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Results: 6

1.
Fig. 3.

Fig. 3. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

PLZF and RARα-PLZF recruit repressor and activator complexes, respectively. (Left) Chromatin prepared from transiently transfected 293T cells (PLZF and/or RARα-PLZFFlag) were subjected to the ChIP procedure with anti-PLZF or anti-Flag antibodies, shown on the left, and were again immunoprecipitated using antibodies shown at the top of the image. (Right) Expression of RARα-PLZF and PLZF was confirmed by Western blotting.

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.
2.
Fig. 1.

Fig. 1. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

CRABPI is regulated by PLZF and RARα-PLZF through a remote intronic BS. (A) Western blot showing overexpression of CRABPI in PLZF-RARα/RARα-PLZF transgenic APL blasts compared with blasts from PML-RARα transgenic mice and KG1 cells. β-actin was used as a loading control. (B) Schematic representation of the genomic structure of the human CRABPI gene. Black boxes represent exons, and dark gray boxes represent intronic and promoter regions. In silico search of the human genomic CRABPI gene revealed only one putative PLZF DNA BS, which was located at the end of intron 3–4 (asterisk and underlined text). The location of primers used to amplify the region containing the BS are denoted in bold italic text.

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.
3.
Fig. 6.

Fig. 6. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

RARα-PLZF confers retinoid resistance in a CRABPI-dependent fashion. (A and B) RARα-PLZF blocks RA-induced differentiation of HL60 cells in a CRABPI-dependent fashion. HL60 cells were plated in selection medium with or without RA at 10−6 M concentration and were treated with antisense (CrI AS) and missense (CrI MS) CRABPI oligonucleotides for 7–10 days; the number of CFU-G colonies formed was determined after 7–10 days of culture. In the presence of culture medium alone, HL60 cells spontaneously produce CFU-G (A1 and B). RA led to a marked increase in CFU-G (A2 and B), which was not influenced by CRABPI antisense or missense oligonucleotides. (A and D) Expression of RARα-PLZF blocked RA-induced differentiation (A6), which was restored (A7) after knockdown of CRABPI by the presence of the antisense oligonucleotide (D), but was unaffected by the missense oligonucleotide (A8 and D). (C) Methylation status of CRABPI promoter in HL60 cells according to expression of RARα-PLZF. The CRABPI promoter is methylated in wild-type HL60 cells but is demethylated in the presence of RARα-PLZF. CRABPI antisense (CrI AS) was confirmed to have no impact on the methylation status of the CRABPI locus. (D) (Left) Western blot showing that expression of RARα-PLZF (detected by anti-Flag antibody) leads to induction of CRABPI protein levels that were effectively knocked down by CRABPI antisense (CrI AS). β-Actin was used as a loading control. (Right) Corresponding levels of CRABPI expression normalized to the ABL housekeeping gene.

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.
4.
Fig. 4.

Fig. 4. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

PLZF propagates a repressive chromatin environment from a distant BS to silence the CRABPI promoter, which is overcome by RARα-PLZF. (A) Expression vectors for PLZF and RARα-PLZF (200 ng) were transfected into 293T cells. Levels of transiently expressed proteins were monitored at various time points (0, 2, 5, 12, and 24 h) by Western blot analysis using an anti-Flag (Anti-Flag Western) antibody, and their binding activities were monitored by ChIP using an anti-Flag antibody (Anti-Flag ChIP) at the same time points. Levels of expressed CRABPI protein throughout the time-course experiment were assessed by Western blotting using an anti-CRABPI antibody (Anti-CRABPI Western). The anti-Flag antibody was used to pull down the Flag-tagged proteins attached to the genomic DNA of the 293T cells and was tested to determine whether the overexpressed proteins were able to bind the endogenous genomic CRABPI sequence. (B) The effect of PLZF and its chimeric protein RARα-PLZF on the acetylation levels of histone H3 and the recruitment of MBD1 were assessed throughout the length of the CRABPI gene. Various segments of genomic DNA situated in the intronic regions 3–4 and 2–3, as well as in the promoter region of the CRABPI gene, were tested at various time points after PLZF and RARα-PLZF transfection into 293T cells for their respective levels of histone H3 acetylation and the abundance of MBD1 proteins interacting with DNA (Anti-AcH3 and Anti-MBD1, respectively). Results are shown as gel pictures and as quantification bar graphs (gray bars, histone H3 acetylation; black bars, MBD1 recruitment to DNA). All ChIP experiments were performed from a single chromatin preparation for each time point. Throughout the ChIP experiment, efficiency of the precipitation was assessed by using an anti-histone H3 (Anti-H3) antibody as a positive control.

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.
5.
Fig. 2.

Fig. 2. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

Relative binding of PLZF and RARα-PLZF at the CRABPI intronic regulatory element. (A) CRABPI(intron3–4)-tkLuc plasmid was used to assess the effects of PLZF (black square), RARα-PLZF (gray circle), and the two transcription factors (PLZF+RARα-PLZF, gray square) on the transcriptional activity of the reporter. Increasing amounts of each expression vector were cotransfected into 293T cells in the presence of the luciferase reporter (same amount for each point). Luciferase activity was determined 16 h later. (B) ChIP assay was performed in parallel with the luciferase assay to assess the acetylation level in the proximity of the PLZF BS (Anti-AcH3), together with the binding of the chimeric RARα-PLZF (Anti-Flag) and PLZF (Anti-PLZF) proteins. The level of histone H3 protein in the vicinity of this site (Anti-H3) was used as a positive control for the ChIP procedure and to normalize the quantity of input DNA at each transfection point. Lane A, CRABPI(intron3–4)-tkLuc reporter alone; lane B, luciferase reporter and RARα-PLZFFlag; lane C, luciferase reporter and PLZF; lane D, luciferase reporter, PLZF, and RARα-PLZFFlag. (C) Endogenous PLZF DNA-binding activity in the hematopoietic KG1 cell line. (Left) ChIP assay shows binding of PLZF protein to the endogenous genomic CRABPI-BS present in KG1 cells (lanes 3 and 7, immunoprecipitation done with polyclonal anti-PLZF antibody) in the presence of transiently expressed PLZF-RARαFlag or RARα-PLZFFlag. Binding of the chimeric proteins was assessed using a monoclonal anti-Flag antibody (lanes 4 and 8). As expected, RARα-PLZFFlag, but not PLZF-RARαFlag, was able to bind to the CRABPI-BS. It appears that only the RARα-PLZFFlag chimeric protein is competing with wild-type PLZF for the BS on genomic DNA. An irrelevant polyclonal antibody was used as a negative control in the ChIP procedure (lanes 1 and 5). (Right) Expression of the chimeric fusion proteins was confirmed by Western blotting.

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.
6.
Fig. 5.

Fig. 5. From: RAR?-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

Methylation status of the CRABPI promoter correlates with RARα-PLZF expression in primary APL cells. Methylation status was determined by digesting the genomic DNA from cell lines and patient samples by AvaI and AvaII endonucleases. These two enzymes behave differently in the presence of methylated DNA, being sensitive and insensitive, respectively. (A) Schematic representation of the genomic structure of the human CRABPI gene. Black boxes represent exons; dark gray boxes represent intronic and promoter regions. The CpG islands located at the beginning of the CRABPI gene are indicated by a light gray box, and AvaI and AvaII endonuclease sites are indicated by arrowheads and circles, respectively. (B) Effect of overexpression of PLZF on the methylation status of the CRABPI promoter. Ectopic expression of PLZF in 293T cells induced methylation in this region, as indicated by the appearance of a specific band in lane avaI at 24 h after transfection, reflecting the inability of the AvaI endonuclease to cut the genomic DNA at this specific location. Lane −, no enzyme input control; lane avaI, AvaI digest; lane avaII, AvaII digest (as a control for digestion). (C) Effect of overexpression of RARα-PLZF and PLZF-RARα in the PLZF expressing the KG1 cell line. In the untransfected, empty vector and PLZF-RARα-transfected KG1 cells, the CRABPI promoter is found to be methylated because of the endogenously expressed PLZF (lane −, untransfected). Interestingly, only overexpression of the chimeric RARα-PLZF protein induced specific demethylation of the promoter (lane avaI, RARα-PLZF). (D) Western blot detection of the CRABPI and -II proteins in KG1 cells. PLZF-RARαFlag and RARα-PLZFFlag were overexpressed for 24 h, and levels of protein expression were assessed. Up-regulation of CRABPI was only observed in the KG1 cells expressing the RARα-PLZFFlag chimeric protein (lane RARα-PLZF) relative to levels of CRABPII and β-actin, which remained unchanged. (E) Methylation status of the CRABPI promoter in primary PLZF-RARA+ leukemic blasts from APL patients, according to expression status of the reciprocal RARA-PLZF fusion transcript. As shown, cases 1 and 2 expressing the reciprocal RARA-PLZF had low levels of DNA methylation compared with cases in which PLZF-RARA was the sole transcript expressed (cases 4 and 5; all avaI lanes).

Fabien Guidez, et al. Proc Natl Acad Sci U S A. 2007 November 20;104(47):18694-18699.

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