1. Volume 10, Issue 3, Pages 509-521 (September 2002)
Negative Regulation of the Mammalian UV Response by Myc through Association with Miz-1 
Steffi Herold, Michael Wanzel, Vincent Beuger, Carsten Frohme, Dorothee Beul, Tomi Hillukkala, Juhani Syvaoja, Hans-Peter Saluz, Frank Haenel, Martin Eilers 
Molecular Cell 
Volume 10, Issue 3, Pages (September 2002)
DOI: /S (02)

2. Figure 1
The POZ Domain of Miz-1 Is Required for Miz-1-Induced Growth Arrest and Transcriptional Regulation
(A) Colony formation assay using Rat1 cells. Cells were infected with the indicated viruses. Forty-eight hours after infection, the indicated aliquots were transferred and plated into selective medium. Ten days later, the plates were stained with crystal violet to visualize growing colonies. A diagram of Miz-1 is shown on the top.
(B) Activation of the p15INK4B promoter by Miz-1 and Miz-1ΔPOZ in transient transfection assays. HeLa cells were transfected with the indicated plasmids; shown are specific luciferase activities 48 hr after transfection.
(C) Western blot documenting expression of Miz-1ΔPOZ in retrovirally infected Rat1 cells (right lanes). Cells were infected and selected as in (A); extracts were prepared 10 days after the start of the selection. An asterisk designates an unspecific band recognized by the antibody. The left lanes document equal expression of Miz-1 and Miz-1ΔPOZ after transient transfection in HeLa cells.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
TopBP1 Binds to the POZ Domain of Miz-1 and Inhibits Transactivation by Miz-1
(A) Summary of two-hybrid assays using a GAL4-POZ chimera as bait.
(B) In vitro GST binding assays demonstrating POZ-dependent binding of Miz-1 to GST-TopBP1 but not to GST alone. Miz-1 and the indicated mutants were synthesized in a reticulocyte lysate in the presence of 35S-cystein and incubated with equal amounts of either GST or GST-TopBP1.
(C) Coimmunoprecipitation of endogenous Miz-1 and TopBP1 from HeLa cell lysates with either control beads, anti-Miz-1 monoclonal antibody, or an anti-TopBP1 polyclonal serum. Precipitates were blotted with the indicated antibodies.
(D) Inhibition of Miz-1-mediated transactivation of the p15INK4B promoter by coexpression of TopBP1. Transient transfection assays were carried out in HeLa cells using the reporter plasmid depicted in Figure 1B.
(E) A carboxy-terminal deletion mutant lacking BRCT repeats 7 and 8 (TopBP1ΔCT) fails to repress transactivation by Miz-1. Shown are transient transfection assays using the p15INK4B promoter and the indicated expression plasmids.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Regulation of Miz-1 and TopBP1 in Response to UV Irradiation of Human Keratinocytes
(A) Immunofluorescence picture demonstrating the intranuclear distribution of Miz-1 and TopBP1 in nonirradiated HaCaT cells, in UV-irradiated cells, and in cells irradiated in the presence of caffeine.
(B) Reverse-transcriptase-PCR assays showing the expression of MIZ-1, MYC, TOPBP1, and p15INK4B in response to UV irradiation in HaCaT cells. “−” designates cells that were mock treated and incubated for the same time.
(C) Immunoprecipitation demonstrating reduced association of endogenous Miz-1 and TopBP1 proteins 4 hr after UV irradiation. “Con” designates a control immunoprecipitation using an irrelevant antibody.
(D) Quantitation of five independent immunoprecipitation experiments performed as in (C); the amount of TOPBP1 bound to Miz-1 in the absence of UV irradiation was arbitrarily set to one.
(E) Reporter assays documenting initiator-dependent upregulation of p15INK4B expression by UV irradiation in HaCaT cells. The upper panels document p15INK4B promoter activity after UV irradiation. Cells were transiently transfected and irradiated 36 hr after transfection. At the indicated time points, luciferase assays were carried out. Shown is the specific luciferase activity in irradiated cells relative to nonirradiated cells. The lower three panels are controls documenting initiator-dependent upregulation of the p15INK4B promoter by Miz-1 (see also Staller et al., 2001) but not by Sp-1 and Smads 3 and 4.
(F) RT-PCR assays documenting expression of TOPBP1, p15INK4B, and GAPDH 4 hr after UV irradiation with or without the addition to caffeine.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Identification of Myc Mutants Specifically Deficient in Binding to Miz-1
(A) Two-hybrid assays in yeast documenting interaction of either Mycwt or the indicated point mutants with Miz-1 (left panel) and Max (right panel).
(B) Alignment of the HLH domains of human c-Myc, N-Myc, L-Myc, Max, and Mad-1 proteins indicating the position of the isolated point mutants. Arrows designate the mutants that were recovered from the loss-of-interaction screen. Open triangles designate the mutants that were designed.
(C) Conservation of the Myc/Miz-1 interface during evolution. Shown is the crystal structure of the Max homodimer. One of the polypeptide chains is color coded and shows for each amino acid the number of different amino acids found at the homologous position in vertebrate Myc proteins: green, one single amino acid found; blue, two different amino acids; white, more than two different amino acids found. Amino acids, which are conserved in Myc, Max, and/or Mad proteins, are shown in red.
(D) The left panels show reporter assays using the indicated reporter plasmids that demonstrate repression of Miz-1-mediated transactivation by either Mycwt or the indicated mutant alleles. The right panel shows E box-dependent transactivation of an E box-dependent reporter plasmid by increasing amounts of either Mycwt or MycV394D. The insert shows a Western blot documenting equal expression of all proteins after transfection into HeLa cells; note that the antibody also recognizes endogenous protein.
(E) Immunoprecipitation with anti-Miz-1 and anti-Max antibodies of Mycwt and of MycV394D from transiently transfected HeLa cells documenting specific association of MycV394D with Max but not Miz-1.
(F) The left panels show FACS profiles of pools of Rat1 cells growing in 10% FCS infected with either control virus or viruses expressing the indicated alleles of Myc. The right panel illustrates quantitation of a representative experiment showing the cell cycle distribution of pools of cells infected with the indicated viruses either in 10% FCS or 48 hr after shift to medium containing 0.1% FCS.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Negative Regulation of the Mammalian UV Response by Myc through Miz-1
(A) Two-dimensional FACS analysis documenting the response of pools of p19ARF−/− mouse embryo fibroblasts expressing Mycwt to UV irradiation.
(B) Quantitation of the proliferative (left panel) and apoptotic (right panel) response of p19ARF−/− cells expressing either Mycwt or MycV394D to UV irradiation. SubG1 cells were quantitated 48 hr after irradiation.
(C) Quantitation of BrdU incorporation of either wt, p15Ink4b−/−, or p21Cip1−/− MEFs either before or 8 or 12 hours after exposure to UV light.
(D) Western blot documenting the expression of Myc, p21Cip1, and, as loading control, Cdk2 in pools of p19ARF−/− mouse embryo fibroblasts infected with the indicated viruses either before or 4 hr after UV irradiation.
(E) Gel shift assays documenting binding of Miz-1 and TopBP1 to the start site of the p21Cip1 promoter. The left panel shows a gel shift assay using nuclear extracts from nonirradiated HeLa cells using the indicated antibodies. Arrows designate specifically binding complexes (as judged by competition with cold competitors; data not shown). Stars designate supershifted bands observed in the presence of α-Myc and α-TopBP1 antibodies. The right panels show the shift from either nonirradiated HeLa cells or cells irradiated with increasing doses of UV light; all shifts shown are preincubated with α-TopBP1 antibodies.
(F) Chromatin-immunoprecipitation assay (ChIP) documenting binding of Myc and Miz-1 to the p21 initiator in vivo; control panels show ChIP reactions for the E box of the prothymosin-α gene and the coding region of the p15Ink4b gene as negative control (Staller et al., 2001).
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Regulation of p21Cip1 Expression by Myc and Miz-1 in Response to UV Irradiation
(A) Myc does not interfere with UV-induced accumulation and phosphorylation of p53. Top: RT-PCR assays documenting the requirement for intact p53 in p21Cip1 induction in this experiment. Bottom: Western blots using the indicated antibodies documenting accumulation and phosphorylation of p53 in cells expressing Mycwt and MycV394D.
(B) RT-PCR assays documenting expression of p21Cip1 and Gapdh in p19ARF−/− MEFs treated with the indicated siRNA oligonucleotides in response to UV irradiation. A diagram of the experiment is shown at the top.
(C) RT-PCR assays documenting expression of p21Cip1 (left) and Gapdh (right) in Rat1 cells of the indicated genotype before and at the indicated times after UV irradiation.
(D) Repression of p21Cip1 expression by Myc in p19ARF+/+ cells. wtMEFs were infected with either control viruses or viruses expressing the indicated proteins, and resistant pools were selected. Shown are Western blots of lysates before or 4 hr after UV irradiation probed with the indicated antibodies.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7 A Model to Describe Our Findings
The two-input model was originally suggested to describe TGF-β regulation of p15INK4B expression by Seoane et al. (2001). We now extend this model to p53-dependent regulation of p21Cip1 in response to UV irradiation. For details, see text.
Molecular Cell  , DOI: ( /S (02) )