Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL enhances PRC2 activity in vitro. A, Coomassie Blue staining of PRC2 complexes (containing EZH2, SUZ12, and EED) purified from Sf9 cells. B, MNase digestion of reconstituted oligonucleosomes resolved by 2% agarose gel. Left panel: lane 1 shows the pure pG5E4 plasmid DNA, and lane 2 shows band shift of pG5E4 DNA assembly into oligonucleosomes. Right panel: equimolar amounts of reconstituted oligonucleosomes were digested with increasing amounts of MNase (Sigma). The DNA was isolated and subjected to electrophoresis on a 2% agarose gel in the presence of ethidium bromide. Partial MNase digestion (oligonucleosome: MNase = 2 μg: 0.5 μl) generated a nucleosomal DNA ladder with visible mono-, di-, and trinucleosomal fragments, which are indicated by corresponding numbers of asterisks. Mononucleosomal DNA runs as a 147 bp fragment. C, CDYL stimulates PRC2 activity in vitro. Reconstituted recombinant oligonucleosomes were incubated with EZH2/SUZ12/EED complexes (PRC2-core) in the absence or presence of increasing amounts of baculovirus generated CDYL and histone methyltransferase activity was determined by standard HMT assays. The reaction products were examined by Western blotting with the antibodies indicated on the right. Ponceau staining of histones is shown in the bottom panel to show equal amounts of substrates used in each reaction. D, CDYL only stimulates PRC2 methyltransferase activity toward oligonucleosome, but not mononucleosome substrates. Reconstituted Xenopus oligonucleosomes were digested with MNase (oligonucleosome: MNase = 1 μg: 1 μl) at room temperature for 5 min. This treatment yielded mainly mononucleosomes (see Fig. 4B). Equal amounts of mononucleosomes were used as substrates for the HMT assay in the top panel, whereas equal amounts of undigested oligonucleosomes were used as substrates in the bottom panel. Commercially available EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (PRC2-full) were used to provide methyltransferase activity as indicated. The mild increase of PRC2 activity seen upon CDYL addition in the top panel was mainly due to incomplete digestion of oligonucleosomes (see Fig. 4B). E, binding affinity between CDYL and H3K27me3 is much stronger than the affinity between EED and H3K27me3. In the top panel, histone peptide binding assays show that CDYL, but not EED, binds to H3K27me2 when the same amounts of FLAG-tagged proteins (0.5 μg) were used in the assay. To compare the binding affinity for H3K27me3, 0.2, 0.4, or 1 μg of baculovirus-expressed FLAG-CDYL and 0.6, 1.2, or 3 μg of FLAG-EED proteins were used in the peptide binding assay. Ten percent of total proteins were used as loading controls (middle panel). Peptide-protein complexes were pulled down by streptavidin beads and bound proteins were examined by Western blotting using anti-FLAG antibodies (bottom panel). Ponceau staining of baculovirus-expressed FLAG-CDYL and FLAG-EED is shown in the right panel.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Activity Assay, In Vitro, Staining, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Isolation, Electrophoresis, Generated, Recombinant, Incubation, Western Blot, HMT Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL is physically associated with the PRC2 complex. A, in vivo immunoprecipitation. Top two panels: endogenous IP of MCF-7 cell lysates using antibodies against CDYL. Antibodies (EZH2 or SUZ12) used for Western blotting are indicated on the right. Bottom panel: MCF-7 cells were transfected with a FLAG-CDYL construct and subjected to co-IP assays after 48 h. Cell protein extracts were immunoprecipitated with polyclonal antibodies against EED, and blotted with monoclonal anti-FLAG antibodies. An immunoglobulin G (IgG) control is included in each experiment. B, GST pull-down assays. Purified GST or GST-CDYL proteins immobilized on glutathione Sepharose 4B beads were incubated with in vitro translated EZH2, SUZ12, or EED. Bound proteins were detected with monoclonal anti-EZH2 antibodies (top panel) or anti-MYC tag antibodies (lower two panels). C, Superose 6 gel filtration analysis of the MCF-7 nuclear extracts. Migration of molecular markers is indicated above the panels and the antibodies for Western blotting are indicated on the right. Equal volumes from each fraction were analyzed. D, similar FPLC experiments as in C using a Superdex 200 10/300 GL column. The chromatographic fractions were analyzed by Western blotting using the indicated antibodies. Bottom panel: HMT assays were performed using the indicated eluted fractions. Recombinant Xenopus H3 proteins were used as substrates, and the reaction products were analyzed by Western blotting with anti-H3K27me3 antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Transfection, Construct, Co-Immunoprecipitation Assay, Purification, Incubation, In Vitro, Filtration, Migration, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Mapping the domains responsible for the interaction between CDYL and EZH2. A, schematic drawing of CDYL protein. CDYL deletion mutants including del1 (1–309 aa), del2 (1–60 aa, the chromodomain), del3 (61–545 aa), del4 (310–545 aa, the coAP domain), and del5 (61–309 aa) were fused to GST. B, GST pull-down experiments were performed with in vitro translated FLAG-EZH2 and purified GST or GST-CDYL deletion mutants. The precipitated complexes were examined by Western blotting using monoclonal anti-EZH2 antibodies (the upper panel). Only CDYL mutants containing the middle region from 61–309 aa (del1, del3, del5) efficiently pulled down EZH2. The lower panel shows the Ponceau staining of purified GST fusion proteins added to the reaction. The arrows indicate the positions of the respective GST fusion proteins as labeled on the top. C, schematic drawing of EZH2 protein. EZH2 deletion mutants (del1 to del5) were cloned into the pGBKT7 plasmid, which contains a c-Myc epitope tag and can be transcribed/translated in vitro. Del8 and Del9 were fused to GST. D, GST pull-down experiments were performed with in vitro translated Myc-EZH2 deletion mutants and purified GST or GST-CDYL in the left panels. Right panel: GST pull-down assays were performed with in vitro translated Myc-CDYL, which was incubated with purified GST, GST-del8, GST-del9, or GST-SET8 (negative control protein). Bound proteins were examined by Western blotting using monoclonal anti-Myc antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vitro, Purification, Western Blot, Staining, Labeling, Clone Assay, Plasmid Preparation, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Validation of common target genes of CDYL and PRC2. A, quantitative ChIP assays were performed in MCF-7 cells with primer pairs specific to indicated gene promoters (see supplemental Table S1). Normal rabbit IgG, as well as polyclonal antibodies against CDYL, EZH2, and H3K27me3 were used to immunoprecipitate the protein-DNA complex. B, conventional semi-quantitative ChIP assays performed at the MYT1 and BASE promoters. C, CDYL and PRC2 exist in the same protein complex at the MYT1 and BASE promoters. ChIP and re-ChIP experiments were performed with the indicated antibodies and primer pairs. D, CDYL expression was efficiently knocked down by specific siRNAs. Non-silencing or CDYL specific siRNAs were transfected into MCF-7 cells. Total proteins were extracted and the expression of CDYL and EZH2 proteins were examined by Western blotting. Actin protein levels were measured to indicate equal loading of protein lysates. E, CDYL is required for PRC2 chromatin targeting at the MYT1 and BASE promoters. MCF-7 cells were transfected with control siRNA or CDYL-specific siRNA. 48 hours after the transfection, cell lysates were collected, and ChIP experiments were performed using the indicated antibodies. Real-time PCR assays were performed for the measurement. F, CDYL mainly represses the expression of target genes. MCF-7 cells were transfected with control or CDYL-specific siRNAs. Total RNAs were prepared and the mRNA levels of the indicated genes were examined by real-time RT-PCR. The data were normalized against the expression of GAPDH. Each bar represents the mean ± S.D. for triplicate measurements.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Human Mutation

Article Title: Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

doi: 10.1002/humu.22946

Figure Lengend Snippet: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro . Histone methyltransferase reactions were performed using 2 μg purified core histones and 0.67 μM 3 H‐S‐adenosyl‐methionine ( 3 H‐SAM). Each reaction was incubated with 250 ng of either wild‐type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3 H‐labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for each complex: EZH2 WT • , p.(Phe672Ile) × , p.(Pro132Ser) ★ , p.(Tyr153del) △, p.(His694Tyr) ▽, p.(Glu745Lys) ▴, p.(Ala682Thr) ▾, p.(Arg684Cys) ▪, p.(Tyr133Cys) □, and p.(Asp185His) ◇. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t‐test showed statistically significant difference between the two groups (P value < 0.0001). B : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One‐way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Article Snippet: To test our hypothesis, we designed recombinant human EZH2 proteins, had them preassembled into PRC2 complexes (BPS Bioscience, San Diego CA), and tested their activity in vitro using a well‐accepted in vitro assay [Ernst et al., ; Yap et al., ; Score et al., ].

Techniques: Activity Assay, In Vitro, Purification, Incubation, Mutagenesis, Labeling, Standard Deviation, Positive Control, Negative Control

Journal: Practical Laboratory Medicine

Article Title: Complete blood count in neonatal Intensive care Unit (NICU): Performance comparison between POCT Sight OLO® and Sysmex XN-9100™ hematology analyzers

doi: 10.1016/j.plabm.2025.e00453

Figure Lengend Snippet: Regression analysis in the newborn population. Results of the method comparison study between the Sight OLO® and the Sysmex XN™ hematology analyzers. Graphs indicate Pearson correlation, slope and intercept for the main parameters. WBC: White Blood Cells, RBC: Red Blood Cells, PLT: Platelets, HGB: Hemoglobin, NEUT: Neutrophils, LYMPHO: Lymphocytes, MONO: Monocytes, EOS: Eosinophils, BASO: Basophils.

Article Snippet: Flags of the OLO® system were in partial accordance with the Sysmex XNTM system, but the POCT showed good performance compared to the final report, after revision carried out by the laboratory specialist.

Techniques: Comparison

Journal: Practical Laboratory Medicine

Article Title: Complete blood count in neonatal Intensive care Unit (NICU): Performance comparison between POCT Sight OLO® and Sysmex XN-9100™ hematology analyzers

doi: 10.1016/j.plabm.2025.e00453

Figure Lengend Snippet: Bias plot in the newborn population. Bland Altman of the results of a method comparison study between the Sight OLO® and the Sysmex XN™ hematology analyzers for the main measurements in the newborn population. WBC: White Blood Cells, RBC: Red Blood Cells, PLT: Platelets, HGB: Hemoglobin, NEUT: Neutrophils, LYMPHO: Lymphocytes, MONO: Monocytes, EOS: Eosinophils, BASO: Basophils.

Article Snippet: Flags of the OLO® system were in partial accordance with the Sysmex XNTM system, but the POCT showed good performance compared to the final report, after revision carried out by the laboratory specialist.

Techniques: Comparison

Journal: Practical Laboratory Medicine

Article Title: Complete blood count in neonatal Intensive care Unit (NICU): Performance comparison between POCT Sight OLO® and Sysmex XN-9100™ hematology analyzers

doi: 10.1016/j.plabm.2025.e00453

Figure Lengend Snippet: Bias plot in the adult population. Bland Altman of the results of method comparison study between the Sight OLO® and the Sysmex XN™ hematology analyzers for the main measurands in the adult population. WBC: White Blood Cells, RBC: Red Blood Cells, PLT: Platelets, HGB: Hemoglobin, NEUT: Neutrophils, LYMPHO: Lymphocytes, MONO: Monocytes, EOS: Eosinophils, BASO: Basophils.

Article Snippet: Flags of the OLO® system were in partial accordance with the Sysmex XNTM system, but the POCT showed good performance compared to the final report, after revision carried out by the laboratory specialist.

Techniques: Comparison

Journal: Practical Laboratory Medicine

Article Title: Complete blood count in neonatal Intensive care Unit (NICU): Performance comparison between POCT Sight OLO® and Sysmex XN-9100™ hematology analyzers

doi: 10.1016/j.plabm.2025.e00453

Figure Lengend Snippet: Instrumental analytical alarms (flags) concordance between Sight OLO® and Sysmex XN™ in the newborn population.

Article Snippet: Flags of the OLO® system were in partial accordance with the Sysmex XNTM system, but the POCT showed good performance compared to the final report, after revision carried out by the laboratory specialist.

Techniques:

Journal: Practical Laboratory Medicine

Article Title: Complete blood count in neonatal Intensive care Unit (NICU): Performance comparison between POCT Sight OLO® and Sysmex XN-9100™ hematology analyzers

doi: 10.1016/j.plabm.2025.e00453

Figure Lengend Snippet: Instrumental analytical alarms (flags) concordance between Sight OLO® and Sysmex XN™ in the adult population.

Article Snippet: Flags of the OLO® system were in partial accordance with the Sysmex XNTM system, but the POCT showed good performance compared to the final report, after revision carried out by the laboratory specialist.

Techniques: