epz5676 targetmol cat  (TargetMol)

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor HOXA9 induces expression of the chromatin modifier SMYD3 to drive leukemogenesis

doi: 10.1016/j.jbc.2025.110320

Figure Lengend Snippet: Combinational inhibition of SMYD3 and DOT1L induces enhanced growth arrest and differentiation in AML . A , synergistic effect of the combination of BCI-121 and EPZ5676 in MOLM-13 and THP-1 cells was analyzed by MTS assay after incubating cells with a serial diluted mixture at a fixed ratio of the two inhibitors. CI is plotted against the fraction effect. The reference line indicates CI = 1. CI values below 1 indicate synergism between the two inhibitors. B–D , co-treatment with BCI-121 and EPZ5676 led to enhanced inhibitory activity against the growth of AML cells. After being incubated with BCI-121 with or without EPZ5676 for 72 h, MOLM-13 and THP-1 cells were harvested, and then subjected to Western blotting analysis ( B ), dynamic cell counting ( C ), or colony formation ( D ), respectively. E– G , co-treatment with BCI-121 and EPZ5676 led to enhanced differentiation in AML cells. After exposed to BCI-121 with or without EPZ5676 for 72 h, MOLM-13 and THP-1 cells were subjected to Wright-Giemsa staining ( E ) and flow cytometry analysis for CD11b ( F ) or CD14 ( G ). Data are mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001, one-way ANOVA, Multiple comparison, Tukey's test was applied in C–G . All the experiments were repeated at least thrice.

Article Snippet: BCI-121 (T5322) and EPZ5676 (T3099) were purchased from TargetMol Chemicals (Shanghai, China).

Techniques: Inhibition, MTS Assay, Activity Assay, Incubation, Western Blot, Cell Counting, Staining, Flow Cytometry, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor HOXA9 induces expression of the chromatin modifier SMYD3 to drive leukemogenesis

doi: 10.1016/j.jbc.2025.110320

Figure Lengend Snippet: Combination of SMYD3 depletion and DOT1L inhibition induces enhanced growth arrest and differentiation in AML . A , MOLM-13 and THP-1 cells stably expressing Scramble or shSMYD3 were exposed to increasing concentrations of EPZ5676 for 72 h, and subjected to MTS assay. B–D , SMYD3 depletion sensitized AML cells to EPZ5676 treatment. After SMYD3 depletion, MOLM-13 and THP-1 cells were exposed to EPZ5676 for 72 h or not, and then subjected to Western blotting analysis ( B ), dynamic cell counting ( C ), or colony formation ( D ), respectively. E–G , SMYD3 depletion combined with EPZ5676 treatment led to enhanced differentiation in AML cells. After SMYD3 depletion, MOLM-13 and THP-1 cells were exposed to EPZ5676 for 72 h or not, and then subjected to Wright-Giemsa staining ( E ) or analyzed by flow cytometry for CD11b ( F ) or CD14 ( G ). H , a proposed model to describe the fundamental role of SMYD3 in AML. Data are mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001, one-way ANOVA, Multiple comparison, Tukey's test was applied in C–G . All the experiments were repeated at least thrice.

Article Snippet: BCI-121 (T5322) and EPZ5676 (T3099) were purchased from TargetMol Chemicals (Shanghai, China).

Techniques: Inhibition, Stable Transfection, Expressing, MTS Assay, Western Blot, Cell Counting, Staining, Flow Cytometry, Comparison

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 1. Dot1l deficiency activates retrotransposons. ( A ) qPCR analysis of the expression of Dot1l in ESCs treated with control (Ctrl) shRNA or Dot1l shRNAs. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of MERVL after the depletion of Dot1l in ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Schematic of mutation sites in Dot1l −/ −ESCs. Black dashes: deleted bases; red bases: sgRNA target sequences; blue bases: protospacer adjacent motif (PAM) sequences. ( D ) qPCR analysis of the expression of Dot1l in WT ESCs and Dot1l −/ −ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) Western blot analysis of Dot1l protein in WT ESCs and Dot1l −/ −ESCs. -Actin was used as a loading control. ( F ) Western blot analysis of H3K79me1 / 2 / 3 le v el in WT ESCs and Dot1l −/ −ESCs. H3 was used as a loading control. ( G ) qPCR analysis of the expression of retrotransposons in WT ESCs and Dot1l −/ −ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, *** P < 0.001 in Student’s t -test.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Expressing, Control, shRNA, Mutagenesis, Western Blot

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 2. Loss of Dot1l transformed ESCs to 2CLCs. ( A ) The expression of Dot1l in the entry to 2CLC state. D0 2C −refers to MERVL-negati v e population before Dux induction, D1 2C −and D1 2C + r epr esent MERVL-negati v e and MERVL-positi v e populations after 1 day Dux induction respecti v ely. ( B ) The expression of Dot1l during the exit from 2CLC state. D1 2C + r epr esents 2C-like cells after 1 day of Dux induction. D4 2C −r epr esents D1 2C + cells cultured for 3 days to exit the 2C-like sta te. 2C −, MERVL-nega ti v e cells; 2C + , MERVL-positi v e 2CLCs. ( C ) Western b lot analysis of MERVL-gag le v el in WT ESCs and Dot1l −/ −ESCs. -Actin was used as a loading control. ( D ) Flow cytometry analysis of the MERVL-gag + population in WT ESCs and Dot1l −/ −ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) qPCR analysis of the expression of TSC genes in WT ESCs and Dot1l −/ −ESCs dif ferentia ted into TSCs respecti v ely. qPCR data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of 2-cell embryo genes in WT ESCs and Dot1l −/ −ESCs. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent e xperiments). ( G ) Immunob lot analysis of the expression of Dot1l after ov ere xpression of HA-Dot1l in WT ESCs and Dot1l −/ −ESCs. -Actin was included as a loading control. Ctrl OE: control vector over expr ession. ( H, I ) qPCR analysis of the expression of Dot1l (H) and MERVL (I) after ov ere xpression of Dot1l in Dot1l −/ −ESCs. Ctrl OE: control vector over expr ession. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001 in Student’s t -test.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Transformation Assay, Expressing, Cell Culture, Western Blot, Control, Flow Cytometry, Plasmid Preparation

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 4. Dot1l interacts with Npm1 to r epr ess MERVL. ( A ) qPCR analysis of the expression of Mllt10 , Mllt6 and Mllt3 after transfected with control (Ctrl) shRNA and shRNAs against Mllt10 , Mllt6 and Mllt3 . Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of MERVL after transfected with control (Ctrl) shRNA and shRNAs against Mllt10 , Mllt6 and Mllt3 . qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) The expression level of Npm1 during earl y embryo genesis according to published RNA-seq data ( 71 ). ( D ) qPCR analysis of the expression of Npm1 in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) qPCR analysis of the expression of MERVL after the depletion of Npm1 in E14 ESCs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of 2-cell embryo genes in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( G, H ) ChIP-qPCR analysis of Dot1l (G) and Npm1 (H) binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. (n = 3 independent experiments). ( I, J ) Visualization the Dot1l (I) and Npm1 (J) ChIP-seq peaks mapped to MERVL consensus sequence in IGV. ( K ) Enrichment heatmap of Npm1 around the binding regions of Dot1l. The ChIP-seq signal was calculated as the log 2 ratio of normalized r eads r elati v e to the input. ( L ) A ChIP-reChIP assa y was perf ormed to detect the co-occupancy of HA-Dot1l and Npm1 on MERVL. ChIP DN A was anal yzed by qPCR using primers of MERVL. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001 in Student’s t -test.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Expressing, Transfection, Control, shRNA, RNA Sequencing, ChIP-qPCR, Binding Assay, ChIP-sequencing, Sequencing

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 5. Distinct roles of individual Dot1l domains. ( A ) A schematic summary of Dot1l mutants used for functional rescue. , deletion. ( B ) qPCR analysis of MERVL expression after the rescue with the ov ere xpression of Dot1l mutants in Dot1l −/ −ESCs. qPCR results were normalized to Gapdh and compared with the ov ere xpressed control v ector in Dot1l −/ −ESCs to anal yze w hether the differ ences ar e sta tistically significant. Da ta ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Sequences of WT (top) and CI (bottom) Dot1l alleles at exon 5. Mutated bases and amino acid r esidues ar e in red. WT: wild-type; CI: catalytic inacti v e; AA: amino acid. ( D ) qPCR analysis of MERVL expr ession after r escue of Dot1l C-terminal (CI) mutant in Dot1l −/ −ESCs. Ctrl OE: control vector over expr ession; CI: ca talytic inactive. qPCR da ta are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E–G ) qPCR analysis of the expression of MERVL in E14 ESCs (E), J1 ESCs (F) and E3 ESCs (G) treated with Dot1l inhibitor (EPZ5676). DMSO tr eated samples wer e included as a control. qPCR data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student’s t -test. ( H ) Western blot analysis of HA-Dot1l / Npm1 co-immunoprecipitation in ESCs ov ere xpressing control empty vector (Ctrl OE) or HA- tagged Dot1l (HA-Dot1l OE). IP was done with anti-HA magnetic beads. IP, immunopr ecipitation. OE, over expr ession. ( I ) Co-IP experiments to detect the interactions of Fla g-ta gged Dot1l mutants with HA-tagged Npm1 in HEK293T cells respecti v ely, IP was done with anti-Flag magnetic beads. The resulting co-IP samples were subjected to western blot.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Functional Assay, Expressing, Control, Mutagenesis, Plasmid Preparation, Western Blot, Immunoprecipitation, Magnetic Beads, Co-Immunoprecipitation Assay

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 6. Dot1l and Npm1 regulate histone H1 protein le v el and its loading onto chromatin. ( A ) qPCR analysis of the expression of H1.0 and H1.2 in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of H1.0 and H1.2 in WT ESCs and Dot1l −/ −ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Western blot analysis of H1.0, H1.2 and Npm1 protein in WT ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. -Actin was used as a loading control. ( D ) Western blot analysis of H1.0, H1.2 and Dot1l protein in WT ESCs and Dot1l −/ −ESCs. -Actin was used as a loading control. ( E , F ) ChIP-qPCR analysis of H1.0 (E) and H1.2 (F) binding on different retrotransposons. ChIP-qPCR data were normalized to input and that of the control region. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, ** P < 0.01, *** P < 0.001 in Student’s t -test. ( G ) The volcano plot shows transcriptome analysis of TEs expression after H1 TKO (triple-knockout of H1.2 , H1.3 and H1.4 ). The result from DEseq2 was used to plot the diagram. Differ ent color ed dots r epr esent differ ent r etroelement families. Color ed dots indicate TEs with significant expr ession change (adjusted P < 0.05, Wald test). ( H ) The RNA-seq reads mapped to MERVL in WT ESCs and ESC with H1 TKO (triple knockout of H1.2 , H1,3 and H1.4 ) were visualized using IGV.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Expressing, Control, shRNA, Western Blot, ChIP-qPCR, Binding Assay, Triple Knockout, RNA Sequencing

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 7. Dot1l regulates interaction between Npm1 and histone H1. ( A ) Western blot analysis of H1.2 protein after rescue with catalytic-acti v e or inacti v e (CI) Dot1l mutants in Dot1l −/ −ESCs (left) and quantification (right). -Actin was used as a loading control. The ratio of H1.2 / -actin protein from Dot1l −/ −samples was normalized to that from WT ESCs + Ctrl OE sample, which is set as 1. Data are presented as mean ± s.e.m. (n = 3 independent experiments). Ctrl OE: control vector over expr ession; CI: catalytic inactive; , deletion. ( B ) ChIP-qPCR analysis of H1.2 binding on MERVL. ChIP- qPCR data were normalized to input and that of the control region. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) ChIP- qPCR analysis of Dot1l and Dot1l (CI)-Flag binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( D ) ChIP-qPCR analysis of Npm1 binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) Western blot analysis of Npm1 / H1.2 co-immunoprecipitation in WT ESCs or Dot1l −/ −ESCs (left) and quantification (right). IP, immunoprecipitation. IP was done with anti-Npm1 antibody. 1.25% input was loaded as a control. The ratio of IP protein / input protein from Dot1l −/ −sample was normalized to that from WT ESC sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of MERVL after the depletion of Dot1l and Npm1 individually or simultaneously in ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant. ( G ) ChIP-qPCR analysis of Dot1l binding on MERVL upon the Npm1 depletion. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student’s t -test.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Western Blot, Control, Plasmid Preparation, ChIP-qPCR, Binding Assay, Immunoprecipitation, Expressing

Journal: Nucleic acids research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Figure 8. Histone H1.2 was degraded by ubiquitination in Dot1l −/ −ESCs. ( A ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody in WT ESCs or Dot1l −/ −ESCs (left) and quantification (right). IP, immunoprecipitation. IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/ −ESCs is adjusted to ma tch tha t in WT ESCs and IP protein loading was adjusted proportionally according to the input. The ratio of IP protein / input protein from Dot1l −/ −sample was normalized to that from WT ESC sample, which is set as 1. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent e xperiments). ( B ) Western b lot analysis of H1.2 le v els in Dot1l −/ −ESCs treated with the inhibitor of NAE (MLN4924) (left) and quantifica tion (right). DMSO trea ted sample was included as a control. -Actin was used as a loading control. The ratio of H1.2 / -actin pr otein fr om Dot1l −/ −samples was normalized to that from WT ESC + DMSO sample, which is set as 1. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody in Dot1l −/ −ESCs treated with the inhibitor of NAE (MLN4924) (left) and quantification (right). IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/ −ESCs and treated with NAE inhibitor (MLN4924) are adjusted to match that in WT ESCs. The ratio of IP / input protein from Dot1l −/ −samples was normalized to that from WT ESC + DMSO sample, which is set as 1. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ( D ) qPCR analysis of the expression of MERVL in Dot1l −/ −ESCs treated with NAE inhibitor (MLN4924). DMSO treated sample was included as a control. Data ar e pr esented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student’s t -test. ( E ) qPCR analysis of the expression of MERVL after ov ere xpression of H1.2K64R in Dot1l −/ −ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). *** P < 0.001 in Student’s t -test. Ctrl OE: control v ector ov ere xpression; K, Lysine; R, Arginine. ( F ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody after ov ere xpression of H1.2K64R in Dot1l −/ −ESCs (left) and quantification (right). IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/ −ESCs and H1.2K64R OE are adjusted to match that in WT ESCs and IP protein loading was adjusted proportionally according to the input. The ratio of IP protein / input protein from Dot1l −/ −sample was normalized to that from WT ESC sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( G ) Schematic of Dot1l r epr essing MERVL in ESCs. Histone H1 is loaded to chromatin by Dot1l and Npm1 to maintain the r epr ession of MERVL. Conversely, in the absence of Dot1l, histone H1 dissociates from chromatin and its protein le v el decreases, resulting in de-repression of MERVL transcription.

Article Snippet: For inhibitor treatment, SCs were treated with 1 M Dot1l inhibitor EPZ5676 T3099, TargetMol) or 0.1 M NAE inhibitor MLN4924 S81085, MedMol) during cell passage and treated continously for 48 h. hRNA knockdown or gene knockdown, the designed shRNAs were cloned nto pSuper-puro vector and sequenced.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Control, Expressing

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Dot1l deficiency activates retrotransposons. ( A ) qPCR analysis of the expression of Dot1l in ESCs treated with control (Ctrl) shRNA or Dot1l shRNAs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of MERVL after the depletion of Dot1l in ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Schematic of mutation sites in Dot1l −/− ESCs. Black dashes: deleted bases; red bases: sgRNA target sequences; blue bases: protospacer adjacent motif (PAM) sequences. ( D ) qPCR analysis of the expression of Dot1l in WT ESCs and Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) Western blot analysis of Dot1l protein in WT ESCs and Dot1l −/− ESCs. β-Actin was used as a loading control. ( F ) Western blot analysis of H3K79me1/2/3 level in WT ESCs and Dot1l −/− ESCs. H3 was used as a loading control. ( G ) qPCR analysis of the expression of retrotransposons in WT ESCs and Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, *** P < 0.001 in Student's t -test.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Expressing, Control, shRNA, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Loss of Dot1l transformed ESCs to 2CLCs. ( A ) The expression of Dot1l in the entry to 2CLC state. D0 2C − refers to MERVL-negative population before Dux induction, D1 2C − and D1 2C + represent MERVL-negative and MERVL-positive populations after 1 day Dux induction respectively. ( B ) The expression of Dot1l during the exit from 2CLC state. D1 2C + represents 2C-like cells after 1 day of Dux induction. D4 2C − represents D1 2C + cells cultured for 3 days to exit the 2C-like state. 2C − , MERVL-negative cells; 2C + , MERVL-positive 2CLCs. ( C ) Western blot analysis of MERVL-gag level in WT ESCs and Dot1l −/− ESCs. β-Actin was used as a loading control. ( D ) Flow cytometry analysis of the MERVL-gag + population in WT ESCs and Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) qPCR analysis of the expression of TSC genes in WT ESCs and Dot1l −/− ESCs differentiated into TSCs respectively. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of 2-cell embryo genes in WT ESCs and Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( G ) Immunoblot analysis of the expression of Dot1l after overexpression of HA-Dot1l in WT ESCs and Dot1l −/− ESCs. β-Actin was included as a loading control. Ctrl OE: control vector overexpression. ( H, I ) qPCR analysis of the expression of Dot1l (H) and MERVL (I) after overexpression of Dot1l in Dot1l −/− ESCs. Ctrl OE: control vector overexpression. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001 in Student's t -test.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Transformation Assay, Expressing, Cell Culture, Western Blot, Control, Flow Cytometry, Over Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Transcriptome regulated by Dot1l in ESCs. ( A ) The volcano plot of gene expression in Dot1l −/− ESCs versus WT ESCs. Red, up-regulated genes; blue, down-regulated genes; black, other genes. Genes with expression change ≥1.5-fold and adjusted P < 0.05 are shown. ( B ) The volcano plot shows transcriptome analysis of TEs expression after Dot1l knockout. Red, upregulated TEs; blue, downregulated TEs; black, other TEs; adjusted P < 0.05, Wald test. ( C ) The TEs with the highest number of loci upregulated in Dot1l −/− ESCs. ( D ) Gene set enrichment analysis (GSEA) of 2-cell genes in the transcriptome of Dot1l −/− ESCs. Red, up-regulated genes; blue, down-regulated genes; NES , normalized enrichment scores; FDR , false discovery rate. The Kolmogorov–Smirnov statistic was used for the calculation of the P -value. ( E ) The volcano plot of all expressed genes in WT ESCs and Dot1l −/− ESCs. Genes fused with MERVL are labelled in red. ( F, G ) Gene ontology (GO) analysis of upregulated genes (F) and downregulated genes (G) after Dot1l knockout. The analysis was done with clusterProfiler.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Expressing, Knock-Out

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Dot1l interacts with Npm1 to repress MERVL. ( A ) qPCR analysis of the expression of Mllt10, Mllt6 and Mllt3 after transfected with control (Ctrl) shRNA and shRNAs against Mllt10, Mllt6 and Mllt3 . Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of MERVL after transfected with control (Ctrl) shRNA and shRNAs against Mllt10, Mllt6 and Mllt3 . qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) The expression level of Npm1 during early embryogenesis according to published RNA-seq data . ( D ) qPCR analysis of the expression of Npm1 in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) qPCR analysis of the expression of MERVL after the depletion of Npm1 in E14 ESCs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of 2-cell embryo genes in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( G, H ) ChIP-qPCR analysis of Dot1l (G) and Npm1 (H) binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. (n = 3 independent experiments). ( I, J ) Visualization the Dot1l (I) and Npm1 (J) ChIP-seq peaks mapped to MERVL consensus sequence in IGV. ( K ) Enrichment heatmap of Npm1 around the binding regions of Dot1l. The ChIP-seq signal was calculated as the log 2 ratio of normalized reads relative to the input. ( L ) A ChIP-reChIP assay was performed to detect the co-occupancy of HA-Dot1l and Npm1 on MERVL. ChIP DNA was analyzed by qPCR using primers of MERVL. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001 in Student's t -test.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Expressing, Transfection, Control, shRNA, RNA Sequencing Assay, Binding Assay, ChIP-sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Distinct roles of individual Dot1l domains. ( A ) A schematic summary of Dot1l mutants used for functional rescue. Δ, deletion. ( B ) qPCR analysis of MERVL expression after the rescue with the overexpression of Dot1l mutants in Dot1l −/− ESCs. qPCR results were normalized to Gapdh and compared with the overexpressed control vector in Dot1l −/− ESCs to analyze whether the differences are statistically significant. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Sequences of WT (top) and CI (bottom) Dot1l alleles at exon 5. Mutated bases and amino acid residues are in red. WT: wild-type; CI: catalytic inactive; AA: amino acid. ( D ) qPCR analysis of MERVL expression after rescue of Dot1l ΔC-terminal (CI) mutant in Dot1l −/− ESCs. Ctrl OE: control vector overexpression; CI: catalytic inactive. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E–G ) qPCR analysis of the expression of MERVL in E14 ESCs (E), J1 ESCs (F) and E3 ESCs (G) treated with Dot1l inhibitor (EPZ5676). DMSO treated samples were included as a control. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student's t -test. ( H ) Western blot analysis of HA-Dot1l/Npm1 co-immunoprecipitation in ESCs overexpressing control empty vector (Ctrl OE) or HA-tagged Dot1l (HA-Dot1l OE). IP was done with anti-HA magnetic beads. IP, immunoprecipitation. OE, overexpression. ( I ) Co-IP experiments to detect the interactions of Flag-tagged Dot1l mutants with HA-tagged Npm1 in HEK293T cells respectively, IP was done with anti-Flag magnetic beads. The resulting co-IP samples were subjected to western blot.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Functional Assay, Expressing, Over Expression, Control, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Magnetic Beads, Co-Immunoprecipitation Assay

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Dot1l and Npm1 regulate histone H1 protein level and its loading onto chromatin. ( A ) qPCR analysis of the expression of H1.0 and H1.2 in E14 ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. qPCR data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) qPCR analysis of the expression of H1.0 and H1.2 in WT ESCs and Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Western blot analysis of H1.0, H1.2 and Npm1 protein in WT ESCs treated with control (Ctrl) shRNA or Npm1 shRNAs. β-Actin was used as a loading control. ( D ) Western blot analysis of H1.0, H1.2 and Dot1l protein in WT ESCs and Dot1l −/− ESCs. β-Actin was used as a loading control. ( E , F ) ChIP-qPCR analysis of H1.0 (E) and H1.2 (F) binding on different retrotransposons. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, ** P < 0.01, *** P < 0.001 in Student's t -test. ( G ) The volcano plot shows transcriptome analysis of TEs expression after H1 TKO (triple-knockout of H1.2, H1.3 and H1.4 ). The result from DEseq2 was used to plot the diagram. Different colored dots represent different retroelement families. Colored dots indicate TEs with significant expression change (adjusted P < 0.05, Wald test). ( H ) The RNA-seq reads mapped to MERVL in WT ESCs and ESC with H1 TKO (triple knockout of H1.2, H1,3 and H1.4 ) were visualized using IGV.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Expressing, Control, shRNA, Western Blot, Binding Assay, Triple Knockout, RNA Sequencing Assay

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Dot1l regulates interaction between Npm1 and histone H1. ( A ) Western blot analysis of H1.2 protein after rescue with catalytic-active or inactive (CI) Dot1l mutants in Dot1l −/− ESCs (left) and quantification (right). β-Actin was used as a loading control. The ratio of H1.2/β-actin protein from Dot1l −/− samples was normalized to that from WT ESCs + Ctrl OE sample, which is set as 1. Data are presented as mean ± s.e.m. (n = 3 independent experiments). Ctrl OE: control vector overexpression; CI: catalytic inactive; Δ, deletion. ( B ) ChIP-qPCR analysis of H1.2 binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) ChIP-qPCR analysis of Dot1l and Dot1l (CI)-Flag binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( D ) ChIP-qPCR analysis of Npm1 binding on MERVL. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( E ) Western blot analysis of Npm1/H1.2 co-immunoprecipitation in WT ESCs or Dot1l −/− ESCs (left) and quantification (right). IP, immunoprecipitation. IP was done with anti-Npm1 antibody. 1.25% input was loaded as a control. The ratio of IP protein/input protein from Dot1l −/− sample was normalized to that from WT ESC sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( F ) qPCR analysis of the expression of MERVL after the depletion of Dot1l and Npm1 individually or simultaneously in ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant. ( G ) ChIP-qPCR analysis of Dot1l binding on MERVL upon the Npm1 depletion. ChIP-qPCR data were normalized to input and that of the control region. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student's t -test.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Western Blot, Control, Plasmid Preparation, Over Expression, Binding Assay, Immunoprecipitation, Expressing

Journal: Nucleic Acids Research

Article Title: Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells

doi: 10.1093/nar/gkad640

Figure Lengend Snippet: Histone H1.2 was degraded by ubiquitination in Dot1l −/− ESCs. ( A ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody in WT ESCs or Dot1l −/− ESCs (left) and quantification (right). IP, immunoprecipitation. IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/− ESCs is adjusted to match that in WT ESCs and IP protein loading was adjusted proportionally according to the input. The ratio of IP protein/input protein from Dot1l −/− sample was normalized to that from WT ESC sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( B ) Western blot analysis of H1.2 levels in Dot1l −/− ESCs treated with the inhibitor of NAE (MLN4924) (left) and quantification (right). DMSO treated sample was included as a control. β-Actin was used as a loading control. The ratio of H1.2/β-actin protein from Dot1l −/− samples was normalized to that from WT ESC + DMSO sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( C ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody in Dot1l −/− ESCs treated with the inhibitor of NAE (MLN4924) (left) and quantification (right). IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/− ESCs and treated with NAE inhibitor (MLN4924) are adjusted to match that in WT ESCs. The ratio of IP/input protein from Dot1l −/− samples was normalized to that from WT ESC + DMSO sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( D ) qPCR analysis of the expression of MERVL in Dot1l −/− ESCs treated with NAE inhibitor (MLN4924). DMSO treated sample was included as a control. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, *** P < 0.001 in Student's t -test. ( E ) qPCR analysis of the expression of MERVL after overexpression of H1.2K64R in Dot1l −/− ESCs. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). *** P < 0.001 in Student's t -test. Ctrl OE: control vector overexpression; K, Lysine; R, Arginine. ( F ) Western blot analysis of ubiquitination after immunoprecipitation with H1.2 antibody after overexpression of H1.2K64R in Dot1l −/− ESCs (left) and quantification (right). IP was done with anti-H1.2 antibody. 1.25% input was loaded as a control. H1.2 input protein amount of Dot1l −/− ESCs and H1.2K64R OE are adjusted to match that in WT ESCs and IP protein loading was adjusted proportionally according to the input. The ratio of IP protein/input protein from Dot1l −/− sample was normalized to that from WT ESC sample, which is set as 1. Data are presented as mean ± s.e.m. ( n = 3 independent experiments). ( G ) Schematic of Dot1l repressing MERVL in ESCs. Histone H1 is loaded to chromatin by Dot1l and Npm1 to maintain the repression of MERVL. Conversely, in the absence of Dot1l, histone H1 dissociates from chromatin and its protein level decreases, resulting in de-repression of MERVL transcription.

Article Snippet: For inhibitor treatment, ESCs were treated with 1 μM Dot1l inhibitor EPZ5676 (T3099, TargetMol) or 0.1 μM NAE inhibitor MLN4924 (S81085, MedMol) during cell passage and treated continuously for 48 h.

Techniques: Western Blot, Immunoprecipitation, Control, Expressing, Over Expression, Plasmid Preparation