Journal: Molecular Therapy

Article Title: miR-1293 , a Candidate for miRNA-Based Cancer Therapeutics, Simultaneously Targets BRD4 and the DNA Repair Pathway

doi: 10.1016/j.ymthe.2020.04.001

Figure Lengend Snippet: miR-1293 Downregulated the Expression of APEX1, RPA1, and POLD4 by Directly Targeting Their 3′ UTRs

Article Snippet: 19 The following primary antibodies were used for western blotting: antibodies against BRD4 (#13440), cleaved PARP (#9541), phospho-histone H2AX (Ser139) (#9718), and RPA70 (#2267) were purchased from Cell Signaling Technology; the antibody against APEX1 (10203-1-AP) was purchased from Proteintech (Chicago, IL); anti-POLD4 antibody (MBS9609765) was obtained from MyBioSource.com (San Diego, CA, USA); and anti-β-actin (A5441) and anti-Vinculin (V9131) were obtained from Sigma-Aldrich (Tokyo, Japan).

Techniques: Expressing

Journal: Nature genetics

Article Title: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

doi: 10.1038/s41588-019-0413-z

Figure Lengend Snippet: a , BRD4 interactomes in MEG-01, K-562, MV4-11and MOLM-13 cell lines. Proteins are represented as circles, colors indicate the number of cell lines in which a particular interacting protein was detected. b , Western blot confirmation of the BRD4-MTHFD1 interaction in leukemia cell lines. The experiment was repeated twice with similar results. c , Upper panel: Western blot following nuclear vs cytoplasmic fractionation in HAP1, KBM7 and HEK293T cell lines. RCC1 was used as nuclear loading control while tubulin was used as cytosolic loading control. Lower panel: Western blot following MTHFD1 pull-down in the different cell fractions. The experiment was repeated three times with similar results. d , Western blot performed on chromatin-associated protein samples extracted from HAP1 cells treated with the indicated compounds for 2 h (dBET1: 0.5 μM; dBET6: 0.5 μM; MTX: 1 μM) or 24 h (dBET1: 0.5 μM; dBET6: 0.05 μM; MTX: 1 μM). H2B was used as loading control. The experiment was repeated three times with similar results. e , Western blot for nuclear vs cytoplasmic protein levels in HAP1 cells treated for 24 h as above. The experiment was repeated twice with similar results. f , Western blot from chromatin fractions of MEG-01, K-562, MV4-11 and MOLM-13 cells treated with dBET6 for 2 h. The experiment was repeated twice with similar results. g , Immunofluorescence images of HeLa cells treated with the indicated compounds and stained for MTHFD1, BRD4, and DAPI (small inserts). Scale bar 10 μm.

Article Snippet: HAP1 cells were transiently transfected with PX459 containing the gRNAs by using TurboFectin (OriGene) according to manufacturer’s instructions.

Techniques: Western Blot, Fractionation, Immunofluorescence, Staining

Journal: Nature genetics

Article Title: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

doi: 10.1038/s41588-019-0413-z

Figure Lengend Snippet: a , Validation of MTHFD1 knock-out HAP1 cell lines. The experiment was repeated three times with similar results. b , Representative genome browser view of BRD4, MTHFD1, and H3K27ac binding in the promoters of TFAP4 (left) and KEAP1 (right). All ChIP tracks were normalized to 1X genome coverage. All the IPs were performed in biological duplicate. Specifically for MTHFD1 knock-out cells, MTHFD1 KO_1 and MTHFD1 KO_3 were used as independent biological replicates. c , Enrichment of BRD4 and MTHFD1 ChIP signal. Peaks were sorted by total abundance and data represent merged replicates normalized to 1× coverage. d , Principal component analysis of RNA-seq data of two MTHFD1 knock-out clones and of WT HAP1 cells treated with 0.1 μM dBET6, 1 μM ( S )-JQ1, 1 μM MTX, shRNAs targeting BRD4 or MTHFD1. Equal amount of DMSO, or non-targeting hairpins were used as respective control conditions and two biological replicates were performed for each experimental condition. e , Heatmap of relative transcription changes in HAP1 cells compared to respective control cells. f , Integration of ChIP-seq and RNA-seq data in HAP1 cells. BRD4 and MTHFD1 binding at sites associated with genes which are significantly up- or down-regulated upon knockdown of BRD4 and/or MTHFD1 and in MTHFD1 knock-out cells compared to HAP1 WT cells. Values represent estimated factor abundance normalized by matched IgG signal and equality of distributions was assessed with with a one-sided Mann–Whitney U test. Boxplot boxes represent interquartile range with center on median, and whiskers represent values 1.5× outside the respective interquartile range.

Article Snippet: HAP1 cells were transiently transfected with PX459 containing the gRNAs by using TurboFectin (OriGene) according to manufacturer’s instructions.

Techniques: Knock-Out, Binding Assay, RNA Sequencing Assay, Clone Assay, ChIP-sequencing, MANN-WHITNEY

Journal: Nature genetics

Article Title: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

doi: 10.1038/s41588-019-0413-z

Figure Lengend Snippet: a , Representation of the folate pathway. Enzyme names are reported inside the geometric shapes, connecting the different metabolites. Enzymes that were found associated with chromatin in HAP1 and K-562 cells by mass spectrometry analysis are indicated in red and blue, respectively. Two biological replicates were done. b , Western blot for folate pathway enzymes in the cytoplasmic (C) and chromatin (Ch) fractions of HAP1 cells. The experiment was repeated twice with similar results. c , Recombinant enzyme assays for MTHFD1 activity to convert THF and formate to 5,10-methenyl-THF and vice versa in the presence or absence of full-length BRD4 or its first bromodomain. Mean ± SD from n = 2 independent samples. d , Scatter plot representing metabolite changes in the pyrimidine, purine and methionine biosynthetic pathways upon downregulation of BRD4 or MTHFD1 by shRNA. Two biological replicates were done for each experimental condition. r -value indicates the Pearson correlation coefficient. e , Incorporation of labeled formate into RNA. HAP1 WT and MTHFD1 knock-out cells were treated with 13 C-labeled formate for 24 h, followed by RNA extraction and LC-MS-MS analysis of nucleotides for the 13 C/ 12 C ratio. Two biological replicates were performed for each experimental condition. f , Incorporation of labeled formate into RNA using the same procedure with MTHFD1 knock-out cells transiently transfected with full-length MTHFD1, or the protein with either a nuclear localization signal (NLS) or a nuclear export signal (NES). Percent of control is calculated considering the 13 C incorporation in HAP1 WT and MTHFD1 knock-out respectively as 100% and 0. Two biological replicates were performed for each experimental condition.

Article Snippet: HAP1 cells were transiently transfected with PX459 containing the gRNAs by using TurboFectin (OriGene) according to manufacturer’s instructions.

Techniques: Mass Spectrometry, Western Blot, Recombinant, Activity Assay, shRNA, Labeling, Knock-Out, RNA Extraction, Liquid Chromatography with Mass Spectroscopy, Transfection

Journal: Nature genetics

Article Title: MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

doi: 10.1038/s41588-019-0413-z

Figure Lengend Snippet: a , Dose response matrix displaying REDS1 and REDS3 RFP-positive cells treated with the indicated concentrations of (S)-JQ1 and MTX alone or in combination. Means from two biological replicates. b , Dose response matrices displaying cell viability of HAP1, NOMO-1, K-562 and A549 treated for 72 h with ( S )-JQ1 and MTX alone or in combination. Means from two biological replicates. Differential volume indicates the sum of all deviations from Bliss additivity over the dose response matrix. c , Tumor volumes from a A549 xenograft mouse model treated five times per week with 30 mg/kg ( S )-JQ1 and/or twice weekly with 25 mg/kg MTX from day 19. Means and standard deviations from eight mice per group. Asterisks indicate significance of 1-way ANOVA adjusted by Tukey’s multiple comparison test (* P < 0.05; ** P < 0.005; *** P < 0.0001) d , Weight and of tumors at the end of the experiment (day 43). Means and standard deviations from eight mice per group. Asterisks indicate significance of 1-way ANOVA adjusted by Tukey’s multiple comparison test ( P = 0.00000050). e , Images of tumors at the end of the experiment (day 43).

Article Snippet: HAP1 cells were transiently transfected with PX459 containing the gRNAs by using TurboFectin (OriGene) according to manufacturer’s instructions.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 2. Knockdown of HAP1 causes a loss of synaptic AMPAR responses. A and B, cumulative distribution plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, HAP1 siRNA, or a scrambled control siRNA. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 3. Dominant negative HAP1 (DN-HAP1) occludes the reducing effect of polyQ-htt on synaptic AMPAR responses. A and B, cumulative distribu- tion plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, polyQ-htt, DN-HAP1, or DN-HAP1 plus polyQ-htt. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Dominant Negative Mutation, Transfection

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 4. The polyQ-htt-induced impairment of synaptic AMPAR responses is occluded by knockdown of KIF5, but not KIF-17. A, representative Western blots in HEK293 cells transfected with FLAG-tagged rat KLC1 in the absence or presence of a control siRNA or a KLC1 siRNA. B–D, representative mEPSC traces (B) and cumulative distribution plot of the mEPSC amplitude (C) and inter-event interval (D) from cortical neurons transfected with control siRNA, polyQ-htt, KLC1 siRNA, or KLC1 siRNA plus polyQ-htt. Scale bar: 25 pA, 1 s. E and F, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA. NS, no significance.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 5. The WT-htt-induced enhancement of synaptic AMPAR responses is blocked by knockdown of KIF5. A–C, representative mEPSC traces (A) and cumulative distribution plot of the mEPSC amplitude (B) and inter-event interval (C) from cortical neurons transfected with control siRNA, WT-htt, KLC1 siRNA, or KLC1 siRNA plus WT-htt. Scale bar: 25 pA, 1 s. D and E, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA. NS, no significance.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 7. HAP1/GluR2/KIF5/microtubule complex is disrupted in the mouse model of HD. A–C, co-immunoprecipitation assays showing the asso- ciation between tubulin and KIF5 (A), GluR2 and KIF5 (B), tubulin and GluR2 (B), GluR2 and HAP1 (C) from striatal slices of WT versus HD mice (4-month- old). Each experiment was repeated in 3–5 pairs of mice. D, immunoblots and quantification analysis showing the surface and total GluR2 and GluR1 sub- units in lysates of striatal slices taken from WT versus HD mice (4-month-old). *, p 0.01, ANOVA. E, co-immunoprecipitation assays showing the associa- tion between KIF5 and GluR1 or GluR2 in brain lysates.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 8. A schematic model demonstrating the potential mechanism for huntingtin regulation of AMPAR trafficking. A, in normal conditions, WT-htt binds to HAP1 and enables the binding of HAP1 to KIF5 motor protein. KIF5 heavy chain interacts with GRIP1/GluR2-containing vesicles, and the wholecomplexisanterogradelytransportedalongthemicrotubule(MT)rails. B, in HD conditions, polyQ-htt interacts abnormally with HAP1 (Step 1), thereby interrupting the binding of GluR2/KIF5 (Step 2), and KIF5/MT (Step 3). Consequently, KIF5 is unable to transport the GluR2 cargo along MTs (Step 4), leading to insufficient synaptic delivery of AMPAR channels.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Binding Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Analysis of mRNA that encode store-operated calcium entry (SOCE) proteins in day in vitro 21 (DIV21) medium spiny neuron (MSN) cultures from YAC128 and wildtype mice. (A) Gene expression analysis of mRNA that encode SOCE proteins and proteins that are involved in Ca 2+ homeostasis in MSNs from wildtype (control) and YAC128 mice using real-time polymerase chain reaction (PCR). The gene expression results for all genes were normalized to Gapdh . Data from wildtype MSNs were normalized to 1. (B) Relative mRNA levels of genes that encode SOCE proteins, measured as 2 ∧ -dCt. (C) Immunoblots of HTT-associated protein-1 (HAP1) normalized to GAPDH and CacyBP/SIP (both monomers and dimers) normalized to Vinculin in YAC128 and wildtype MSN cultures. On the bottom of each panel, the expression levels of HAP1 and CacyBP/SIP proteins are plotted. The results are expressed as mean ± SEM and represent data from three independent mRNA and protein preparations of three different MSN cultures. ns, not significant.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Parameters of Ca 2+ homeostasis in YAC128 MSNs that overexpress HAP1A. (A) Immunoblots of HAP1 and green fluorescent protein (GFP) in YAC128 MSN cultures that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP and pLenti-GFP. (B) GFP fluorescence that indicates the overexpression of HAP1A-pLenti-GFP and HAP1B-pLenti-GFP and pLenti-GFP in YAC128 MSNs. (C) Protocol to induce SOCE in YAC128 MSNs that overexpressed HAP1 isoforms using the eight-well system. (D) Protocol to induce SOCE in wildtype MSNs that overexpressed HAP1 isoforms. Cultures of MSNs on DIV14 from YAC128 and wildtype mice (control) that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP, or pLenti-GFP using lentiviruses were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the endoplasmic reticulum (ER) was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (E) Ca 2+ release from the ER in YAC128 MSNs that overexpressed HAP1 isoforms. (F) Ca 2+ release from the ER in wildtype MSNs that overexpressed HAP1 isoforms. (G) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1 isoforms. (H) Ca 2+ influx via DHPG-induced SOCE in wildtype MSNs that overexpressed HAP1 isoforms. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of at least three independent MSN culture preparations are shown. * p < 0.05, *** p < 0.001. ns, not significant.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Western Blot, Fluorescence, Over Expression, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Parameters of Ca 2+ homeostasis in YAC128 MSNs with silencing of HAP1A. (A) Immunoblots of HAP1 and beta-actin in YAC128 MSN cultures that overexpressed short-hairpin RNA (shRNA) against HAP1. shRNAs against HAP1 are named a, b, c, d, control scrambled shRNA, and control MSN cultures as indicated on the blot. (B) Protocol to induce SOCE in YAC128 MSNs with silencing of HAP1 using the eight-well system. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed different shRNAs against HAP1 in pLenti-GFP were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (C) Ca 2+ release from the ER in YAC128 MSNs that overexpressed different shRNAs against HAP1. (D) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed different shRNAs against HAP1. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of three independent MSN culture preparations are shown. *** p < 0.001.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Western Blot, shRNA, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Effect of HAP1A on ER Ca 2+ content in YAC128 MSNs. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP, or pLenti-GFP using lentiviruses that were loaded with the Ca 2+ indicator Fura-2AM. (A) The figure shows the protocol to induce ionomycin-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed HAP1 isoforms using the eight-well system. YAC128 MSNs that overexpressed HAP1A isoforms were incubated in Ca 2 -free medium (0.1 mM EGTA), followed by 15 μM ionomycin application to induce the release of Ca 2+ . KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (B) Ionomycin-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1 isoforms. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. * p < 0.05. ns, not significant. The results were obtained from three independent MSN culture preparations.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Incubation