MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-6

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>, MOZ-TIF2, and vector alone. After 36 hours, the cells were lysed and co-immunoprecipitation conducted by an anti His-tag antibody conjugated to agarose beads. The co-immunoprecipitated proteins were separated by SDS-PAGE and western analyses were performed with an anti-CBP antibody. The input represents 5% of the protein used in the co-immunoprecipitation and was from cells transfected with MOZ-TIF2. . Western blot showing MOZ and MOZ-TIF2 bound to the agarose beads. . Transfected cells subjected to SDS-PAGE to demonstrate the presence of CBP in the transfected HEK293 cells. . Western blot analysis to demonstrate CBP co-immunoprecipitating with MOZ and MOZ-TIF2. and To determine the presence of MOZ-TIF2 in ER activation complexes, HEK 293 cells were transiently co-transfected with pEGFP-MOZ-TIF2 or pEGFP-TIF2 and with an expression plasmid for the estrogen receptor, RSV-hER. After the addition of 50 nM estradiol for 36 hours, the cells were lysed and co-immunoprecipitation was performed with 500 μg protein using an anti-EGFP antibody. The co-immunoprecipitated proteins were subjected to 12% SDS-PAGE fractionation and ER was detected by western blot with anti-ER antibody. The input represents 5% of the protein used in the co-immunoprecipitation. . The expression level of EGFP fusions in cells transfected with MOZ-TIF2 (GFP-MT2) and TIF2 (GFP-TIF2). . The co-immunoprecipitation of ER by MOZ-TIF2 or TIF2. Input 1 is from cells transfected with pEGFP-TIF2 and input 2 is from cells transfected with pEGFP-MOZ-TIF2. . Co-localization between endogenous MOZ and CBP in HEK293 cells during the cell cycle. Top panel, co-localization of MOZ and CBP in the nucleus during G1-S-G2 stage. Middle panel, the co-localization disassociated from chromosomes during the metaphase. Bottom panel, the restoration of co-localization with chromatin

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-4

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>erase activity was measured as described after incubation of the transfected cells for 36 hours of cells with 50 nM estradiol. . The forced expression of CBP does not antagonize MOZ-TIF2 inhibition of ER-mediated transcription. . The inhibition of ER-mediated transcription by MOZ-TIF2 was partially released by increased expression of wild type of TIF2. Open bars, the presence of 50 nM estradiol; dark bars, without estradiol. Double stars represent a significant difference at P < 0.01 by the two tailed Student T- test compared to the transfection with MOZ-TIF2 in the ligand added condition. Dark circles represent a significant difference at P < 0.05 compared to the transfection with TIF2 alone in the ligand added condition. The numbers on top of the open bars indicate the ratio of light units as described in Figure 3

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-0

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p> 1117 and for TIF2 is amino acid 939. Expression of MOZ-TIF2 RNA. Left panel, schematic representation of PCR primers (thick arrows) in fusion region of MOZ-TIF2. F, forward primer and R, reverse primer. Right panel, RNA expression of MOZ-TIF2 as detected by RT-PCR in transduced or transfected cells. Lane 1, NIH-3T3 cells transduced with retrovirus control. Lane 2, NIH-3T3 cells transduced with a retrovirus expressing MOZ-TIF2 fusion protein. Lane 3 and Lane 4, HEK 293 cells (Lane 3) and K562 cells (Lane 4) transfected with pcDNA3.1-MOZ-TIF2. Lane 5, K562 cells transfected with pcDNA3.1 alone. Lane M, molecular weight markers. Expression of EGFP-tagged MOZ, MOZ-TIF2, and TIF2 proteins in HEK293 cells. Cells were transiently transfected with various pEGFP fusion constructs, cell lysates extracted 36 hours later and separated by SDS-PAGE (4–20% polyacrylamide). Western blot analysis was performed to detect EGFP tagged proteins of MOZ, MOZ-TIF2, and TIF2 with mouse monoclonal antibody to EGFP. MW, molecule markers in kilodaltons (kD). The localization of EGFP-tagged MOZ-TIF2 in HEK 293, CV-1, and K562 cells with the nuclei stained with DAPI

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-9

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p> 1117 and for TIF2 is amino acid 939. Expression of MOZ-TIF2 RNA. Left panel, schematic representation of PCR primers (thick arrows) in fusion region of MOZ-TIF2. F, forward primer and R, reverse primer. Right panel, RNA expression of MOZ-TIF2 as detected by RT-PCR in transduced or transfected cells. Lane 1, NIH-3T3 cells transduced with retrovirus control. Lane 2, NIH-3T3 cells transduced with a retrovirus expressing MOZ-TIF2 fusion protein. Lane 3 and Lane 4, HEK 293 cells (Lane 3) and K562 cells (Lane 4) transfected with pcDNA3.1-MOZ-TIF2. Lane 5, K562 cells transfected with pcDNA3.1 alone. Lane M, molecular weight markers. Expression of EGFP-tagged MOZ, MOZ-TIF2, and TIF2 proteins in HEK293 cells. Cells were transiently transfected with various pEGFP fusion constructs, cell lysates extracted 36 hours later and separated by SDS-PAGE (4–20% polyacrylamide). Western blot analysis was performed to detect EGFP tagged proteins of MOZ, MOZ-TIF2, and TIF2 with mouse monoclonal antibody to EGFP. MW, molecule markers in kilodaltons (kD). The localization of EGFP-tagged MOZ-TIF2 in HEK 293, CV-1, and K562 cells with the nuclei stained with DAPI

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-3

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>g. The reporter plasmid PL was used as described in Figure 3. Del CID: The CID domain was deleted from MOZ-TIF2 (MT2 delCID) (amino acids 1182–1312) and TIF2 (TF2 delCID) (amino acids 1002–1132). DBL : mutations were created in the leucine-repeat regions of CID of MOZ-TIF2 (MT2DBL) and TIF2 (TF2 DBL) as shown in panel . The letters in bold type in C represent the mutated amino acids. Open bars, 50 nM estradiol (A) or DHT (B). Dark bars, no added estradiol (A) or DHT (B). Double stars represent a significant difference at P < 0.01 by the two-tailed Student T- test compared to the transfection with MOZ-TIF2 in the ligand added condition. Solid circles represent a significant difference at P < 0.05 by two-tailed Student T- test compared to the transfection with TIF2 in the ligand added condition. The numbers on top of the open bars indicate the ratio of light units in the presence and absence of ligand as described in Figure 3. The fold increase given in the text was calculated as light units with the mutated MOZ-TIF2 compared to the wild type MOZ-TIF2 in absence or presence of ligand and fold decrease was calculated as the light units with the wild type TIF2 compared to the light units with the CID deleted TIF2

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-8

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>ted with the pcDNA3.1 plasmid carrying the full length MOZ-TIF2 cDNA. After selection with G418, two clones were established, MT2-1 and MT2-2, and expression of MOZ-TIF2 (MT2) mRNA was examined by RT-PCR in the two clones, in the patient's blasts, and in U937 cells stably transfected with vector alone. . Expression of C/EBPβ protein in RA treated U937 cells. Cells were treated with 1 μM RA for 72 hours, whole cell protein extracted with RIPA buffer, and proteins fractionated by SDS-PAGE. Western blot analyses were conducted with anti-C/EBPβ antibody. Top panel, western blot of C/EBPβ and β-tubulin. Bottom panel, relative level of C/EBPβ protein in control and MOZ-TIF2 expressing U937 cells after standardization to β tubulin levels used as a loading control. . Expression of C/EBPβ RNA in RA treated U937 cells. Cells were treated with RA for 24 and 48 hours. RNA was extracted and RT-PCR was conducted as described in Experimental Procedures. The fold-induction by RA was calculated as RA treated sample over non-treated sample for 24 hours (open bars) and 48 hours (dark bars) of RA treatment. . Fold-induction of CD11b RNA in RA treated U937 cells. Cells were treated with 1 μM RA for 8 and 48 hours. RNA was extracted, real-time RT-PCR conducted, and fold-induction by RA calculated comparing the RA treated sample to the non-treated sample. Open and dark bars, 8 and 48 hours of RA treatment respectively. . Percentage of CD11b positive U937 cell after RA treatment. Cells were treated with 1 μM RA for 72 hours and were stained with APC-conjugated anti CD 11b antibody (eBiosciences, San Diego, CA.) After washing, the stained cells were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ) and the percentage of CD11b positive cells calculated with FlowJo 6.3 software. Top and middle panel, a representative histograph of CD11b stained U937 cells stably transfected with pcDNA3 (top) and MOZ-TIF2 (middle). Y-axis, % cell number and X-axis, APC stain intensity. The horizontal line scale represents the range of CD11b positive cells. The dashed and solid lines represent basal and RA treated U937 cells, respectively. The number above the line scale indicates percentage of CD11b positive cells after treatment with RA. Bottom panel, percentage of CD11b positive U937 cells stably transfected with control (pcDNA3), MOZ-TIF2 (MT2), MT2 del 82–892, and MT2 del CID. The experiment was conducted in triplicates with the open bars representing the basal level of CD11b positive cells and the dark bars representing the level of CD11b after RA treatment. Double stars represent a significant difference at P < 0.01 by a two tailed Student T- test compared to the RA treated U937 cells with MT2-2. The percentage inhibition cited in the text was calculated from the amount of RNA or protein expressed in MOZ-TIF2 expressing cells compared to that in pcDNA-transfected control cells in presence of RA subtracted from 100%

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-1

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>on protein in HEK 293 cells was observed by confocal microscopy. Nuclei were stained in live HEK 293 cells with DRAQ5™ (AXXORA, LLC, San Diego, CA)

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-5

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>leukemic blasts of the patient exhibiting the MOZ-TIF2 fusion. RT-PCR was conducted as described in Experimental Procedures using the primers listed in Table 1. Shown are the results as relative amounts of RNA in the non-MOZ-TIF2 leukemic blasts (clear bars) compared to the MOZ-TIF2 blasts (shaded bars). Stars represent a significant difference at P < 0.05 by the two-tailed Student T-test comparing RNA levels in the blasts of the patients without the MOZ-TIF2 fusion to the levels in the blasts with the MOZ-TIF2 fusion

MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2-2

<p><b>Copyright information:</b></p><p>Taken from "MOZ-TIF2 repression of nuclear receptor-mediated transcription requires multiple domains in MOZ and in the CID domain of TIF2"</p><p>http://www.molecular-cancer.com/content/6/1/51</p><p>Molecular Cancer 2007;6():51-51.</p><p>Published online 13 Aug 2007</p><p>PMCID:PMC2048977.</p><p></p>into CV-1 cells with an estrogen receptor expression vector and pCDNA3.1-MOZ, pCDNA3.1-MOZ-TIF2 (MT2), pCDNA3.1-TIF2 or vector alone. and . MOZ-TIF2 and AR activity. Two androgen response element-driven luciferase reporter systems were employed. shows the effect of a plasmid that contains a full length PSA promoter (PL) with multiple androgen response element sites and shows the effect of a plasmid containing a minimum PSA promoter (PC) with only two androgen response elements. Both ARE-containing reporter plasmids were co-transfected with an androgen receptor expression vector and either pCDNA3.1-MOZ, pCDNA3.1-MOZTIF2 (MT2), pCDNA3.1-TIF2 or vector alone. 5α-dihydrotestosterone (DHT) was added to a final concentration of 50 nM, the cells collected and luciferase activity measured as described above. Double or single stars represent a significant difference at P < 0.01 or P < 0.05 level respectively by the two tail Student T- test compared to the transfection with MOZ-TIF2 in the ligand added condition. Open bars, 50 nM estradiol (A) or DHT (B and C). Dark bars, no added estradiol (A) or DHT (B and C). The numbers on top of the open bars, i.e. added ligand, are the ratios of light units in presence of the ligand to light units in the absence of ligand. The percentage inhibition noted in the text was calculated from the percent of light units resulting from induction by estrogen in MOZ-TIF2 expressing cells compared to pcDNA-transfected control cells subtracted from 100%