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1.
FIG. 7.

FIG. 7. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Analysis of PRMT1 and PAD4 mRNA levels in different cell lines upon retinoid treatment. (A) PRMT1 mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR measurements ± standard deviations. (B) PAD4 mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR measurements ± standard deviations. (C) mRNA levels of PAD4 in 9-cis retinoic acid-treated naive and DMSO-primed HL-60 cells. Relative increases of mRNA levels during priming or retinoid treatment of primed cells are shown on the arrows. PAD4 mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR measurements ± standard deviations.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
2.
FIG. 5.

FIG. 5. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Histone tail modifications during priming. (A) H4 arginine 3 methylation levels detected by chromatin immunoprecipitation on the enhancer element (HR1) during priming with DMSO. (B) H3 lysine 4 methylation levels detected by chromatin immunoprecipitation on the core promoter during priming with DMSO. Values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in at least three independent chromatin preps.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
3.
FIG. 10.

FIG. 10. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Proposed model of gene expression modulation by changes in H4 arginine 3 methylation. PRMT1 and PAD4 regulate arginine 3 methylation. The naive state is characterized by lack of H4 arginine methylation on the enhancer region. Priming results in increased H4 R3 methylation, while retinoid treatment leads to decreased H4R3 methylation and to the indicated changes in lysine acetylation. Preacetylation of histone tails prior to priming by TSA is likely to reduce the affinity of histone tails toward PRMT1 and, by this, prevents the enhanced retinoid responsiveness caused by priming. The activation of PAD4 after priming by calcium ionophore treatment leads to the removal of the methyl mark from H4R3 and abolition of the enhanced retinoid responsiveness caused by priming.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
4.
FIG. 3.

FIG. 3. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Priming produces changes at the chromatin level on the promoter of TGM2. (A) DNase I sensitivity of the RARβ promoter in naive and primed cells. DNase I hypersensitivity was measured as described in Materials and Methods. Values are the means of three independent QPCR measurements ± standard deviations of a representative experiment. (B) DNase I sensitivity of the TGM2 promoter in naive and primed cells. DNase I hypersensitivity was measured as described in Materials and Methods. Values are the means of three independent QPCR measurements ± standard deviations of a representative experiment. (C) Chromatin immunoprecipitation analysis of HR1 enhancer element of TGM2. H4 acetylation was measured as described in Materials and Methods. The acetylation level is expressed as a percentage of input DNA. All no-antibody controls were lower than 0.2% of input DNA. Values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in at least three independent chromatin preps.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
5.
FIG. 4.

FIG. 4. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Detailed map of histone tail modifications on the promoter/enhancer of TGM2. The bar graphs on the left show the effect of priming on the histone tails bound to the enhancer region HR1 (except H3K4, which is shown on the core promoter). The line graphs show the changes in histone tail modifications along the 1.8-kb studied fragment of the promoter/enhancer in primed cells after 9-cis retinoic acid treatment for the indicated period (in hours). Copy numbers are expressed as percentages of input. All no-antibody controls were lower than 0.2% of input DNA. Values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in at least three independent chromatin preps. The scheme of the 1.8-kb human TGM2 promoter and the location of the various QPCR assays are shown at the bottom.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
6.
FIG. 9.

FIG. 9. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Effect of PRMT1 and PAD4 on retinoid-regulated gene expression. (A) Transfection of PRMT1 expression vector into HL-60 cells increases the TGM2 induction after retinoid treatment in primed cells, while the catalytic mutant has no effect on retinoid responsiveness. Cells were treated with 1 μM 9-cis retinoic acid for 10 h. mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR measurements ± standard deviations. (B) Cotransfection of PAD4 and PRMT1 expression vectors and their catalytic mutants into HL-60 cells modulates TGM2 induction. Cells were treated with 1 μM 9-cis retinoic acid for 24 h. mRNA copy numbers were normalized for cyclophilin transcript levels. Values are the means of two independent biological replicates and three independent QPCR measurements for each sample ± standard deviations. (C) Cotransfection of PAD4 and PRMT1 expression vectors and their catalytic mutants into 293T cells modulates the luciferase expression on a βRARE tkLuc reporter system. Values were normalized to measured β-galactosidase values and expressed in arbitrary units (AU) as described in Materials and Methods. Values are the mean of three independent biological replicates ± standard deviations. +, present; −, absent.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
7.
FIG. 6.

FIG. 6. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Epigenetic changes on TGM2 promoter and mRNA expression upon priming in methylation mute cells (methyltransferases were blocked with 10 μM ADOX for 16 h as described in Materials and Methods). Values are the means of three parallel QPCR measurements ± standard deviations, and results were confirmed from at least three independent biological samples. All chromatin immunoprecipitation values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in at least three independent chromatin preps. (A) H4 arginine 3 methylation levels determined by chromatin immunoprecipitation on the HR1 enhancer element. (B) Arginine methylation levels determined by chromatin immunoprecipitation on the HR1 enhancer element with an anti-Pan methyl arginine antibody. (C) H3 lysine 4 methylation studied by chromatin immunoprecipitation on the core promoter. (D) Chromatin immunoprecipitation analysis of H4 acetylation levels in naive, primed, and primed methylation mute cells on the enhancer element HR1. (E) TGM2 mRNA levels normalized to 36B4 copy numbers in naive, primed, and primed methylation mute cells (the structure of ADOX is shown in the inset). (F) TGM2, CD38, and CYP27 transcript levels after blocking methylation with ADOX in naive and primed cells. ADOX was used in increasing concentrations as shown. The mRNA copy numbers were normalized to 36B4 transcript levels. Values are the means of three parallel QPCR measurements ± standard deviations, and results were confirmed from at least three independent biological samples.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
8.
FIG. 8.

FIG. 8. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Modulation of TGM2 expression levels in HL-60 cells. (A) TSA pretreatment for 1 h of HL-60 cells enhances the retinoid response in naive cells (50, 100, and 150 nM TSA was used). TSA pretreatment for 1 h of HL-60 cells before DMSO priming reduces the effect of priming on TGM2 induction (50, 100, and 150 nM TSA was used). Arrows indicate the effect of pretreatment with 150 nM TSA on primed versus naive retinoid-treated cells. mRNA copy numbers were normalized to 36B4 transcript levels. Values are the means of three independent QPCR measurements ± standard deviations. (B) Activation of PAD4 after priming by a 15-min treatment with 1 μM calcium ionophore A23187 reduces the priming effect. mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of three independent QPCR measurements ± standard deviations. (C) H4R3 methylation levels are changed during priming and modulated by pretreatment with 100 nM TSA for 1 hour or a 15-min treatment with 1 μM calcium ionophore A23187. H4R3 methylation was determined by chromatin immunoprecipitation on the HR1 enhancer element. Copy numbers are expressed as percentages of the input. (D) H4 acetylation levels in primed, retinoid-treated cells and TSA-pretreated, primed retinoid-treated cells. Cells were pretreated with 100 nM TSA for 1 hour, and H4 acetylation levels were determined by chromatin immunoprecipitation on the HR1 enhancer element. Copy numbers are expressed as percentages of the input. (E) H4R3 methylation levels in primed, retinoid-treated cells and TSA-pretreated, primed retinoid-treated cells. Cells were pretreated with 100 nM TSA for 1 hour, and H4R3 methylation levels were determined by chromatin immunoprecipitation on the HR1 enhancer element. Copy numbers are expressed as percentages of the input. The values of the no-antibody controls were subtracted. Values are the means of three independent QPCR measurements of a representative experiment. Chromatin immunoprecipitation results were confirmed in two independent chromatin preps with two independent immunoprecipitations, respectively. +, present; −, absent.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
9.
FIG. 1.

FIG. 1. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

Priming enhances retinoid response in HL-60 cells. (A) Experiment outline. HL-60/CDM-1 cells were primed with 1.25% DMSO overnight. The control (naive) cells received no priming. After overnight incubation, cells were washed, resuspended in fresh medium, and treated with 1 μM 9-cis retinoic acid. (B) Priming with DMSO or vitamin D enhances 9-cis retinoic acid-dependent expression of integrin CD11b. Cells were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the priming agent, cells were treated with 1 μM 9-cis retinoic acid and analyzed on a flow cytometer at the indicated time points as described in Materials and Methods. Isotype controls had no change (not shown). (C) Priming with DMSO or, to a lesser extent, with vitamin D enhances 9-cis retinoic acid-dependent expression of integrin CD11c. Cells were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the priming agent, cells were treated with 1 μM 9-cis retinoic acid and analyzed with a flow cytometer at the indicated time points as described in Materials and Methods. Isotype controls had no change (not shown). (D) Priming with DMSO but not with vitamin D enhances 9-cis retinoic acid-dependent expression of integrin CD18. Cells were primed with 1.25% DMSO or 100 nM vitamin D. After overnight incubation and washing out of the priming agent, cells were treated with 1 μM 9-cis retinoic acid and analyzed with a flow cytometer at the indicated time points as described in Materials and Methods. Isotype controls had no change (not shown). (E) Relative increase of TGM2 mRNA induction compared to that of naive, retinoid-treated cells. Assessment of the maintenance in time of the priming effect: TGM2 mRNA was measured by real-time QPCR after priming with 1.25% DMSO and compared to naive, retinoid-treated cells. After overnight incubation and washing out of the priming agent, cells were treated with 1 μM 9-cis retinoic acid at different time points after the priming, as indicated. The retinoid treatment was 12 h long in all cases. Values are the means of the results from three independent QPCR measurements ± standard deviations. (F) Relative increase of TGM2 induction compared to that of naive, retinoid-treated cells. Assessment of the maintenance in time of the priming effect: TGM2 mRNA was measured by real-time QPCR after priming with 100 nM vitamin D and compared to naive, retinoid-treated cells. After overnight incubation and washing out of the priming agent, cells were treated with 1 μM 9-cis retinoic acid at different time points after the priming, as indicated. The retinoid treatment was 12 h long in all cases. Values are the means of the results from three independent QPCR measurements ± standard deviations.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.
10.
FIG. 2.

FIG. 2. From: Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells.

TGM2 expression is enhanced by DMSO priming at both the RNA and protein levels, and this effect is not cell type specific. (A) 9-cis retinoic acid induction of TGM2 mRNA as measured by real-time QPCR. The expression of TGM2 mRNA in primed and naive cells is shown on the same graph with two different y axes with a difference of 2 orders of magnitude of the scales. Transcript copy numbers were normalized to 36B4 transcript levels. Values are the means of the results from three independent QPCR measurements ± standard deviations. (B) Relative difference in gene expression in naive and primed cells. Fold changes are indicated above the bars. Copy numbers of TGM2 mRNA are determined by real-time QPCR and normalized to 36B4 transcript levels. Values are the means of the results from three independent QPCR measurements ± standard deviations. (C) Intracellular immunostaining and flow cytometric analysis of retinoid-treated HL-60 cells as described in Materials and Methods. The expression of TGM2 protein in naive cells (shaded) and its isotype control (white) after retinoid induction. The percentage of cells expressing TGM2 protein is shown on the graph along with the mean fluorescence intensity of the positive cells. Values are expressed in arbitrary units (AU). (D) Intracellular immunostaining and flow cytometric analysis of retinoid-treated HL-60 cells as described in Materials and Methods. TGM2 expression in DMSO-primed cells (shaded) along with its isotype control (white). The percentage of cells expressing TGM2 is shown on the graph along with the mean fluorescence intensity of the positive cells, expressed in arbitrary units (AU). At least three independent determinations have been carried out. Data from a representative experiment are shown. (E) Effect of priming upon TGM2 induction by retinoids in MonoMac6 (MM6) cells. TGM2 copy numbers were normalized to cyclophilin transcript levels. Values are the means of the results from three independent QPCR measurements ± standard deviations. (F) TGM2 induction in NB4 and RARα mutant NB4R2 cells in naive and primed states. The mRNA copy numbers were normalized to cyclophilin transcript levels. Values are the means of the results from three parallel QPCR measurements ± standard deviations, and results were confirmed from at least three independent biological samples. +, present; −, absent.

Balint L. Balint, et al. Mol Cell Biol. 2005 July;25(13):5648-5663.

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