Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 1. Identification of candidate interacting partners of LEDGF/p75 using transcription factor protein arrays. A, Coomassie blue–stained SDS-PAGE gel showing purified His-LEDGF/p75. E. coli BL21 strain was transformed with pET28a-dfs70 encoding His-LEDGF/p75 and induced with IPTG. Lysate was passed through a nickel column to purify His- LEDGF/p75. B, immunoblot showing the specificity of the human autoantibody against LEDGF/p75 used as detection reagent in the transcription factor protein arrays. The autoantibody reacts specifically with LEDGF/p75 in a PC3 prostate cancer cell lysate. WB, Western blotting. C, transcription factor arrays were used to identify candidate interacting transcription factors of LEDGF/p75. Purified His-LEDGF/p75 was incubated with transcription factors spotted on membranes. Protein interactions were detected with human anti-LEDGF/p75 autoantibody and chemiluminescence. A section of the transcription factor array membrane containing MeCP2 is shown.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Staining, SDS Page, Transformation Assay, Nickel Column, Western Blot, Incubation, Membrane

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 6. The N-terminus of LEDGF/p75 interacts with MeCP2. A, diagram of LEDGF/p75 deletion constructs used to map interaction regions. ATH, AT-hook. CR, charged region. IBD, integrase-binding domain. NLS, nuclear localization signal. B, Flag-MeCP2 binds to eGFP-LEDGF/p75 but not to eGFP-tagged truncated LEDGF/p75 constructs. 293T cells ectopically overexpressing the tagged proteins labeled in the blot were immunoprecipitated (IP) with GFP antibody and visualized by immunoblotting with antibodies to GFP and Flag. C, Flag-MeCP2 binds to eGFP-tagged LEDGF/p75 and LEDGF/p52 but not to truncated constructs. Proteins ectopically overexpressed in U2OS cells were immunoprecipitated with Flag antibody and visualized with both anti-GFP and anti-Flag antibodies. , degradation product of LEDGF/p75. D, recombinant Flag-LEDGF/p75, Flag-PWWP-CR1 (1–141 aa), and Flag-PWWP (1–101 aa) were incubatedwithU2OScelllysate.Proteinspulleddownwithanti-Flagaffinitymatrixweredetectedbyimmunoblotting.EndogenousMeCP2inthecelllysatewas pulled down (PD) by Flag-LEDGF/p75 and Flag-PWWP-CR1. Absence of recombinant proteins (U2OS only) served as negative control. WB, Western blotting.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Construct, Binding Assay, Labeling, Immunoprecipitation, Western Blot, Recombinant, Negative Control

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 7. LEDGF/p75, LEDGF/p52, and MeCP2 transactivate Hsp27 promoter. U2OS cells were cotransfected with pGL3-Hsp27pr-luc and the indicated amount of (A) empty pcDNA vector or pcDNA-Flag-MeCP2, or (B) pCruzHA, pCruzHA-LEDGF/p75, or pCruzHA-p52 (1 ¼ 1.66 mg DNA). Promoter activity determined as luciferase light units/protein is expressed as fold activation compared with control activity (cotransfection of pGL3-Hsp27pr-luc with empty expression vectors). Data from each graph are representative of at least 3 independent experiments. Representative immunoblots corresponding to the reporter assays show protein expression. C, schematic diagram of Hsp27pr showing AT-rich regions, and HSE and STRE sites. PCR primers targeted Hsp27pr regions A (bp 271 to þ18), B (bp 480 to 220), C (bp 803 to 382), and D (bp 1,071 to 781). D, ChIP analysis of MeCP2 and LEDGF/p75 binding to Hsp27pr. Formaldehyde-fixed chromatin from U2OS cells was precipitated with nonspecific IgG or antibodies specific for MeCP2 or LEDGF/p75. PCR amplifications of immunoprecipitated DNA derived from U2OS cells were carried out with primer sets specific for Hsp27pr regions A to D. Hsp27pr primers amplified DNA fragments precipitated by LEDGF/p75 antibody or MeCP2 antibody but not by IgG. Primers that target human b-actin controlled for optimal enzymatic digestion of chromatin. Data represent the average of at least 3 independent experiments.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Plasmid Preparation, Activity Assay, Luciferase, Activation Assay, Control, Cotransfection, Expressing, Western Blot, Binding Assay, Immunoprecipitation, Derivative Assay

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 8. LEDGF/p75 and p52 influence MeCP2-induced transactivation of Hsp27pr. A, U2OS cells were cotransfected with pGL3-Hsp27pr-luc, pCruzHA, or pCruzHA-LEDGF/p75, and increasing amounts of pcDNA-Flag-MeCP2 (1 ¼ 1.66 mg DNA). B, U2OS cells were cotransfected with pGL3-Hsp27pr-luc, pCruzHA, or pCruzHA-LEDGF/p52, and increasing amounts of pcDNA-Flag-MeCP2. C, transient knockdown of LEDGF/p75 in U2OS cells was achieved using specific siRNA oligos. Cells were then cotransfected with pcDNA-Flag-MeCP2 and pGL3-Hsp27pr-luc. Promoter activity determined as luciferase light units/protein is expressed as fold activation compared with control activity. D, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were cotransfected with pGL3-Hsp27pr-luc and pMAX-GFP (transfection control). E, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were cotransfected with pGL3-Hsp27pr-luc and pcDNA-Flag-MeCP2. F, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were transfected with siRNA oligos to knockdown this protein. Cells were then cotransfected with pGL3- Hsp27pr-luc and pcDNA-Flag-MeCP2. Promoter activity determined as luciferase light units/GFP is expressed as fold activation compared with control activity. Data represent the average of at least 3 independent experiments. Representative immunoblots corresponding to the reporter assays show protein expression. All protein bands in (F) were from the same blot. , P < 0.05; , P < 0.01.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Knockdown, Activity Assay, Luciferase, Activation Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Nature Communications

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

doi: 10.1038/s41467-018-03854-0

Figure Lengend Snippet: SHARP1 is a downstream target of MLL-AF6 and DOT1L. a SHARP1 mRNA expression in human AML cell lines assessed by qPCR. The cell lines analyzed are: ML-2 (MLL-AF6), CTS (MLL-AF6), SHI-1 (MLL-AF6), MOLM-14 (MLL-AF9), MV4-11 (MLL-AF4), and Kasumi-1 (AML1-ETO). b ChIP-seq profiles of ML-2 cells using MLL N , MEN1, LEDGF, H3K79me2, and H3K79me3 antibodies at the loci of the HOXA gene cluster (left panel) and SHARP1 gene (right panel). c ChIP-seq profiles of SHI-1 cells using MLL N antibody and input at the loci of the HOXA gene cluster (left) and SHARP1 gene (right). d qPCR for MLL-AF6 (left panel) and SHARP1 (right panel) mRNA expression in ML-2 cells upon MLL knockdown. Shown is the relative expression value to ML-2 transduced with shGFP. e Western blot of H3K79me2 in MOLM-14 (MLL-AF9) and ML-2 (MLL-AF6) cells treated with the DOT1L inhibitor, EPZ5679 (1 μM) or DMSO vehicle for 96 h. f qPCR for SHARP1 mRNA in MOLM-14 or ML-2 cells treated with EPZ5679 or DMSO vehicle for 6 days. Relative expression is the value compared to ML-2 cells treated with DMSO vehicle. g Western blot of SHARP1 and β-actin in ML-2 cells treated with EPZ5679 or DMSO vehicle for 10 days. All Western blots are representative of three independent experiments. All quantitation data include three independent experiments and are presented as mean ± s.e.m

Article Snippet: The antibodies used included SHARP1 (a mixture of H-72 Santa-Cruz, 12688-1-AP Proteintech, and ab175544 Abcam), H3K79me3 (Diagenode, pAb-068-050), MLL1 (Bethyl, A300-086A), H3K4me3 (Active Motif, 39159), H3K27me3 (Millipore, 07-499), H3K27ac (Millipore, 07-360), LEDGF (Bethyl, A300-848A), and MEN1 (Bethyl, A300-105A).

Techniques: Expressing, ChIP-sequencing, Knockdown, Transduction, Western Blot, Quantitation Assay

Journal: Nature Communications

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

doi: 10.1038/s41467-018-03854-0

Figure Lengend Snippet: SHARP1 interacts with MLL-AF6 and regulates gene targets. a Enriched gene sets in ML-2 shGFP cells over shSHARP1 on RNA-seq. b Venn diagram showing overlapping of SHARP1-bound genes with MLL-AF6 target genes (MLL N+ H3K79me2) in ML-2 cells. c Heatmap images representing the relative expression levels of 14 MLL-AF6/SHARP1 co-target genes downregulated upon SHARP1 knockdown obtained from RNA-seq data. d Genome view of MLL N , MEN1, LEDGF, H3K79me2, H3K79me3, and SHARP1 peak binding on three MLL-AF6 + SHARP1 target genes in ML-2 shGFP: MEF2C , CDK6 and RUNX2 gene. e Co-immunoprecipitation studies of SHARP1 and MLL-AF6 with an anti-HA antibody in 293 T cells transfected with plasmids encoding MLL-AF6 and/or HA-tagged SHARP1. Proteins present in immunoprecipitates (IP, lane 1–4) or whole cell lysates of transfected cells (input, lane 5–8) were separated by SDS-PAGE and immunoblotted with antibodies specific for MLL N and SHARP1. Interaction of SHARP1 and MLL-AF6 was detected (lane 4) and not observed in negative control lanes with either empty vector, SHARP1 or MLL-AF6 only (lane 1–3). f Schematic showing a series of MLL deletion mutants. Interaction with SHARP1 is indicated by + sign and loss of interaction by − sign. Boxes indicate AT hook motifs (blue), nuclear translocation sequences (NTS1 and NTS2) (orange), subnuclear localization domains (SNL1 and SNL2) (purple), and DNA methyltransferase domain (MT) (yellow). g Domain mapping analysis of MLL required for interaction with SHARP1. 293 T cells are transfected with plasmids encoding FLAG-tagged MLL deletion mutants and HA-tagged SHARP1. Whole cell lysates were prepared from the transfected cells and subjected to immunoprecipitation with anti-FLAG antibody. Proteins present in immunoprecipitates (IP, lane 1–7) or whole cell lysates of transfected cells (input, lane 8–14) were separated by SDS-PAGE and immunoblotted with antibodies specific for FLAG-tagged MLL deletion mutants and HA-tagged SHARP1. The arrows indicate MLL mutant proteins. Western blots are representative of at least three independent experiments

Article Snippet: The antibodies used included SHARP1 (a mixture of H-72 Santa-Cruz, 12688-1-AP Proteintech, and ab175544 Abcam), H3K79me3 (Diagenode, pAb-068-050), MLL1 (Bethyl, A300-086A), H3K4me3 (Active Motif, 39159), H3K27me3 (Millipore, 07-499), H3K27ac (Millipore, 07-360), LEDGF (Bethyl, A300-848A), and MEN1 (Bethyl, A300-105A).

Techniques: RNA Sequencing, Expressing, Knockdown, Binding Assay, Immunoprecipitation, Transfection, SDS Page, Negative Control, Plasmid Preparation, Translocation Assay, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

doi: 10.1038/s41467-018-03854-0

Figure Lengend Snippet: SHARP1 binds to actively transcribed genes and positively regulates target genes. a Integrated view of SHARP1 binding sites in conjunction with H3K4me3, H3K27ac, and H3K27me3 profiles across promoters, introns and intergenic regions. Top enriched motifs within SHARP1 ChIP-seq peaks in ML-2 cells are shown according to their genomic location discovered by peak-motifs module from the RSAT suite, using oligomer length ranging from 6 to 8 nucleotides and the 'merge lengths for assembly' option. b Box plot showing the expression levels in microarray analysis of ML-2 cells for the all genes (25293 genes) and genes enriched with SHARP1 + H3K4me3 (6459 genes), SHARP1 + H3K27ac (5840 genes), and SHARP1 + H3K27me3 (1055 genes) identified in ChIP-seq analysis. The box extends from the 25 th to 75 th percentiles and the whisker extends from the minimum level to the maximum. Median value is plotted in the box. c Representative SHARP1 binding peaks in the known target gene loci (circadian clock genes and MLH1 ). d Pathway analysis for the genes in the SHARP1 and H3K4me3 co-bounded regions within the promoter and gene body

Article Snippet: The antibodies used included SHARP1 (a mixture of H-72 Santa-Cruz, 12688-1-AP Proteintech, and ab175544 Abcam), H3K79me3 (Diagenode, pAb-068-050), MLL1 (Bethyl, A300-086A), H3K4me3 (Active Motif, 39159), H3K27me3 (Millipore, 07-499), H3K27ac (Millipore, 07-360), LEDGF (Bethyl, A300-848A), and MEN1 (Bethyl, A300-105A).

Techniques: Binding Assay, ChIP-sequencing, Expressing, Microarray, Whisker Assay

Journal: Nature Communications

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

doi: 10.1038/s41467-018-03854-0

Figure Lengend Snippet: SHARP1 is a downstream target of MLL-AF6 and DOT1L. a SHARP1 mRNA expression in human AML cell lines assessed by qPCR. The cell lines analyzed are: ML-2 (MLL-AF6), CTS (MLL-AF6), SHI-1 (MLL-AF6), MOLM-14 (MLL-AF9), MV4-11 (MLL-AF4), and Kasumi-1 (AML1-ETO). b ChIP-seq profiles of ML-2 cells using MLL N , MEN1, LEDGF, H3K79me2, and H3K79me3 antibodies at the loci of the HOXA gene cluster (left panel) and SHARP1 gene (right panel). c ChIP-seq profiles of SHI-1 cells using MLL N antibody and input at the loci of the HOXA gene cluster (left) and SHARP1 gene (right). d qPCR for MLL-AF6 (left panel) and SHARP1 (right panel) mRNA expression in ML-2 cells upon MLL knockdown. Shown is the relative expression value to ML-2 transduced with shGFP. e Western blot of H3K79me2 in MOLM-14 (MLL-AF9) and ML-2 (MLL-AF6) cells treated with the DOT1L inhibitor, EPZ5679 (1 μM) or DMSO vehicle for 96 h. f qPCR for SHARP1 mRNA in MOLM-14 or ML-2 cells treated with EPZ5679 or DMSO vehicle for 6 days. Relative expression is the value compared to ML-2 cells treated with DMSO vehicle. g Western blot of SHARP1 and β-actin in ML-2 cells treated with EPZ5679 or DMSO vehicle for 10 days. All Western blots are representative of three independent experiments. All quantitation data include three independent experiments and are presented as mean ± s.e.m

Article Snippet: The antibodies used included SHARP1 (a mixture of H-72 Santa-Cruz, 12688-1-AP Proteintech, and ab175544 Abcam), H3K79me3 (Diagenode, pAb-068-050), MLL1 (Bethyl, A300-086A), H3K4me3 (Active Motif, 39159), H3K27me3 (Millipore, 07-499), H3K27ac (Millipore, 07-360), LEDGF (Bethyl, A300-848A), and MEN1 (Bethyl, A300-105A).

Techniques: Expressing, ChIP-sequencing, Transduction, Western Blot, Quantitation Assay

Journal: Nature Communications

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

doi: 10.1038/s41467-018-03854-0

Figure Lengend Snippet: SHARP1 interacts with MLL-AF6 and regulates gene targets. a Enriched gene sets in ML-2 shGFP cells over shSHARP1 on RNA-seq. b Venn diagram showing overlapping of SHARP1-bound genes with MLL-AF6 target genes (MLL N+ H3K79me2) in ML-2 cells. c Heatmap images representing the relative expression levels of 14 MLL-AF6/SHARP1 co-target genes downregulated upon SHARP1 knockdown obtained from RNA-seq data. d Genome view of MLL N , MEN1, LEDGF, H3K79me2, H3K79me3, and SHARP1 peak binding on three MLL-AF6 + SHARP1 target genes in ML-2 shGFP: MEF2C , CDK6 and RUNX2 gene. e Co-immunoprecipitation studies of SHARP1 and MLL-AF6 with an anti-HA antibody in 293 T cells transfected with plasmids encoding MLL-AF6 and/or HA-tagged SHARP1. Proteins present in immunoprecipitates (IP, lane 1–4) or whole cell lysates of transfected cells (input, lane 5–8) were separated by SDS-PAGE and immunoblotted with antibodies specific for MLL N and SHARP1. Interaction of SHARP1 and MLL-AF6 was detected (lane 4) and not observed in negative control lanes with either empty vector, SHARP1 or MLL-AF6 only (lane 1–3). f Schematic showing a series of MLL deletion mutants. Interaction with SHARP1 is indicated by + sign and loss of interaction by − sign. Boxes indicate AT hook motifs (blue), nuclear translocation sequences (NTS1 and NTS2) (orange), subnuclear localization domains (SNL1 and SNL2) (purple), and DNA methyltransferase domain (MT) (yellow). g Domain mapping analysis of MLL required for interaction with SHARP1. 293 T cells are transfected with plasmids encoding FLAG-tagged MLL deletion mutants and HA-tagged SHARP1. Whole cell lysates were prepared from the transfected cells and subjected to immunoprecipitation with anti-FLAG antibody. Proteins present in immunoprecipitates (IP, lane 1–7) or whole cell lysates of transfected cells (input, lane 8–14) were separated by SDS-PAGE and immunoblotted with antibodies specific for FLAG-tagged MLL deletion mutants and HA-tagged SHARP1. The arrows indicate MLL mutant proteins. Western blots are representative of at least three independent experiments

Article Snippet: The antibodies used included SHARP1 (a mixture of H-72 Santa-Cruz, 12688-1-AP Proteintech, and ab175544 Abcam), H3K79me3 (Diagenode, pAb-068-050), MLL1 (Bethyl, A300-086A), H3K4me3 (Active Motif, 39159), H3K27me3 (Millipore, 07-499), H3K27ac (Millipore, 07-360), LEDGF (Bethyl, A300-848A), and MEN1 (Bethyl, A300-105A).

Techniques: RNA Sequencing Assay, Expressing, Binding Assay, Immunoprecipitation, Transfection, SDS Page, Negative Control, Plasmid Preparation, Translocation Assay, Mutagenesis, Western Blot