hdac1  (Proteintech)

Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1) is a key interactor of histone deacetylase 1 (HDAC1) and serves as a prognostic marker in acute leukaemia. (A) Expression levels of HDACs classes from The Cancer Genome Atlas (TCGA) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) datasets. Transcript per million (TPM) was used to quantify gene expression. (B) Venn diagram (left) showing the overlap of the top 100 genes interacting with HDACs identified from the STRING dataset (confidence > .7) and the HDAC scores derived from single‐sample gene set enrichment analysis (ssGSEA) of the TCGA dataset (False discovery rate [FDR] < .01, correlation coefficient > .3). Heatmap (right) illustrates the correlation between eight genes and the HDACs score (indicated in orange), as well as the interaction network confidence with HDACs (indicated in red). (C) Cox proportional hazards regression analysis reveals the prognostic value of the genes. Dots represent log 2 ‐transformed hazard ratio (HR) and horizontal lines indicate 95% confidence intervals (95% CIs). Genes are ranked according to the magnitude of their HR. (D) Pearson correlation analysis demonstrating correlations between DNTTIP1 expression and HDACs score ( R = .38, p = 1.5e−06). (E) GSEA showing DNTTIP1 enrichment in HDAC‐related pathways (Normalized enrichment score [NES] = 1.61, p .adjust = .00508). (F) Schematic representation of the mitotic deacetylase complex (MiDAC). (G) Expression analysis of DNTTIP1 in dataset GSE48558 . (H and I) Survival analysis of DNTTIP1 in TARGET (K) and TCGA (L) datasets. Survival differences were evaluated using the log‐rank test, with a significance threshold of p < .05. (J) Western blotting (WB) analysis of DNTTIP1 protein expression in acute leukaemia cell lines, and healthy donor‐derived bone marrow mononuclear cells (BMNCs) as a normal control. A representative blot is shown. (K) Western blot analysis of DNTTIP1 and HDAC1 in BMNCs from acute leukaemia patients ( n = 4) and healthy donors ( n = 2). A representative blot is shown.

Article Snippet: Briefly, 5 μg GST‐tagged HDAC1 (SignalChem, H83‐30G) and 5 μg His‐tagged DNTTIP1 (Solarbio, P05485 ) were mixed with 20 μL pre‐washed beads (GST‐labelled magnetic beads for GST pull‐down; NTA‐Ni beads for His pull‐down) in pull‐down buffer.

Techniques: Histone Deacetylase Assay, Marker, Expressing, Gene Expression, Derivative Assay, Transformation Assay, Western Blot, Control

Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Histone deacetylase 1/2 (HDAC1/2) are critical for deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1)‐mediated leukaemogenesis. (A and B) Co‐immunoprecipitation (Co‐IP) analysis in RS4;11 (A) and MV4;11 (B) cells stably expressing HA‐tagged DNTTIP1. Cell lysates were immunoprecipitated with anti‐HA (top) or anti‐HDAC1 (bottom) antibodies, followed by immunoblotting with the indicated antibodies. Input lanes represent 5% of total lysate ( n = 3). (C) Proximity ligation assay (PLA, red) detecting endogenous DNTTIP1‒HDAC1 interaction in RS4;11 (top) and MV4‐11 (bottom) cells. Scale bar = 20 µM ( n = 3). (D and E) GST/His‐tagged pull‐down assay showing the interaction between DNTTIP1 and HDAC1. (F) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating HDAC1 knockdown using two independent short hairpin RNAs (shRNAs) (shHDAC1_1 and shHDAC1_2). Cell counting of RS4;11 cells expressing shHDAC1_1 and shHDAC1_2 compared with control (pLKO) (right) ( n = 3). (G) Flow cytometric analysis of Annexin V/propidium iodide (PI) staining in RS4;11 cells with HDAC1 knockdown (shHDAC1_1 and shHDAC1_2) versus control (pLKO) ( n = 3). (H) Flow cytometric analysis was performed to assess cell cycle distribution in RS4;11 cells with HDAC1 knockdown (shHDAC1_1 and shHDAC1_2) versus control (pLKO) ( n = 3). (I) Immunoblot analyses demonstrating the rescue effect of DNTTIP1 overexpression (DNTTIP1_5) upon HDAC1 knockdown (shHDAC1_2) cells (left). Cell counting analysis demonstrating that overexpression of DNTTIP1 (DNTTIP1_5) partially rescues the growth inhibition induced by HDAC1 knockdown (shHDAC1_2) in RS4;11 cells (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in RS4;11 cells with DNTTIP1 overexpression on HDAC1 knockdown ( n = 3). shHDAC1_2 + DNTTIP1_5 versus shHDAC1_2 + EV is denoted by symbol (#). All groups versus pLKO + EV is denoted by symbol (*). (K) Reciprocal Co‐IP analysis in RS4;11 cells demonstrated endogenous interaction between DNTTIP1 and HDAC2 ( n = 3). (L) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating HDAC2 knockdown using two independent shRNAs (shHDAC2_1 and shHDAC2_3). Cell counting of RS4;11 cells expressing shHDAC2_1 and shHDAC2_3 compared with control (Scr) (right) ( n = 3). (M) Flow cytometric analysis of Annexin V/PI staining in RS4;11 cells with HDAC2 knockdown (shHDAC2_1 and shHDAC2_3) versus control (Scr) ( n = 3). (N) Flow cytometric analysis was performed to assess cell cycle distribution in RS4;11 cells with HDAC2 knockdown (shHDAC2_1 and shHDAC2_3) versus control (Scr) ( n = 3). (O) Immunoblot analysis demonstrating the efficiency of HDAC1/2 double knockdown (left). Cell counting analysis showing that HDAC1/2 double knockdown inhibited proliferation more potently than individual knockdown of either HDAC1 or HDAC2 (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in RS4;11 cells with HDAC1/2 double knockdown ( n = 3). All groups versus pLKO + Scr is denoted by symbol (*). (P) Schematic diagram of DNTTIP1 domains (top). HEK‐293T cells were transiently transfected with HA‐tagged DNTTIP1 deletion constructs and cultured for 24 h. Western blot analysis was performed on total cell lysates and immunoprecipitated fractions using HA‐specific antibodies (bottom) ( n = 3). (Q) Cell counting of RS4;11 cells transfected with HA‐tagged DNTTIP1 deletion constructs ( n = 3).

Article Snippet: Briefly, 5 μg GST‐tagged HDAC1 (SignalChem, H83‐30G) and 5 μg His‐tagged DNTTIP1 (Solarbio, P05485 ) were mixed with 20 μL pre‐washed beads (GST‐labelled magnetic beads for GST pull‐down; NTA‐Ni beads for His pull‐down) in pull‐down buffer.

Techniques: Histone Deacetylase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Proximity Ligation Assay, Pull Down Assay, Quantitative RT-PCR, Knockdown, Cell Counting, Control, Staining, Over Expression, Inhibition, Transfection, Construct, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1)‒histone deacetylase 1/2 (HDAC1/2)‐mediated chromatin regulation modulates autophagy and apoptosis. (A) Heatmap illustrating the expression profiles of differentially expressed genes (DEGs) in RS4;11 cells following DNTTIP1 knockdown or MS‐275 treatment. (B‒D) Heatmap visualisation of coordinated changes in assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) (B), H3K27ac (C) and HA‐DNTTIP1 (D) binding across responsive loci in RS4;11 leukaemia cells. (E) Venn diagram integrating multi‐omics data to delineate high‐confidence DNTTIP1 effector genes in RS4;11 cells. The intersection comprises four criteria: genes consistently upregulated across two RNA‐seq datasets, ATAC‐seq‐accessible loci upon DNTTIP1 knockdown (shDNT_4), H3K27ac‐targeted genes upon DNTTIP1 knockdown (shDNT_4) and direct DNTTIP1‐targeted genes. (F) Multi‐omics‐identified overlapping differentially expressed genes (DEGs) were visualised using bubble plots to show the top 10 Gene Ontology (GO) pathways (top) and bar plots to show the top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (bottom). (G) Chord diagrams illustrating the interconnected autophagy and apoptosis networks among multi‐omics overlapping genes. (H) Integrative genomic viewer (IGV) snapshot of cleavage under targets and tagmentation (CUT&Tag) (top: DNTTIP1; middle: H3K27ac) and ATAC‐seq (bottom) signals at the promoter regions of key BH3‐only genes (BCL2‐modifying factor [BMF], BCL2 binding component 3 [PUMA] and Phorbol‐12‐myristate‐13‐acetate‐induced protein 1 [NOXA]).

Article Snippet: Briefly, 5 μg GST‐tagged HDAC1 (SignalChem, H83‐30G) and 5 μg His‐tagged DNTTIP1 (Solarbio, P05485 ) were mixed with 20 μL pre‐washed beads (GST‐labelled magnetic beads for GST pull‐down; NTA‐Ni beads for His pull‐down) in pull‐down buffer.

Techniques: Histone Deacetylase Assay, Expressing, Knockdown, Sequencing, Binding Assay, Biomarker Discovery, RNA Sequencing

Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1) interacts with SP1 to repress the transcription of BCL2‐modifying factor (BMF). (A) Motif analysis identifies the top 15 transcription factors enriched within DNTTIP1‐bound genomic regions. (B) Venn diagram showing the overlap of multi‐omics binding motifs (assay for transposase‐accessible chromatin using sequencing [ATAC‐seq] and cleavage under targets and tagmentation [CUT&Tag]) and predicated JASPAR motifs anchored to the DNTTIP1‐bound BMF promoter in RS4;11 cells (left). Table presenting the SP1 motif logo and position‐weight matrix (right). (C‒E) Reciprocal co‐immunoprecipitation (Co‐IP) analysis in RS4;11 cells demonstrated binding of endogenous SP1 to DNTTIP1 and HDAC1/2 ( n = 3). (F) Correlation analysis of SP1 with DNTTIP1 and HDAC1/2 in The Cancer Genome Atlas (TCGA)‐LAML. Pearson correlation coefficients were computed using GEPIA 2 ( http://gepia2.cancer‐pku.cn/#index ). (G) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating SP1 knockout using two different single‐guide RNAs (sgSP1_1 and sgSP1_2). (H) Cell counting of RS4;11 cells expressing sgSP1_1 and sgSP1_2 compared with control (lacZ) (right) ( n = 3). (I) Flow cytometric analysis of Annexin V/propidium iodide (PI) staining in RS4;11 cells with SP1 knockout (sgSP1_1 and sgSP1_2) versus control (lacZ) ( n = 3). (J‒L) RT‐qPCR analysis of mRNA expression of BH3‐only genes (BMF, PUMA and NOXA) in leukaemia cells following DNTTIP1 knockdown (J), MS‐275 treatment (K) or FK228/Merck60 treatment (L). Data represent mean ± standard deviation (SD) of three independent experiment. * p < .05, ** p < .01, *** p < .001, **** p < .0001. (M) Schematic of chromatin immunoprecipitation quantitative PCR (ChIP‐qPCR) locus‐specific BMF primer locations (left). ChIP‐qPCR analysis showing the HA (DNTTIP1) enrichment on BMF promoter in HA‐tagged DNTTIP1 overexpressed RS4;11 cells (right). (N) ChIP‐qPCR analysis showing the HDAC1 enrichment at BMF promoter in control (pLKO) and DNTTIP1 knockdown (shDNT_4) RS4;11 cells. (O) ChIP‐qPCR analysis showing the HDAC2 (left) and H3K27ac (right) enrichment at BMF promoter in control (pLKO) and DNTTIP1 knockdown (shDNT_4) RS4;11 cells. (P) ChIP‐qPCR analysis showing the SP1 enrichment at BMF promoter in control (pLKO) and DNTTIP1 knockdown (shDNT_4) RS4;11 cells. (Q) ChIP‐qPCR analysis showing the HDAC1 (left) and H3K27ac (right) enrichment at BMF promoter in control (lacZ) and SP1 knockout (sgSP1_2) RS4;11 cells.

Article Snippet: Briefly, 5 μg GST‐tagged HDAC1 (SignalChem, H83‐30G) and 5 μg His‐tagged DNTTIP1 (Solarbio, P05485 ) were mixed with 20 μL pre‐washed beads (GST‐labelled magnetic beads for GST pull‐down; NTA‐Ni beads for His pull‐down) in pull‐down buffer.

Techniques: Biomarker Discovery, Binding Assay, Sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Knock-Out, Cell Counting, Expressing, Control, Staining, Knockdown, Standard Deviation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR

Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1)‒histone deacetylase 1/2 (HDAC1/2)‒BCL2‐modifying factor (BMF) axis drives acute leukaemia progression by suppressing apoptosis and autophagy. (A) Levels of BMF and H3K27ac detected by Western blotting after knockdown of DNTTIP1 in RS4;11 cells and MV4‐11 cells. (B and C) Levels of BMF and H3K27ac detected by Western blotting following treatment with MS‐275 (B) or Merck60 (C) in RS4;11 cells and MV4‐11 cells. (D and E) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating BMF knockdown using two independent short hairpin RNAs (shRNAs) (shBMF_1 and shBMF_2). Cell counting of RS4;11 and MV4‐11 cells expressing shBMF_1 and shBMF_2 compared with control (scramble) (right) ( n = 3). (F) Immunoblot analyses demonstrating the rescue effect of BMF knockdown (shBMF_1) upon DNTTIP1 knockdown (shDNT_4) (left). Cell counting of DNTTIP1‐deficient RS4;11 cells with BMF knockdown (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in DNTTIP1‐deficient RS4;11 cells with BMF knockdown (right) ( n = 3). shDNT_4 + shBMF_1 versus shDNT_4 + scramble is denoted by symbol (#). All groups versus pLKO + scramble is denoted by symbol (*). # p < .05, ## p < .01, ### p < .001, #### p < .0001, * p < .05, ** p < .01, *** p < .001, **** p < .0001. (G) Immunoblot analyses demonstrating the rescue effect of BMF overexpression (BMF_1) upon DNTTIP1 overexpression (DNT_5) (left). Cell counting of RS4;11 cells with DNTTIP1 overexpression and concurrent BMF overexpression (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in RS4;11 cells with concurrent DNTTIP1 and BMF overexpression (right) ( n = 3). DNT_5 + BMF_1 versus DNT_5 + EV_2 is denoted by symbol (#). All groups versus EV_1 + EV_2 denoted by symbol (*). # p < .05, ## p < .01, ### p < .001, #### p < .0001, * p < .05, ** p < .01, *** p < .001, **** p < .0001. (H and I) Western blot analysis of the apoptosis‐related (H) and autophagy‐related (I) protein levels after DNTTIP1 knockdown in RS4;11 cells and MV4‐11 cells. (J and K) Western blot analysis showing the apoptosis‐related (J) and autophagy‐related (K) protein changes in DNTTIP1‐deficient RS4;11 cells with BMF knockdown.

Article Snippet: Briefly, 5 μg GST‐tagged HDAC1 (SignalChem, H83‐30G) and 5 μg His‐tagged DNTTIP1 (Solarbio, P05485 ) were mixed with 20 μL pre‐washed beads (GST‐labelled magnetic beads for GST pull‐down; NTA‐Ni beads for His pull‐down) in pull‐down buffer.

Techniques: Histone Deacetylase Assay, Western Blot, Knockdown, Quantitative RT-PCR, Cell Counting, Expressing, Control, Over Expression