Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of the screening strategy. epi, epigenetic; FACS, fluorescence-activated cell sorting; NGS, Next-generation sequencing. ( B and C ) Scatterplot of the screen results. Control sgRNAs (dark blue) are scattered randomly across the diagonal. EZH2 (light blue) and KDM1A (green) represent positive control sgRNAs. NT, non-targeting. ( D ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells transduced with the indicated sgRNAs. ( E ) CD47 or PD-L1 mRNA levels in HuH-7 cells upon DOT1L depletion with or without IFNγ treatment for 48 h. Results are shown as mean ± SD (n = 3). ( F ) CD47 or PD-L1 flow cytometry analyses of WT and DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Fluorescence, FACS, Next-Generation Sequencing, Control, Positive Control, Western Blot, Transduction, Flow Cytometry

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A and B ) Volcano plot of RNA-seq analysis in WT and DOT1L-depleted HuH-7 cells. NT, non-targeting. ( C ) Gene ontology (GO) analysis of RNA-seq data showing top 10 pathways that are up-regulated upon DOT1L depletion in HuH-7 cells. ( D ) Gene Set Enrichment Analysis (GSEA) of top 5 hallmark gene sets enriched in DOT1L-depleted HuH-7 cells. ( E ) GSEA of RNA-seq data showing IFNα (top) and IFN γ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( F and G ) Heatmaps of selected immune-related genes whose expression is higher in DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h. ( H ) Flow cytometry histograms of MHC I in sgDOT1L HuH-7 cells with or without IFNγ treatment for 48 h. ( I and J ) Binding density heatmap showing H3K4me3 distribution at the promoters of all genes (I) or the up-regulated genes (J) caused by DOT1L KO in HuH-7 cells. ( K and L ) Binding density heatmap showing H3K27ac distribution at the promoters of all genes (K) or the up-regulated genes (L) caused by DOT1L KO in HuH-7 cells.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells with EPZ-5676 treatment for 7 days. ( B ) RT-qPCR analysis of CD47 and PD-L1 mRNA levels of HuH-7 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( C ) Flow cytometry histograms of CD47 and PD-L1 in HuH-7 cells with EPZ-5676 treatment for 7 days. ( D ) Volcano plot of RNA-seq analysis in DMSO and EPZ-5676 treatment HuH-7 cells. ( E ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in HuH-7 cells with EPZ-5676 treatment. ( F ) Heatmaps of selected immune-related genes whose expression is higher in EPZ-5676 treatment for 7 days. ( G ) Expression levels of immune-related genes by RT-qPCR analysis. Results are shown as mean ± SD (n=3). ( H ) Flow cytometry histograms of MHC I in HuH-7 cells with EPZ-5676 treatment for 7 days. ( I ) GSEA of RNA-seq data showing IFNα (top) and IFNγ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( J ) Immunoblotting analysis of the DOT1L expression after overexpression of WT or catalytic inactive (CI) HA-DOT1L in DOT1L-depleted HuH-7 cells. ( K ) RT-qPCR analysis of immune-related genes after rescue of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). ( L ) Flow cytometry histograms of CD47 and PD-L1 after overexpression of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Western Blot, Quantitative RT-PCR, Flow Cytometry, RNA Sequencing, Expressing, Over Expression

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Volcano plot shows transcriptome analysis of transposon elements (TEs) expression after DOT1L KO in HuH-7 cells. ( B ) The expression changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( C ) RT-qPCR analyses of TEs in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). n.s., not significant, * P < 0.05, ** P < 0.01, unpaired Student’s t -test. ( D ) The ATAC-seq signal changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( E ) Binding density heatmap of ATAC-seq, H3K4me3, and H3K27ac ChIP-seq signals of up-regulated TEs caused by DOT1L KO in HuH-7 cells. ( F ) Flow cytometry histograms of dsRNA staining of WT or DOT1L KO HuH-7 cells. DAC, Decitabine served as a positive control. ( G and H ) RT-qPCR analysis of immune-related genes in DOT1L-depleted HuH-7 cells transduced with shRNA against scramble, MDA5 (G), or RIG-I (H). Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( I ) Heatmaps of IgG and DOT1L signals by fanChIP in HuH-7 cells. ( J ) Genomic annotations of DOT1L binding peaks by fanChIP in HuH-7 cells. ( K ) DOT1L fanChIP signals, ATAC-seq, and RNA-seq profiles in HuH-7 cells of LINEs (top) or LTR (bottom).

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Flow Cytometry, Staining, Positive Control, Transduction, shRNA, RNA Sequencing

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Venn diagrams of H3K79me2-associated genes and down-regulated genes in HuH-7 cells. ( B ) Expression levels of ZEB1 by RNA-seq in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=2). ** P < 0.01, unpaired Student’s t -test. RPKM, Reads Per Kilobase per Million mapped reads. ( C ) RT-qPCR analysis of ZEB1 upon DOT1L depletion in HuH-7 cells. Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( D ) Immunoblotting analysis of ZEB1 protein levels using whole-cell lysates from WT and DOT1L KO HuH-7 cells. ( E ) DOT1L fanChIP tracks, H3K79me2, H3K4me3, and H3K27ac ChIP-seq signals, and ATAC-seq profiles of WT and DOT1L-depleted HuH-7 cells in the ZEB1 locus. ( F ) Enrichment of ZEB1 motifs in the accessible chromatin regions specific to DOT1L-deficient HuH-7 cells by HOMER analysis. ( G ) Cistrome toolkit analysis of ATAC-seq data revealed that DNA-binding sites of ZEB1 were more open in the DOT1L-deficient HuH-7 cells. ( H ) Binding density heatmap of ZEB1 ChIP-seq signals in the DOT1L-deficient HuH-7 cells. ( I ) ChIP-seq tracks of ZEB1 in WT and DOT1L-depleted HuH-7 cells in the JAK2 , STAT1 , and PD-L1 loci. ( J ) Immunoblotting analysis using whole-cell lysates from WT and ZEB1 KO HuH-7 cells. ( K ) Expression levels of immune-related genes by RT-qPCR analysis upon ZEB1 depletion in HuH-7 cells. Results are shown as mean ± SD (n=3).

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from WT and Dot1l KO Hepa1-6 cells. ( B ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in Hepa1-6 cells upon Dot1l deficiency. ( C ) GSEA of RNA-seq data showing IFN (top) and IL2_STAT5 (bottom) gene sets that are up-regulated upon DOT1L depletion in Hepa1-6 cells. ( D ) RT-qPCR analyses of TEs in WT and Dot1l-depleted Hepa1-6 cells. Results are shown as mean ± SD (n=3). n.s., not significant, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. ( E ) Flow cytometry histograms of dsRNA staining of WT or Dot1l KO Hepa1-6 cells. ( F ) Immunoblotting analysis using whole-cell lysates from Hepa1-6 cells with EPZ-5676 treatment for 7 days. ( G ) RT-qPCR analysis of immune-related genes of Hepa1-6 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( H ) Schematic of ICB treatments in mice injected with Hepa1-6 WT or Dot1l KO cells. ( I ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 WT or Dot1l KO cells reconstituted as in (H) and subsequently treated with isotype control or anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( J ) Survival analysis conducted on the C57BL/6 mice described in (H). n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Logrank test. ( K ) Schematic of Dot1l inhibitor and ICB treatments in mice injected with Hepa1-6 cells. ( L ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 cells reconstituted as in (K) and subsequently treated with 20 mg/kg EPZ-5676 and anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( M ) Survival analysis conducted on the C57BL/6 mice described in (L). n.s., not significant, *** P < 0.001, Logrank test. ( N to R ), The intra-tumoral abundance of CD4+ T cells, CD8+ T cells, Tregs, NK, and Macrophages cells upon Dot1l inhibition alone or in combination (Combo) with anti-PD-L1 blockade. Results are shown as mean ± SD (n=5). P values were calculated by unpaired Student’s t -test.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Western Blot, RNA Sequencing, Quantitative RT-PCR, Flow Cytometry, Staining, Injection, Control, Inhibition

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of DOT1L inhibitor treatment in huHSC-NCG mice injected with HuH-7 cells. ( B ) Analysis of tumor volume in huHSC-NCG mice subcutaneously injected with HuH-7 cells reconstituted as in (A) and subsequently treated with 20 mg/kg EPZ-5676 (n=5). *** P < 0.001, unpaired Student’s t -test. ( C ) The intra-tumoral abundance of hCD8+ T cells upon DOT1L inhibition treatment in huHSC-NCG mice (n=5). * P < 0.05, unpaired Student’s t -test. ( D to G ) GSEA of published RNA-seq data showing immune-related gene sets that are up-regulated upon inhibition the methyltransferase activity of DOT1L in multiple cancer cell lines. ( H ) Analysis of DOT1L expression in tumors and normal tissues from TCGA patients with the indicated cancer types. n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, wilcox.test. ( I ) Correlation analysis between DOT1L expression levels and z-scores of the IFN gene sets in different tumor types of TCGA. ( J ) DOT1L mRNA expression levels of responder and non-responder in ICB treatment clinical trials with anti-PD-1 treatment in melanoma. P values were calculated by unpaired Student’s t -test. ( K ) A proposed model for the dual function of DOT1L in regulating the immune response of tumors.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Injection, Inhibition, RNA Sequencing, Activity Assay, Expressing, Clinical Proteomics

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of the screening strategy. epi, epigenetic; FACS, fluorescence-activated cell sorting; NGS, Next-generation sequencing. ( B and C ) Scatterplot of the screen results. Control sgRNAs (dark blue) are scattered randomly across the diagonal. EZH2 (light blue) and KDM1A (green) represent positive control sgRNAs. NT, non-targeting. ( D ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells transduced with the indicated sgRNAs. ( E ) CD47 or PD-L1 mRNA levels in HuH-7 cells upon DOT1L depletion with or without IFNγ treatment for 48 h. Results are shown as mean ± SD (n = 3). ( F ) CD47 or PD-L1 flow cytometry analyses of WT and DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h.

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Fluorescence, FACS, Next-Generation Sequencing, Control, Positive Control, Western Blot, Transduction, Flow Cytometry

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A and B ) Volcano plot of RNA-seq analysis in WT and DOT1L-depleted HuH-7 cells. NT, non-targeting. ( C ) Gene ontology (GO) analysis of RNA-seq data showing top 10 pathways that are up-regulated upon DOT1L depletion in HuH-7 cells. ( D ) Gene Set Enrichment Analysis (GSEA) of top 5 hallmark gene sets enriched in DOT1L-depleted HuH-7 cells. ( E ) GSEA of RNA-seq data showing IFNα (top) and IFN γ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( F and G ) Heatmaps of selected immune-related genes whose expression is higher in DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h. ( H ) Flow cytometry histograms of MHC I in sgDOT1L HuH-7 cells with or without IFNγ treatment for 48 h. ( I and J ) Binding density heatmap showing H3K4me3 distribution at the promoters of all genes (I) or the up-regulated genes (J) caused by DOT1L KO in HuH-7 cells. ( K and L ) Binding density heatmap showing H3K27ac distribution at the promoters of all genes (K) or the up-regulated genes (L) caused by DOT1L KO in HuH-7 cells.

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells with EPZ-5676 treatment for 7 days. ( B ) RT-qPCR analysis of CD47 and PD-L1 mRNA levels of HuH-7 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( C ) Flow cytometry histograms of CD47 and PD-L1 in HuH-7 cells with EPZ-5676 treatment for 7 days. ( D ) Volcano plot of RNA-seq analysis in DMSO and EPZ-5676 treatment HuH-7 cells. ( E ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in HuH-7 cells with EPZ-5676 treatment. ( F ) Heatmaps of selected immune-related genes whose expression is higher in EPZ-5676 treatment for 7 days. ( G ) Expression levels of immune-related genes by RT-qPCR analysis. Results are shown as mean ± SD (n=3). ( H ) Flow cytometry histograms of MHC I in HuH-7 cells with EPZ-5676 treatment for 7 days. ( I ) GSEA of RNA-seq data showing IFNα (top) and IFNγ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( J ) Immunoblotting analysis of the DOT1L expression after overexpression of WT or catalytic inactive (CI) HA-DOT1L in DOT1L-depleted HuH-7 cells. ( K ) RT-qPCR analysis of immune-related genes after rescue of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). ( L ) Flow cytometry histograms of CD47 and PD-L1 after overexpression of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells.

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Western Blot, Quantitative RT-PCR, Flow Cytometry, RNA Sequencing, Expressing, Over Expression

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Volcano plot shows transcriptome analysis of transposon elements (TEs) expression after DOT1L KO in HuH-7 cells. ( B ) The expression changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( C ) RT-qPCR analyses of TEs in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). n.s., not significant, * P < 0.05, ** P < 0.01, unpaired Student’s t -test. ( D ) The ATAC-seq signal changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( E ) Binding density heatmap of ATAC-seq, H3K4me3, and H3K27ac ChIP-seq signals of up-regulated TEs caused by DOT1L KO in HuH-7 cells. ( F ) Flow cytometry histograms of dsRNA staining of WT or DOT1L KO HuH-7 cells. DAC, Decitabine served as a positive control. ( G and H ) RT-qPCR analysis of immune-related genes in DOT1L-depleted HuH-7 cells transduced with shRNA against scramble, MDA5 (G), or RIG-I (H). Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( I ) Heatmaps of IgG and DOT1L signals by fanChIP in HuH-7 cells. ( J ) Genomic annotations of DOT1L binding peaks by fanChIP in HuH-7 cells. ( K ) DOT1L fanChIP signals, ATAC-seq, and RNA-seq profiles in HuH-7 cells of LINEs (top) or LTR (bottom).

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Flow Cytometry, Staining, Positive Control, Transduction, shRNA, RNA Sequencing

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Venn diagrams of H3K79me2-associated genes and down-regulated genes in HuH-7 cells. ( B ) Expression levels of ZEB1 by RNA-seq in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=2). ** P < 0.01, unpaired Student’s t -test. RPKM, Reads Per Kilobase per Million mapped reads. ( C ) RT-qPCR analysis of ZEB1 upon DOT1L depletion in HuH-7 cells. Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( D ) Immunoblotting analysis of ZEB1 protein levels using whole-cell lysates from WT and DOT1L KO HuH-7 cells. ( E ) DOT1L fanChIP tracks, H3K79me2, H3K4me3, and H3K27ac ChIP-seq signals, and ATAC-seq profiles of WT and DOT1L-depleted HuH-7 cells in the ZEB1 locus. ( F ) Enrichment of ZEB1 motifs in the accessible chromatin regions specific to DOT1L-deficient HuH-7 cells by HOMER analysis. ( G ) Cistrome toolkit analysis of ATAC-seq data revealed that DNA-binding sites of ZEB1 were more open in the DOT1L-deficient HuH-7 cells. ( H ) Binding density heatmap of ZEB1 ChIP-seq signals in the DOT1L-deficient HuH-7 cells. ( I ) ChIP-seq tracks of ZEB1 in WT and DOT1L-depleted HuH-7 cells in the JAK2 , STAT1 , and PD-L1 loci. ( J ) Immunoblotting analysis using whole-cell lysates from WT and ZEB1 KO HuH-7 cells. ( K ) Expression levels of immune-related genes by RT-qPCR analysis upon ZEB1 depletion in HuH-7 cells. Results are shown as mean ± SD (n=3).

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from WT and Dot1l KO Hepa1-6 cells. ( B ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in Hepa1-6 cells upon Dot1l deficiency. ( C ) GSEA of RNA-seq data showing IFN (top) and IL2_STAT5 (bottom) gene sets that are up-regulated upon DOT1L depletion in Hepa1-6 cells. ( D ) RT-qPCR analyses of TEs in WT and Dot1l-depleted Hepa1-6 cells. Results are shown as mean ± SD (n=3). n.s., not significant, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. ( E ) Flow cytometry histograms of dsRNA staining of WT or Dot1l KO Hepa1-6 cells. ( F ) Immunoblotting analysis using whole-cell lysates from Hepa1-6 cells with EPZ-5676 treatment for 7 days. ( G ) RT-qPCR analysis of immune-related genes of Hepa1-6 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( H ) Schematic of ICB treatments in mice injected with Hepa1-6 WT or Dot1l KO cells. ( I ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 WT or Dot1l KO cells reconstituted as in (H) and subsequently treated with isotype control or anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( J ) Survival analysis conducted on the C57BL/6 mice described in (H). n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Logrank test. ( K ) Schematic of Dot1l inhibitor and ICB treatments in mice injected with Hepa1-6 cells. ( L ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 cells reconstituted as in (K) and subsequently treated with 20 mg/kg EPZ-5676 and anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( M ) Survival analysis conducted on the C57BL/6 mice described in (L). n.s., not significant, *** P < 0.001, Logrank test. ( N to R ), The intra-tumoral abundance of CD4+ T cells, CD8+ T cells, Tregs, NK, and Macrophages cells upon Dot1l inhibition alone or in combination (Combo) with anti-PD-L1 blockade. Results are shown as mean ± SD (n=5). P values were calculated by unpaired Student’s t -test.

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Western Blot, RNA Sequencing, Quantitative RT-PCR, Flow Cytometry, Staining, Injection, Control, Inhibition

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of DOT1L inhibitor treatment in huHSC-NCG mice injected with HuH-7 cells. ( B ) Analysis of tumor volume in huHSC-NCG mice subcutaneously injected with HuH-7 cells reconstituted as in (A) and subsequently treated with 20 mg/kg EPZ-5676 (n=5). *** P < 0.001, unpaired Student’s t -test. ( C ) The intra-tumoral abundance of hCD8+ T cells upon DOT1L inhibition treatment in huHSC-NCG mice (n=5). * P < 0.05, unpaired Student’s t -test. ( D to G ) GSEA of published RNA-seq data showing immune-related gene sets that are up-regulated upon inhibition the methyltransferase activity of DOT1L in multiple cancer cell lines. ( H ) Analysis of DOT1L expression in tumors and normal tissues from TCGA patients with the indicated cancer types. n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, wilcox.test. ( I ) Correlation analysis between DOT1L expression levels and z-scores of the IFN gene sets in different tumor types of TCGA. ( J ) DOT1L mRNA expression levels of responder and non-responder in ICB treatment clinical trials with anti-PD-1 treatment in melanoma. P values were calculated by unpaired Student’s t -test. ( K ) A proposed model for the dual function of DOT1L in regulating the immune response of tumors.

Article Snippet: After that, the supernatant was added 1 μg specific antibodies against DOT1L (Cell Signaling Technology, 77087) or isotype control IgG (Abcam, ab172730) with rotations overnight at 4°C.

Techniques: Injection, Inhibition, RNA Sequencing, Activity Assay, Expressing, Clinical Proteomics

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: Upregulated DOT1L expression correlates with PARPi resistance in OCA. The non-BRCA mutated cell lines OVCAR8 were subjected to a gradual increase in the concentration of Olaparib (from 0.5 to 20 µM) to allow for the development of acquired resistance. The IC50 values of Olaparib-resistant OVCAR8 (R8 OlaR) and original parent OVCAR8 (R8) cell lines were detected by CCK8 assay. B . Niraparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. C . Veliparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. D . Talazoparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. E . RNA-seq was performed on R8 OlaR (R) ( n = 3) and R8 (N) ( n = 3). Venn diagram illustrating the overlap between the RNA-seq data (R and N) and the classic epigenetic regulator genes. F . Volcano plot showing differential expression of mRNAs among overlap gene in E. Red dots represent differently expressed mRNAs with P < 0.05 and Log2FC > 1; blue dots represent mRNAs with P < 0.05 and Log2FC<-1; grey dots represent mRNAs with no significance. G . Heatmap showing differentially expressed epigenetic-related genes between R and N (F). H. DOT1L mRNA levels in R8 OlaR and its original parent OVCAR8 cells were analyzed by RT-qPCR. I . R8 OlaR and its original parent OVCAR8 cells were collected and subjected to western blotting to detect with the indicated antibodies. J - K . Analysis of DOT1L protein levels in PARP inhibitor-resistant ( n = 7) and sensitive fresh-frozen (FF) tissue tissues ( n = 7) ( J ). Quantified results are presented as the means ± SD ( n = 3), ** p < 0.01 ( K ). L . DOT1L mRNA levels in PARPi-resistant and sensitive OC tissues were analyzed by RT-qPCR. The data is presented as the means ± SD, * p < 0.05. M IHC staining of DOT1L in PARPi-resistant and sensitive OC tissues. Representative images are shown. Scale bars: 200 μm (upper); 100 μm (lower) (left). Quantification of DOT1L expression in PARPi-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01 (right)

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Expressing, Concentration Assay, CCK-8 Assay, RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L regulates OC sensitivity to Olaparib and contributes to PARPi resistance. A . DOT1L protein levels in a panel of OC cell lines were examined by western blotting. B . PLKO.1 and DOT1L shRNA (shDOT1L) plasmids were stably transfected into SKOV-3 cell line. Western blotting was used to determine DOT1L protein levels. C . PCMV, and PCMV DOT1L plasmids were stably transfected into OVCAR8 cell line. Western blotting was used to determine DOT1L protein levels. D . The CCK8 assay was performed to detect cell viability in SKOV-3 cells treated with Olaparib (Olap) for 96 h. E . The CCK8 assay was performed to detect cell viability in OVCAR8 cells treated with Olaparib for 96 h. F . Clonogenic assays were conducted to assess the colony formation efficiency of SKOV-3 cells in the presence of Olaparib for 7–14 days (left). The number of clones was quantified (right). G . Clonogenic assays were conducted to assess the colony formation efficiency of OVCAR8 cells in the presence of Olaparib for 7–14 days (left). The number of clones was quantified (right). H . A flow cytometry assay was performed to detect cell apoptosis in SKOV-3 cells treated with Olaparib (10 µM) for 48 h. I . A flow cytometry assay was performed to detect cell apoptosis in OVCAR8 cells treated with Olaparib (10 µM) for 48 h. (Data is presented as the mean ± SD; ns, p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001, n = 3). J-L. OVCAR8 and DOT1L stably overexpressed OVCAR8 cells (4 × 10 6 cells) were subcutaneously injected into the left armpit of each mouse. When the tumor volumes reached approximately 50 mm 3 , the mice were randomly divided into four groups (pc + PBS, pc + Olap, oeDOT1L + PBS, oeDOT1L + Olap) and received an intraperitoneal injection of Olaparib (Olap, 50 mg/kg) or PBS three times a week. Three weeks post-injection, the mice were sacrificed, and their body weights and tumor weight were quantified. Tumors from each group are shown in ( J ). Tumor growth curve (K) and tumor weights of each group ( L ) were quantified. M . The nude mice’s body weights of each group before and after administration. (Data are presented as the mean ± SD, ns, p > 0.05; * p < 0.05; ** p < 0.01, n = 5)

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Western Blot, shRNA, Stable Transfection, Transfection, CCK-8 Assay, Clone Assay, Flow Cytometry, Injection

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-mediated transcription regulation directly influences DOT1L expression. A PLKO.1, PARP1 shRNA (shPARP1) plasmids were stably transfected into SKOV-3 cells. Whole cell lysate (WCL) and chromatin-binding protein (CHR) were extracted and analyzed by western blotting with the indicated antibodies. B . RT-qPCR was used to determine the DOT1L and PARP1 mRNA levels. C . PCMV, PCMV PARP1 plasmids were stably transfected into SKOV-3 cells. Whole cell lysate (WCL) and chromatin bind protein (CHR) were extracted and analyzed by western blotting with the indicated antibodies. D . Quantification of PARP1 and DOT1L mRNA levels in ( C ). E . SKOV-3 cells were transfected with control pcDNA, Flag-PARP1(WT), or enzymatically defective Flag-PARP1 (PARP1 E988K). Western blotting was performed to detect DOT1L protein expression levels. F . DOT1L mRNA levels in pcDNA-, PARP1(WT)-, or PARP1 E988K-transfected SKOV-3 cells were analyzed by RT-qPCR. The data represents the means ± SD ( n = 3). * p < 0.05. G - H . OVCAR8 and OVCAR8 OlaR cells were collected, and western blotting and RT-qPCR were performed to detect DOT1L protein expression(G) and mRNA ( H ) levels. I . ChIP–qPCR showing the level of the indicated proteins recruited to the DOT1L promoter regions. The data represents the means ± SD ( n = 3). * p < 0.05. Four independent sets of DOT1L primers were used. J . PARP1-ChIP assay was performed in pcDNA-, PARP1(WT)-, or PARP1 E988K- transfected SKOV-3 cells to examine PARP1 occupancy at DOT1L. K . STAT3-ChIP assay was performed in OVCAR8 and OVCAR8 OlaR cells to examine STAT3 occupancy at DOT1L. L. STAT3-ChIP assay was performed in pcDNA-, PARP1(WT)-, or PARP1 E988K- transfected SKOV-3 cells to examine STAT3 occupancy at DOT1L. M . OVCAR8 and OVCAR8 OlaR cells were transfected with the DOT1L promoter report gene. The luciferase activity was measured 36 h after transfection. N . SKOV-3 cells were transfected with the DOT1L promoter report gene, together with control pcDNA, wild-type Flag-PARP1, and mutant Flag-PARP1 E988K as indicated. The luciferase activity was measured 36 h after transfection. O . The luciferase reporter assays were performed in PARP1 stably knockdown SKOV-3 cells

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Binding Assay, Western Blot, Quantitative RT-PCR, Control, ChIP-qPCR, Luciferase, Activity Assay, Mutagenesis, Knockdown

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L facilitates PARPi resistance via H3K79 methylation. A . Heatmaps of H3K79me2 levels detected by CUT&Tag around gene body regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. 3 kb windows spanning the TSS to TES of all genes were plotted. Genes were arranged by their enrichment of H3K79me2 in shNC and shDOT1L cells. B . The distributions of H3K79me2-binding regions are shown in the pie charts. C . Venn diagram showing the overlap between RNA-seq data and CUT&Tag data. The KEEP analysis revealed the significantly enriched items based on H3K79me2 signature. D . IGV tracks showing the enrichment of H3K79me2 in ABCB1 and PLCG2 gene regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. E - F . ChIP–qPCR showing the level of the indicated proteins recruited to the PLCG2 ( E ) and ABCB1 ( F ) promoter regions in DOT1L-overexpressed OVCAR8 cells. The data represent the means ± SD ( n = 3). * p < 0.05. three independent sets of PLCG2 and ABCB1 primers were used. G . RT-qPCR was performed in DOT1L overexpressed OVCAR8 cells to determine PLCG2 and ABCB1 mRNA levels. H. PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L overexpressed OVCAR8 cells. I . An H3K79me2-ChIP assay was performed in DOT1L knockdown SKOV-3 cells to examine H3K79me2 occupancy at PLCG2 and ABCB1. J . RT-qPCR was performed in DOT1L knockdown SKOV-3 cells to determine PLCG2 and ABCB1 mRNA levels. K . PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L knockdown SKOV-3 cells

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Methylation, Control, Knockdown, Binding Assay, RNA Sequencing, ChIP-qPCR, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-DOT1L-PLCG2/ABCB1 axis contributes to PARPi resistance. A . H3K79me2-ChIP assay was performed with OVCAR8 Olaparib-resistant and parent OVCAR8 cell lines to determine H3K79me2 occupancy at PLCG2 and ABCB1. B . PLCG2 and ABCB1 mRNA levels were determined in R8 OlaR and parent OVCAR8 cells by RT-qPCR. C . Western blotting was performed in Olaparib-resistant OVCAR8 and parent OVCAR8 cell lines to examine PLCG2 and ABCB1 (P-gly) protein expression levels. D . Western blotting was performed in R8 OlaR and parent OVCAR8 cells which were transfected with shNC and DOT1L shRNA respectively with the indicated antibodies. E . R8 OlaR and parent OVCAR8 cells were transfected with shNC, ABCB1 shRNA and PLCG2 shRNA. After 72 h of transfection, cells were collected and analyzed by western blotting with the indicated antibodies. F – G . Colony formation ( F ) and cell apoptosis assay ( G ) were performed in R8 OlaR and parent OVCAR8 stably transfected cell lines. H . Immunohistochemistry (IHC) staining of DOT1L, PARP1, ABCB1 (P-gly), and PLCG2 in PARP inhibitor-resistant human ovarian carcinomas (OC) tissues and sensitive tissues. Representative images are shown. Scale bars: 400 μm (upper); 160 μm (lower). I – K . Correlation analysis between PARP1 and DOT1L(I), DOT1L and P-gly ( J ), and DOT1L and PLCG2 ( K ) were analyzed. L . Quantification of P-gly (right) and PLCG2 (left) expression in PARP inhibitor-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Apoptosis Assay, Stable Transfection, Immunohistochemistry

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: Targeted inhibition of DOT1L sensitizes OC to PARPi in vitro and in vivo. A – B . Bliss synergy analysis of SGC0946 and Olaparib treatment in R8 OlaR ( A ) and SV3 OlaR ( B ) cell lines. Synergy and antagonism degrees between the drugs were determined using SynergyFinder. A positive score represents a synergistic effect. C . A colony formation assay was conducted to detect the effect of combination therapy with SGC0946 (20µM) and Olaparib(10µM) on R8 OlaR and SV3 OlaR proliferation (upper). Quantification of the relative survival rate of clones (lower). D . A flow cytometry assay was performed to detect cell apoptosis in R8 OlaR cells treated with Olaparib (10 µM), SGC0946 (20µM), or Olaparib (10 µM) + SGC0946 (20µM) for 48 h. E . R8 OlaR cells (4 × 10 6 cells) were subcutaneously injected into the left armpit of each mouse. When the tumor volumes reached approximately 50mm 3 , the mice were randomly divided into four groups (ctrl, Olap, SGC, Olap + SGC) and they received an intraperitoneal injection of Olaparib (Ola, 50 mg/kg), SGC0946(SGC, 50 mg/kg), Ola (50 mg/kg) + SGC (SGC, 50 mg/kg) or PBS three times a week. Three weeks post-injection, the mice were sacrificed, and mouse body weights and tumor weights were quantified. Tumors from each group are shown. F – G . The tumor volume curve ( F ) and weight of each group ( G ) were shown. H . The difference in nude mice’s body weights of each group before and after administration. (Data are presented as the mean ± SD, ns, p > 0.05; * p < 0.05, n = 5). I – K . The synergistic effects of SGC0946 and Olaparib on the viability of the indicated PDOs. Organoids were exposed for 5 days to combine treatments with suboptimal doses of SGC0946 (10 and 20 µM), and Olaparib (5 and 10 µM) ( I ). CI values less than 1, which suggest synergism, were calculated for drug combinations relative to the individual drugs and are indicated in the above graphs ( J – K ) (Data is presented as the mean ± SD, *** < 0.001, n = 3). L . Schematic diagram of molecular mechanism. Working model of the role of DOT1L in PARPi resistance

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Inhibition, In Vitro, In Vivo, Colony Assay, Clone Assay, Flow Cytometry, Injection

Journal: Experimental Hematology & Oncology

Article Title: Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins

doi: 10.1186/s40164-024-00488-5

Figure Lengend Snippet: LAMP5-AS1 regulates LAMP5 expression through the binding of DOT1L to the LAMP5 locus. ( a ) Western blotting show that DOT1L was enriched by LAMP5-AS1 pull-down assays. Antisense of LAMP5-AS1 sequence indicates the negative control. FT: flow through. ( b ) Western blotting for the protein levels of H3K79me2, H3K79me3, and LAMP5 in MLL leukemia cells transduced by LAMP5-AS1 siRNA and control. H3 and beta-actin as the internal control. The H3K79me2 or H3K79me3/ H3, LAMP5/Actin densitometric ratio were recorded by ImageJ, respectively. ( c ) ChIP-seq profiles (shown in GSE150483) of H3K79me2 and H3K79me3 at the LAMP5 genomic loci in LAMP5-AS1 knockdown (red) compared with control (blue) MOLM-13 cells. The y-axis scales represent read density per million sequenced reads. ( d, e ) ChIP Quantitative RT-PCR assays show H3K79me2 ( d ) and H3K79me3 ( e ) on the LAMP5 gene locus clearly declined upon LAMP5-AS1 knockdown in MLL leukemia cells. Error bars reflect ± SEM (**, p < 0.01; ***, p < 0.001) from three independent experiments. ( f ) Diagram depicts the procedure of ChIRP. ( g ) Quantitative RT-PCR for the LAMP5-AS1(upper, left) and LAMP5 locus (upper, right) enrichment in ChIRP experiment captured by LAMP5-AS1 probes. GAPDH and NC-probe were used to be the negative control. Western blotting shows the DOT1L and H3K79me2 protein levels that captured by LAMP5-AS1 probes in ChIRP experiment. GAPDH protein as the negative control. ( h ) RNA and DNA FISH and IF experiments showed that LAMP5-AS1 and DOT1L co-localized at LAMP5 locus in the cell nucleus. ( i ) Proposed model for LAMP5-AS1 regulate LAMP5 in MLL leukemia. LAMP5-AS1 regulates LAMP5 by directly interacting with DOT1L

Article Snippet: For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).

Techniques: Expressing, Binding Assay, Western Blot, Sequencing, Negative Control, Control, ChIP-sequencing, Knockdown, Quantitative RT-PCR

Journal: Experimental Hematology & Oncology

Article Title: Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins

doi: 10.1186/s40164-024-00488-5

Figure Lengend Snippet: The LAMP5-AS1-DOT1L-LAMP5 axis suppresses autophagy and maintains MLL fusion protein. ( a ) The resulting decline in MLL-AF9 levels by knocking down either LAMP5-AS1 was reversed upon treatment with the autophagy inhibitor bafilomycin A1 (25 nM, 12 h). MLL-AF9, LC3B-II, and LAMP5 were assayed by western blotting, and beta-tubulin as a loading control. The MLL-AF9, LC3B-II, or LAMP5 /Tubulin densitometric ratio were recorded by ImageJ. ( b ) Proposed model for the LAMP5-AS1-DOT1L-LAMP5 axis regulates autophagy and maintains MLL fusion protein

Article Snippet: For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: Mammalian DOT1L and c-MYC co-regulate genes in triple negative breast cancer cells. A. A schematic of the DOT1L locus and the sgRNA used to target the first exon of DOT1L for CRISPR/Cas9 gene editing. A clonal MDA-MB-231 DOT1L KO/+ line was generated with one allele containing an early stop codon in the DOT1L locus, and the other allele containing a 15 base-pair in-frame deletion. B. DOT1L KO/+ MDA-MB-231 cells have decreased H3K79me2 levels on western blot (N>5). C. Cell counting proliferation assays show that the DOT1L KO/+ MDA-MB-231 cells grow significantly slower than the wild-type counterparts (N=2, with three technical replicates). D. Top upstream regulators affected in the DOT1L KO/+ MDA-MB-231 cells compared to the wild-type counterpart as analyzed by Ingenuity Pathway Analysis. Proteomic analysis was performed on the DOT1L KO heterogenous population and compared to wild-type MDA-MB-231 (N=5). Bulk RNA-seq was done on the clonal DOT1L KO/+ MDA-MB-231 cell line and compared to the wild-type controls (N=4). Blue squares represent a negative activation z-score indicating a downregulated pathway in the DOT1L KO/+ MDA-MB-231 cells compared to wild-type, while orange squares represent a positive activation z-score indicating an upregulated pathway in the DOT1L KO/+ MDA-MB-231 cells compared to wild-type. Proteomic and RNA-seq analyses for the top differentially regulated canonical pathways are mostly in agreement with one another. The MYC pathway is the top pathway downregulated in the differentially regulated canonical pathways in the DOT1L KO/+ MDA-MB-231 cells compared to the wild-type controls as analyzed by Ingenuity Pathway Analysis. E. Overexpressed DOT1L and c-MYC can co-localize in distinct clusters in the nucleus in HCT116 cells. F. Overexpressed DOT1L immunoprecipitates with overexpressed c-MYC in HCT116 cells. Conversely, c-MYC immunoprecipitates with overexpressed DOT1L. G. Overexpressed DOT1L immunoprecipitates with overexpressed c-MYC or YFP-c-MYC in HEK-293T cells. Conversely, overexpressed c-MYC and YFP-c-MYC immunoprecipitate with overexpressed DOT1L.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: CRISPR, Generated, Western Blot, Cell Counting, RNA Sequencing Assay, Activation Assay

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: Bulk RNA-seq and proteomic analyses show that the c-MYC pathway and hallmark c-MYC targets are downregulated in the DOT1L KO/+ cells compared to the control cells. A. IP-western blot of two MDA-MB-231 populations after CRISPR/Cas9 gene editing to knock out DOT1L. The second population (fourth lane) showed successful knockout of the full-length DOT1L protein and was then used to isolate a clonal DOT1L KO/+ line. B. RNA-seq reads of the DOT1L locus show that there are two alleles present in the DOT1L KO/+ cells. There is a 19-bp deletion leading to an early stop codon, and a 15-bp in-frame deletion. C. Principal Component Analysis of RNA-seq data shows that the wild-type and DOT1L KO/+ cells primarily group with their respective genotype (N=4). D. Principal Component Analysis of proteomic data shows that the wild-type population and DOT1L KO primarily group with their respective genotype (N=5). E. Top differentially regulated canonical pathways in the DOT1L KO/+ MDA-MB-231 cells compared to the wild-type counterpart as analyzed by Ingenuity Pathway Analysis. F. Gene set enrichment analysis (GSEA) of proteomic data shows that the cell cycle and protein modification pathways are among the most downregulated in the DOT1L KO population. G. Heatmap shows that c-MYC hallmark targets gene expression is downregulated in DOT1L KO/+ cells. H. Gene set enrichment analysis (GSEA) plots from RNA-seq analysis show that c-MYC Hallmark genes are downregulated in DOT1L KO/+ cells. I. Western blot showing successful immunoprecipitation of overexpressed MYC and YFP-MYC in HEK-293T cells.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: RNA Sequencing Assay, Western Blot, CRISPR, Knock-Out, Modification, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: Depletion in DOT1L leads to decreased c-MYC target gene expression, but increased c-MYC occupancy. A. RT-qPCR of c-MYC target genes show a decrease in mRNA expression in DOT1L KO/+ cells compared to wild-type cells (N= 8). All eight of the selected target genes have decreased mRNA expression. B. A schematic of the c-MYC protein and the corresponding regions of antibody recognition, C. c-MYC ChIP-qPCR of c-MYC target gene promoters shows an increase in c-MYC occupancy despite the decrease in gene expression. All eight target gene promoters had increased c-MYC occupancy (N=4). D. Western blot shows that the DOT1L KO/+ cells have decreased c-MYC protein levels compared to the wild-type controls (N=3). Densitometry analysis of the c-MYC band is shown on the right. E. RT-qPCR shows that acute DOT1L and c-MYC knockdown leads to downregulation of their corresponding gene expression. F & G. c-MYC occupancy at the SNAI1 and miR-17 promoters is increased upon acute depletion of DOT1L via siRNA (N=3). The occupancy of O -GlcNAcylated proteins is also increased upon DOT1L knockdown. H3K79me2 in both promoters is decreased upon siDOT1L and upon treatment with a DOT1L histone methyltransferase (HMT) inhibitor, SGC0946. Inhibition of HMT activity did not increase c-MYC occupancy at the two target promoters to the same extent as DOT1L knockdown. Asterisks *, ** and *** denote P values (Student’s t-test) less than 0.05, 0.01 and 0.001, respectively.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L depletion leads to a decrease in c-MYC target gene expression, but an increase in c-MYC occupancy at the corresponding promoters. A. Endogenous c-MYC tagged with the auxin-inducible degron (AID) is depleted after 50 minutes of auxin treatment (0.1 mg/mL) (N=3). Endogenous c-MYC without the AID in the HCT116 cells containing the auxin receptor F-box protein AtAFB2 is not depleted when treated with auxin. B. Auxin treatment for 4 hours leads to a decrease in c-MYC target gene expression via RT-qPCR (N=3). C. shRNA against DOT1L in HCT116 cells leads to a decrease in c-MYC target gene expression (N=4). D. shRNA against DOT1L in HCT116 cells leads to a decrease in H3K79me2 (N=4) E. A representative biological replicate of c-MYC ChIP-qPCR using the N-terminal c-MYC antibody (Y69) showing that c-MYC occupancy is increased at its target gene promoters in DOT1L KO/+ cells compared to wild-type (N=4). F. A representative biological replicate of c-MYC ChIP-qPCR using the C-terminal c-MYC antibody (9E10) showing that c-MYC occupancy is increased at its target gene promoters in DOT1L KO/+ cells compared to wild-type (N=3). Asterisks *, ** and *** denote P values (Student’s t-test) less than 0.05, 0.01 and 0.001, respectively.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Expressing, Quantitative RT-PCR, shRNA

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: Inhibition of the HMT activity of DOT1L does not alter DOT1L localization. A. Inhibition of HMT activity by SGC0946 (6.4 μM) for 3 (N=2) or 6 days (N=1) leads to the depletion of H3K79me2 in MDA-MB-231 cells. B. Immunocytochemistry for DOT1L (ab64077) in untreated MDA-MB-231 cells shows the nuclear localization of DOT1L. C. Inhibition of HMT activity by SGC0946 (6.4 μM) for 3 (N=2) or 6 days (N=1) does not lead to changes in DOT1L nuclear localization.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Inhibition, Activity Assay, Immunocytochemistry

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L works with the ubiquitin-proteasome system to regulate c-MYC. A. KEGG pathway analysis by EnrichR shows that DOT1L binding partners are enriched in the ribosome, spliceosome, and proteasome components. B. Cycloheximide (CHX) chase assays show that overall c-MYC stability is minimally decreased in DOT1L KO/+ cells compared to control (N=3). C. A schematic of the FLAG-tagged C. elegans MML-1 protein and the MML-1(ok849) protein produced in the mml-1(ok849) mutant. D. Endogenously FLAG-tagged MML-1 is stabilized in the rpn-10(ok1865) worms as evidenced by the smear above the full-length MML-1 protein (N=2). This smearing is not observed with the inactive MML-1(ok849) mutant in the same rpn-10 mutant background. E. 4-hour treatment with 20 μM MG132 leads to the formation of c-MYC multimers in U2OS and HCT116 cells (N=2). F. DOT1L KO/+ cells do not readily form c-MYC multimers compared to the wild-type controls. Both the control and DOT1L KO/+ cells form c-MYC multimers upon 4-hour treatment with 20 μM MG132 (N=2).

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Binding Assay, Produced, Mutagenesis

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L works with the ubiquitin-proteasome system to activate c-MYC driven gene transcription. A. A schematic of the degradation process of c-MYC. This schematic was created with BioRender.com . B. Treatment of wild-type and DOT1L KO/+ cells with 20 μM of MG132 leads to an increase in total ubiquitinated proteins, as well as c-MYC protein levels (full length and ubiquitinated) after 4 hours of treatment (N=5). C. Consistent with non-DMSO treated cells, c-MYC target gene expression is decreased in the DOT1L KO/+ cells compared to the wild-type control cells under 4 hours of DMSO D & E. 4-hour treatment with 20 μM MG132 leads to a decrease in c-MYC target gene expression in the control cells (N=5). DOT1L KO/+ cells have a dampened decrease in c-MYC target gene expression after MG132 treatment, indicating that the proteasome and DOT1L are in the same pathway for c-MYC activation. F. TAK-243 chase experiments show that proteasomal clearing of ubiquitinated substrates is delayed in DOT1L KO/+ cells. c-MYC stabilization after ubiquitination inhibition is also delayed in the mutant cells compared to wild-type. E. Densitometry of c-MYC bands and ubiquitinated smears in F normalized to Actin and the respective DMSO controls.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Expressing, Activation Assay, Inhibition, Mutagenesis

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L cooperates with VCP to regulate c-MYC-driven gene transcription. A. A schematic of the proposed mechanism by which VCP extracts c-MYC from chromatin for degradation. This schematic was created with BioRender.com . B. Overexpressed VCP-GFP immunoprecipitates with overexpressed DOT1L in HCT116 cells (N=2). C. 6-hour treatment with 10 μM of NMS-873 leads to an increase in ubiquitinated proteins and c-MYC protein levels in both the wild-type control and DOT1L KO/+ cells (N=4). D. Consistent with non-DMSO treated cells, c-MYC target gene expression is decreased in the DOT1L KO/+ cells compared to the wild-type control cells under 6 hours of DMSO. E & F. 6-hour treatment with 10 μM NMS-873 leads to a significant decrease in c-MYC target gene expression in the control cells (N=4). This decrease is absent in DOT1L KO/+ cells, indicating that VCP and DOT1L are in the same pathway for c-MYC activation. G. TAK-243 chase experiments show that proteasomal clearing of ubiquitinated substrates is delayed in MDA-MB-231 cells with shDOT1L and shVCP compared to shCtrl. c-MYC stabilization after ubiquitination inhibition is also delayed in the shDOT1l and shVCP cells compared to shCtrl. H. shRNA against VCP and DOT1L leads to a decrease in c-MYC target gene expression.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Expressing, Activation Assay, Inhibition, shRNA

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: VCP and DOT1L cooperate to regulate c-MYC-driven gene transcription. A. Overexpressed VCP-GFP immunoprecipitates with overexpressed DOT1L in HEK-293T cells (N=2). B. Overexpressed VCP-GFP localizes to the cytoplasm and the nucleus, while over expressed DOT1L and c-MYC localize to the nucleus only in HCT116 cells. C. DOT1L depletion does not alter VCP protein levels. D. DOT1L depletion does not alter hexamerization of VCP. Control and DOT1L KO/+ lysates were run on a BN-PAGE gel to see native protein complexes. E. Both shRNAs against VCP leads to an increase in ubiquitinated proteins in the control MDA-MB-231 cells, while only shVCP-1 led to an increase in ubiquitinated proteins in DOT1L KO/+ cells. shDOT1L leads to a decrease in H3K79me2 in control MDA-MB-231 cells. F. shVCP-1 leads to a slight decrease in VCP mRNA expression in DOT1L KO/+ cells, while shVCP-2 does not. G. TAK-243 chase experiments show that proteasomal clearing of ubiquitinated substrates is unchanged in its delay between shCtrl and shVCP DOT1L KO/+ MDA-MB-231 cells.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Expressing

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L has proteolytic activity and shorter c-MYC fragments observed in cells are the result of proteolytic cleavage. A. Transgenic MML::FLAG::GFP in wild-type worms produces a shorter MML::FLAG::GFP product that is stabilized to the full-length transgenic protein in the zfp(ok544) mutant background (N=2). This transgene is not stabilized in the ogt-1(ok430)III null worm. B. Cellular fractionation of MDA-MB-231 cells shows shorter c-MYC fragments in the chromatin fraction, detected by the C-terminal c-MYC antibodies (N=3). C. A consistent 45 kDa c-MYC fragment is produced upon transfection of c-MYC plasmids in HEK-293T cells, which can be pulled down via immunoprecipitation (N=3). D. A schematic of the protease-resistant HmuY constructs used to probe cleavage of different c-MYC fragments. The HmuY fragment aa 1-60 was used as a negative control for cleavage. E. Transfection of the HmuY constructs shows cleavage of c-MYC from aa 143-210, and aa 258-320, as detected by both the HA and FLAG antibodies (N=3). F. Immunoprecipitation of the N-terminal and C-terminal cleavage fragments of c-MYC produced from aa 143-210 and aa 258-320 using the HA and FLAG antibodies (N=2). G. A schematic of DOT-1.1 ( C. elegans ) and DOT1L (mammalian) proteins. A part of the HMT domain (aa 126-192 in human DOT1) of DOT1 family proteins aligns with the catalytic domain of the DDI family of aspartic proteases, which are related to HIV protease. Note the conservation of the catalytic aspartic acid ( D S/T G ) in DDI/HIV proteases and the D L G sequence in DOT1 enzymes. Blue boxes show regions of DOT1L that bind methyl donor S-Adenosyl methionine. Red boxes show the most homologous amino acid residues between DOT1 and DDI protein families. Bold letters indicate identical residues in at least one pair of DOT1 and protease protein family proteins. Grey shade indicates similar amino acid residues. CLUSTAL Omega tool was used to generate the multiple sequence alignment. H. A schematic of the protease-resistant HmuY constructs used for in vitro cleavage assays. Coomassie blue staining shows one predominant band for each in vitro construct. I. Cleavage by the N-terminus, but not C-terminus, of DOT1L occurs between aa 258-367 of c-MYC in vitro (N=3). Commercially available N-terminal DOT1L or purified C-terminal DOT1L expressed in E. coli was incubated with the respective HmuY constructs for 24 hours at 37 °C. J. The region between aa 143-258 of c-MYC is cleaved by the nuclear fraction of U2OS cells. The region between aa 258-367 of c-MYC is cleaved by both the cytoplasmic and nuclear fractions of U2OS cells to produce distinct cytoplasmic and nuclear cleavage bands. The respective HmuY constructs were incubated with 2.5 μg of U2OS cell fractions for 24 hours at 37 °C. K. Western blotting of alpha tubulin and H3 show that the cellular fractionation protocol successfully separated the cytoplasmic and nuclear fractions of U2OS cells.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Activity Assay, Transgenic Assay, Mutagenesis, Cell Fractionation, Produced, Transfection, Immunoprecipitation, Construct, Negative Control, Sequencing, In Vitro, Staining, Purification, Incubation, Western Blot

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: Shorter c-MYC fragments are observed endogenously and through reporters. A. Endogenously FLAG-tagged c-MYC in HCT116 cells show evidence of shorter c-MYC fragments that are stabilized when treated with the proteasome inhibitor MG132 (N=3). B. Transfection of FLAG-tagged c-MYC in HCT116 (N=3) and U2OS (N=4) cells consistently produces a ∼45 kDa c-MYC fragment. C. The 45 kDa c-MYC fragment produced from c-MYC transfections is not a result of an internal start codon (N=3). Transfection of a c-MYC-FLAG construct with the internal Methionine residues mutated to Isoleucine still produced the 45 kDa c-MYC fragment. D. The HmuY protein from Porphyromonas gingivalis can be expressed in HCT116 (N=1) cells. The HmuY protein is expressed in both the cytoplasm and nucleus. E. Transfection of the HmuY constructs shows cleavage of c-MYC at the 143-210 aa region, and 258-320 aa region, as detected by both the HA and FLAG antibodies. These cleavage products are stabilized by proteasomal inhibition with 20 μM MG132. F. A schematic of the nanoluciferase-YFP reporters used to detect proteolytic cleavage in the middle portion of c-MYC. Either a random spacer sequence or amino acids 141-258 of c-MYC was inserted in between the nanoluciferase and YFP construct. G. Western blot using the FLAG and GFP antibodies shows N-terminal (blue dots) and C-terminal (yellow arrow) cleavage fragments arising from the middle portion of c-MYC. The N-terminal fragments have sizes of around 20 kDa and 27.5 kDa, while the C-terminal fragment detected has a size of around 35 kDa (N=3). H. A schematic of DOT-1.1 isoforms ( C. elegans ) and DOT1L (mammalian) protein. Highlighted in yellow is a region in the C-terminus that has homology to serine proteases. I. CLUSTAL Omega alignment of DOT-1.1c and the C-terminus of DOT1L shows conservation of a protein fold of serine proteases.

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Transfection, Produced, Construct, Inhibition, Sequencing, Western Blot

Journal: bioRxiv

Article Title: DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin

doi: 10.1101/2024.02.06.579191

Figure Lengend Snippet: DOT1L cleaves full-length recombinant c-MYC in vitro. A. N- and C-terminal c-MYC cleavage fragments are produced after 24 hours of incubation with N-terminal DOT1L at 37 °C (N=2). Incubation with C-terminal DOT1L does not produce distinct cleavage fragments. B. Incubation of recombinant c-MYC with nuclear or chromatin fractions of U2OS cells produces N- and C-terminal cleavage fragments that match in size to the cleavage fragments produced by the N-terminus of DOT1L. C. Western blotting of alpha tubulin and H3 show that the cellular fractionation protocol successfully separated the cytoplasmic and chromatin fractions of U2OS cells. D. DOT1L works with the ubiquitin-proteasome system to displace c-MYC from target promoters. Unoccupied target promoters are bound by c-MYC. This induces the transcription of target genes and renders c-MYC as a “spent” transcription factor. DOT1L and VCP cooperate in the removal of c-MYC from the target promoter. DOT1L potentially acts by cleaving c-MYC, which may be needed for the removal of c-MYC by VCP. Displaced c-MYC is then degraded by the nuclear proteasome and allows new c-MYC to bind to the target promoter. Depletion of DOT1L disrupts the process of c-MYC displacement leading to prolonged occupancy of “spent” c-MYC, which then blocks new c-MYC from binding and inducing gene expression. This schematic was created with BioRender.com .

Article Snippet: Endogenous proteins were detected by rabbit DOT1L rabbit antibody (Abcam, ab64077, 1:500), VCP antibody (Abcam, EPR3308, 1:5000), mouse alpha tubulin antibody (house-made, E-74, 1:1000), rabbit GAPDH antibody (Abclonal, A10868, 1:1000), rabbit H3 antibody (Millipore, AS3, 1:2000), rabbit H3K79me2 antibody (Millipore, NL59, 1:1000), mouse Pan-ubiquitin antibody (CST, PD41, 1:1000), and mouse Actin antibody (Sigma, C4, 1:5000).

Techniques: Recombinant, In Vitro, Produced, Incubation, Western Blot, Cell Fractionation, Binding Assay, Expressing