIZ-1 (lane 4) using HepG2 nuclear extract as a source of basal factors. (C) Transient transfection experiments used HepG2 cells transfected with an α2 integrin luciferase reporter vector and increasing amounts of a MIZ-1 expression vector Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 5 MIZ-1 Associates with Microtubules In vitro microtubule pelleting assays were performed with (A) purified MIZ-1 and microtubules. (B and C) HepG2 whole-cell lysates with exogenous purified microtubules added or endogenous microtubules stabilized by taxol or (D) in vitro translated 35S-labeled protein representing various MIZ-1 deletion or mutation constructs. After incubations with various forms of MIZ-1 and microtubules, the polymerized microtubules and associated proteins were collected by centrifugation. Equivalent amounts of supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE. (A) Purified recombinant MIZ-1 and SREBP (Näär et al., 1998) were detected by immunoblotting. Reactions shown in lanes 1–4 were conducted in the absence of purified microtubules. Lanes 5–8 contained samples from reactions containing taxol stabilized microtubules and MIZ-1 or SREBP. (B) Coomassie-stained gel shows microtubule pelleting products. Lane 1 shows precleared input (I). Lanes 2 and 4 show supernatant and pellet fractions from microtubule pelleting assays that included exogenous microtubules. Endogenous microtubules were stabilized with taxol (lanes 3 and 5). The asterisk (*) in (B) identifies tubulin bands. (C) Samples shown in (B) were analyzed by immunoblotting using either anti-MIZ or anti-SREBP antibodies. (D) Microtubule pelleting assays were performed as in (A) except 35S-labeled in vitro-translated proteins representing various MIZ-1 deletions and mutations as diagramed were detected by phosphorimager analysis Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 6 X-Ray and Immunofluorescence Microscopy of MIZ-1 and Microtubules (A) HepG2 cells were fixed and stained for endogenous MIZ-1. The right panel is a diagram of the MIZ-1 staining pattern. Scale bar = 1 μm. (B) Triton extracted HepG2 cells were fixed and stained for endogenous MIZ-1 and tubulin and analyzed by indirect immunofluorescence microscopy. Shown is a cytoplasmic region of a single cell Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)

Figure 7 Cytoplasmic to Nuclear Accumulation of MIZ-1 upon T Treatment (A) HepG2 cells were transfected with MIZ-1 expression constructs, treated with vehicle alone or T1, fixed, and stained for MIZ-1, tubulin, and DNA using indirect immunofluorescence. (B) HepG2 cells were transfected with MIZ-1 expression plasmids and treated for 6 hr with DMSO, taxol (tax.), T1 (T), taxol and T1 (tax. + T), colchicine, or vinblastine. Cells were fixed. MIZ-1 was visualized by indirect immunofluorescence, and cells were scored for either cytoplasmic or nuclear localization of MIZ-1. (C) Untransfected HepG2 cells were treated with vehicle alone or T1 for 8 hr, fixed, and stained for endogenous MIZ-1 and tubulin. (D) HepG2 cells were transfected with MIZ-1 expression vectors containing a V5 epitope tag. Cells were treated with T1 for 8 hr, fixed, stained using V5 and tubulin primary antibodies and DAPI. (E) Cells were scored for cytoplasmic or nuclear localization of the V5 signal representing MIZ-1 localization of full-length or ΔPOZ deletion mutant containing amino acids 104–803. (F) Living transfected cells were scored for cytoplasmic or nuclear localization of MIZ-1 during T1 treatment. (G) Images of the same field containing living cells shown at right were taken at the beginning and end of T1 treatment Molecular Cell 2001 8, 339-349DOI: (10.1016/S1097-2765(01)00313-6)