Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Binding affinities and stoichiometry of binding (N) obtained via isothermal titration calorimetry (ITC) for acetylated histone peptide binding to the ATAD2 bromodomain (BRD).

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Binding Assay, Isothermal Titration Calorimetry, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) measurements for the ATAD2 bromodomain interaction with the histone tail ligands. ( A – K ) Exothermic ITC enthalpy plots for binding of ATAD2 bromodomain with mono-, and di-acetylated histone ligands. Calculated binding constants and peptides are indicated for each trace.

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Isothermal Titration Calorimetry, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Chemical shift perturbation of the ATAD2 bromodomain (BRD) residues upon interaction with histone H4 ligands. ( A ) Histogram showing the normalized 1 H, 15 N chemical shift changes in the assigned backbone amides of the ATAD2 BRD upon addition of H4K5ac (1–10), H4K8ac (1–10), and H4K5acK8ac (1–10) histone peptides in a 1:5 protein to peptide, molar ratio. ( B ) Histogram showing the normalized 1 H, 15 N chemical shift changes in the assigned backbone amides of the ATAD2 BRD upon addition of K4K5ac (1–15), H4K12ac (1–15) and H4K5acK12ac (1–15) histone peptides in a 1:5 molar ratio of protein to peptide. Residues highlighted in red correspond to the “RVF shelf” motif and the conserved asparagine 1064. Residues highlighted by the asterisk (*) denote prolines. The gaps in the residue axis denote unassigned amino acids.

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Chemical shift mapping of the ATAD2 bromodomain (BRD) highlighting the histone binding pocket. Chemical shift changes induced by addition of 1:5 molar ratio of BRD to peptides are mapped onto the crystal structure of the apo ATAD2 BRD (PDB ID: 3DAI). Residues that are not assigned are colored grey, residues exhibiting 1 or 2 standard deviations from the average chemical shift change are colored orange and red, respectively. ( A ) Perturbations caused by addition of mono acetylated H4K5ac (1–10) histone peptide. ( B ) Perturbations caused by addition of di-acetylated H4K5acK8ac (1–10) histone peptide. ( C ) Perturbations caused by addition of mono-acetylated H4K5ac (1–15) histone peptide and ( D ) Perturbations caused by addition of mono-acetylated H4K5acK12ac (1–15) histone peptide.

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Molecular weight distribution of apo ATAD2 bromodomain (BRD) and in complex with histone H4 peptides. The molecular weights of the ATAD2 bromodomain by itself (black) and in complex with a 1:1 molar ratio with H4 unmodified (4–17) (dark blue), H4K5ac (1–15) (yellow), H4K8ac (1–10) (red), H4K12ac (1–15) (green), H4K5acK8ac (1–10) (orange) and H4K5acK12ac (1–15) (light blue) histone peptides. All molar masses are consistent with a 1:1 ratio of ATAD2 with one peptide molecule.

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: 15 N, T 1 and T 2 relaxation rate estimates for ATAD2 bromodomain (BRD) alone and in complex with histone H4K5acK12ac (1–15) peptide.

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Binding affinities and stoichiometry of binding (N) obtained via ITC for acetylated histone H4 peptides binding to the ATAD2 bromodomain (residues 966–1112, C1101A).

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Binding Assay, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Data collection and refinement statistics for the crystal structure of the ATAD2 bromodomain (BRD) bound to histone H4K5acK8ac (1–10).

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Coordination of the H4K5acK8ac (1–10) histone ligand by the ATAD2 bromodomain (BRD). ( A ) Ligand coordination for ATAD2 BRD (cyan) with H4K5acK8ac (1–10) (green) (PDBID: 7M98). Hydrogen bonds are indicated as dashed red lines. ( B ) LigPlot + was used to depict coordination of the H4K5ac (1–10) ligand (green) by the ATAD2 BRD. Five polar contacts to D1020, Y1063, N1064, and R1067 are displayed, as well as the hydrophobic interactions assisting in ligand coordination. ( C ) Electron density for the simulated annealing composite omit map (blue) contoured at 1 s for the histone H4K5acK8ac (1–10) ligand (green). Clear electron density is observed for the first five amino acids including the K5ac sidechain. ( D ) Alignment of the ATAD2 BRD structures 4TT2 (magenta), 4QUU (salmon), and 7M98 (cyan). Highlighted residues are labeled in the protein backbone. The first three ligand residues display a different coordination between the overlaid structures and are labeled. Figures were generated with the PyMOL Molecular Graphics System, version 2.3, Schrödinger, LLC .

Article Snippet: The wild type human ATAD2 BRD cDNA was a gift from Dr. Nicola Burgess-Brown (Addgene plasmid #38916).

Techniques: Labeling, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: The association between ATAD2 expression and the clinicopathological variables

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 is upregulated and is associated with poor overall survival of ESCC patients. a , b ATAD2 mRNA expression in 83 paired primary esophageal squamous carcinoma (ESCC) and its adjacent normal tissues in SYSUCC cohort ( a ) and in GEO Database (GES20347) ( b ). c Representative images of positive and negative ATAD2 protein staining in paired ESCC and its adjacent normal tissues from the same patient. d IHC score of ATAD2 protein expression in ESCC and its adjacent normal esophageal tissues. e , f Kaplan-Meier survival curves of overall survival according to ATAD2 mRNA and protein expression (Low vs. High) in ESCC patients. g , h Kaplan-Meier survival curves of overall survival according to ATAD2 protein expression (Low vs. High) in stage I-II and stage III-IV ESCC patients

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 promotes ESCC cell proliferation in vitro and tumorigenesis in vivo. a , b Knockdown of ATAD2 by three shRNAs and restoration of ATAD2 expression in KYSE30 and KYSE510 cells were confirmed by western blot analysis. The following functional experiments in KYSE30 and KYSE510 cells are all grouped in accordance with ( b ). c Effect of altered ATAD2 expression on cell growth by CCK8 assay. d Effect of altered ATAD2 expression on cell colony formation and colonies of each group were quantified. e Effect of altered ATAD2 expression in KYSE510 cells on tumor growth in nude mice ( n = 7 tumors per group), tumor size was measured every 3 days after 1 week after injection. Tumor tissues were confirmed by hematoxylin-eosin staining. Data was presented as mean ± standard deviation (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Functional Assay, CCK-8 Assay, Injection, Staining, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 promotes ESCC cell migration, invasion in vitro and metastasis in vivo . a , b Effect of altered ATAD2 expression on cell migration and invasion ability using transwell assay. The migrated and invasive cells were counted and analyzed. c Macroscopic appearances of lung images of each group, black arrows indicate the tumor nodules. Lung metastasis nodules were confirmed by hematoxylin-eosin staining. Number of metastatic nodules in each group was counted and analyzed. Data was analyzed using unpaired student’s t -test and presented as mean ± SD (* P < 0.05; ** P < 0.01, *** P < 0.001)

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Migration, In Vitro, In Vivo, Expressing, Transwell Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 activates TGF-β signaling pathway and TGF-β1 gene expression. a KEGG pathway analysis using significantly differentially expressed genes between ATAD2 knockdown KYSE510 cells and control cells based on RNA-sequencing data. b TGF-β response element luciferase activity induced by ATAD2 in KYSE30 and KYSE 510 cells. c ATAD2 and TGF-β1 expression in a panel of ESCC cell lines analyzed by western blot. Expression of ATAD2 and TGF-β1 protein were quantified and analyzed using Pearson correlation method. d Effect of ATAD2 knockdown and restoration on TGF-β1 analyzed by western blot. e Effect of ATAD2 overexpression on TGF-β1 analyzed by western blot

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Expressing, RNA Sequencing Assay, Luciferase, Activity Assay, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 exerts pro-metastatic function depending on the TGF-β signaling induced epithelial-mesenchymal transition. A , B Effect of TGF-β1 receptor inhibition on KYSE510 and KYSE30 cell migration and invasion ability using transwell assay. TGF-β1 receptor was inhibited with 5 μM LY2157299. The migrated and invasive cells were counted and analyzed. C Effect of ATAD2 knockdown on transcription factor Snail, epithelial marker E-cadherin and mesenchymal markers Vimentin and N-cadherin analyzed by western blot. D1-4 , E Effect of TGF-β1 receptor inhibition on transcription factor Snail, epithelial marker E-cadherin and mesenchymal markers Vimentin and N-cadherin analyzed by qRT-PCR ( D1-4 ) and western blot ( E ) respectively. For qRT-PCR, TGF-β1 receptor was inhibited with 2.5 μM, 5 μM, 10 μM LY2157299 and SB525334 for 24 h, and 5 μM LY2157299 and SB525334 were applied for 48 h for western blot analysis. Data was presented as mean ± SD (* P < 0.05; ** P < 0.01, *** P < 0.001)

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Inhibition, Migration, Transwell Assay, Marker, Western Blot, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 regulates TGF-β1/Smad3 pathway which is independent of Smad2 and Smad4. a Effect of ATAD2 knockdown and restoration on expression of TGF-β1 and its downstream effectors analyzed by western blot in KYSE30 and KYSE510 cells using the whole cell extracts. b Effect of ATAD2 overexpression on TGF-β1 and its downstream effectors analyzed by western blot in TE-1 and EC9706 cells using the whole cell extracts. c , d Effect of ATAD2 knockdown and restoration on TGF-β1 and its downstream effectors analyzed by Western blot in KYSE30 and KYSE510 using cytoplasmic and nuclear proteins separately. e Effect of ATAD2 knockdown and restoration on TGF-β1 and its downstream effectors in xenograft tumors. f-h Effects of ATAD2 knockdown and restoration on localization of p-Smad2 (green) ( f ), p-Smad3 (green) ( g ), Smad4 (green) ( h ) and DAPI (blue) performed by immunofluorescence staining

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Expressing, Western Blot, Over Expression, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 facilitates nuclear translocation of C/EBPβ which directly activates TGF-β1 gene transcription. a Effect of ATAD2 knockdown on TGF-β1 mRNA expression in KYSE30 and KYSE510 cells by qRT-PCR. b TGF-β1 luciferase activity induced by C/EBPβ in KYSE510 and KYSE30 cells. c Schematic figure summarizing ChIP-PCR primer sets in TGF-β1 promoter region. d C/EBPβ directly bound to the TGF-β1 promoter regions (region #2–4, #6–8, and #11–13). e The interaction between ATAD2 and C/EBPβ was verified by co-immunoprecipitation. Anti-Flag antibody was used to immunoprecipitate Flag-tagged C/EBPβ from whole cell extracts prepared from HEK293T cells co-expressing V5-ATAD2 and Flag-C/EBPβ. f Endogenous interaction of ATAD2 and C/EBPβ in KYSE510 cells verified by co-immunoprecipitation. g Effect of ATAD2 knockdown on C/EBPβ expression by western blot in KYSE30 and KYSE510 cells using cytoplasmic and nuclear proteins separately. Data was presented as mean ± SD (* P < 0.05; ** P < 0.01)

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Translocation Assay, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Immunoprecipitation, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATAD2 interacts with C/EBPβ to promote esophageal squamous cell carcinoma metastasis via TGF-β1/Smad3 signaling

doi: 10.1186/s13046-021-01905-x

Figure Lengend Snippet: ATAD2 is positively correlated with TGF-β1 signaling pathway in ESCC patients. a - e Correlation analysis of ATAD2 with TGF-β1 and epithelial-mesenchymal transition markers in ESCC patients. f Correlation analysis of expression of TGF-β1 and C/EBPβ in ESCC patients. g Mechanistic model of oncogenic function of ATAD2 in ESCC. ATAD2 interacts with C/EBPβ and enhances the latter’s nuclear translocation. ATAD2 interacts with C/EBPβ and enhances the latter’s nuclear translocation. C/EBPβ directly binds to the promoter region of TGF-β1 gene and facilitates its transcription and the subsequent activation of TGF-β signaling pathway induced epithelial-mesenchymal transition

Article Snippet: TE-1 and EC109 cells with ectopic expression of ATAD2 were performed by transfecting with ATAD2 expression plasmid purchased from Addgene.

Techniques: Expressing, Translocation Assay, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: ATAD2 depletion reduces replication-transcription cooccurrence. (A) Example of an S9.6-EdU PLA in GM639-Cas9-EV (UC) cells. (B and C) S9.6-EdU PLA signal quantification as MFI (B) or number of foci (C) in WT24 cells. Cells were incubated with 100 μM cordycepin (or without the drug) for 1 h and then labeled with 20 μM EdU for 20 min with or without cordycepin, respectively. (D and E) S9.6-EdU PLA signal quantification as MFI (D) or number of foci (E) in HDAC+ GM639 cells (WT24). Cells were labeled with 20 μM EdU for 20 min, and EdU was clicked to a mixture of biotin- and Alexa Fluor 488-azides at a 50:1 molar ratio. Data were subsetted by EdU incorporation status. (F) IF in situ demonstrating overexpression of RNase H1 in GM639-Cas9-EV (UC) cells stably transfected with the RNase H1 expression vector. (G) Quantitation of S9.6-EdU PLA results (representative of data from 3 independent experiments) demonstrating a reduction of the nuclear mean fluorescence intensity of the PLA signal in EdU-positive UC cells overexpressing RNase H1 compared to the untransfected parental cells. Cells were labeled with EdU and processed as described above for panels A and B. (H) Quantitation of IF results (representative of data from 2 independent experiments) demonstrating a reduction of nuclear S9.6 fluorescence in UC cells overexpressing RNase H1 compared to the untransfected parental cells. (I and J) Representative data from 2 independent experiments showing a reduction of S9.6-EdU PLA signals as mean PLA fluorescence intensities (I) and numbers of foci (J) in the indicated cells upon depletion of ATAD2 with a mixture of siATAD2-3 and siATAD2-5. Cells were incubated with 20 μM EdU for 30 min prior to harvest, fixed, and clicked to biotin-azide. Cells containing 10 or more foci were considered positive based on the data shown in panels D, E, and G. P values for mean fluorescence intensities were calculated by K-S tests, and those for numbers of foci were calculated by Wilcoxon tests.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: Incubation, Labeling, In Situ, Over Expression, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: The ATAD2 level affects de novo histone H4 acetyls in nascent chromatin. (A) Western blot demonstrating ATAD2 depletion. NCL (nucleolin) is the internal control. (B) Numbers of H4K12ac-EdU PLA foci in cells of the indicated genotypes transfected with nontargeting siRNA or ATAD2 siRNA. Only cells with two or more foci are included. (C) Pairwise comparisons of numbers of H4K12ac-EdU PLA foci in EdU-negative and EdU-positive subsets of cells of the indicated genotypes transfected with nontargeting siRNA or ATAD2 siRNA. Data in panels B and C represent results from 8 independent experiments in total, 2 of which also included 2 biological replicates. (D and E) IF in situ measurements of C-terminally V5-tagged ATAD2 transgene expression in GM639-Cas9-EV (universal control) cells transduced with the ATAD2 construct (ATAD2) or an empty vector without the tag (e.v.). Endogenous ATAD2 was depleted in the indicated cells by 5′-UTR-targeting siRNA. Expression was assessed with ATAD2 antibody (D) and V5 antibody (E). Graphs represent data from 3 independent experiments. (F) Numbers of H4K12ac-EdU PLA foci in the same cells as in panels D and E. Only cells with more than 5 foci are included. (G) H4K5ac-EdU PLA mean fluorescence intensities (left) and numbers of foci (right) in the same cells as in panels D and E. Only cells with more than 10 foci are included. Data in panels F and G represent results from 3 independent experiments for H4K12ac and from 2 independent experiments for H4K5ac PTM. P values throughout were determined by Wilcoxon tests for numbers of foci and by K-S tests for MFIs.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: Western Blot, Transfection, In Situ, Expressing, Transduction, Construct, Plasmid Preparation, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Proteomics of nascent chromatin identifies H4 acetyl PTM reader proteins. (A) Data-independent acquisition (DIA) workflow for iPOND samples. (B) Comparison of two independent label-free LC-MS/MS analyses performed on iPOND samples of HDAC+, hdac1, and hdac2 cells of the GM639 background. Shown are maximum numbers of unique peptides detected for each of the indicated proteins for all three genotypes (experiment 1, Orbitrap Fusion in DDA mode; experiment 2, Q-Exactive HF in DDA and DIA modes). (C) Workflow diagram for label-free iPOND-MS and MaxQuant data for detection of ATAD2 on EdU-labeled DNA in a biotin-specific manner (i.e., pulled down exclusively via the EdU-biotin/streptavidin bead interaction) in wild-type or hdac1- or hdac2-null GM639 cells. (D) Heat map of a cluster of proteins detected in all three genotypes in a biotin-specific manner. ATAD2 is marked by an asterisk. Red, protein is detected; green, protein is absent. Shades of red reflect ion intensities. Note that the experiment was not designed to reliably detect quantitative differences between levels of proteins present on nascent DNA. (E) Sample preparation workflow for a SILAC iPOND experiment. (F) Normalized heavy (H)/light (L) ratios for the wild type (WT) (GM639-Cas9-EV) over hdac2-21 (left) or over hdac2-36 (right) cells were rank ordered and plotted. Data points corresponding to ATAD2 are highlighted in red.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Sample Prep

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: The ATAD2 level in nascent chromatin is affected by HDAC status. (A) Example of an EdU-dependent ATAD2-EdU PLA in GM639-Cas9-EV cells. EdU-labeled cells were clicked to a mixture of biotin- and Alexa Fluor 488-azides. Red and green channels of the same field are shown separately. (B) Western blot demonstrating siRNA-mediated depletion of ATAD2 in hdac1-50 cells and quantitation of ATAD2-EdU PLA fluorescence in the same experiment. Cells with zero foci are included in the plot. (C and D) Numbers of ATAD2-EdU PLA foci in cells of the indicated cell lines. Only cells showing two or more foci are included. (E) Numbers of ATAD2-EdU PLA foci in EdU-negative and EdU-positive subsets of cells of the indicated cell lines. Results shown in panels C through E represent data from 5 independent experiments in total. P values were calculated by Wilcoxon tests. Black lines denote medians of distributions.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: Labeling, Western Blot, Quantitation Assay, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: ATAD2 depletion moderately suppresses fork progression in hdac2 cells. (A) Effects of ATAD2 depletion on percent S-phase cells in cell lines grouped by genotype. (GM639-Cas9-EV, WT1, and WT2 for HDAC+; hdac1-50 and hdac1-47 for hdac1; and hdac2-21 and hdac2-36 for hdac2). Percentages of S-phase cells were determined by calculating the fraction of EdU-positive cells after EdU pulse-labeling for 20 min followed by immunofluorescence in situ. The y axis shows differences between percent S-phase values measured for cells transfected with ATAD2 siRNA and those for cells transfected with control siRNA. Data were derived from over 10 independent experiments, each measuring at least 300 cells. The P value was determined by a Wilcoxon test. (B) Representative experiment showing profiles of EdU incorporation in cells with the indicated genotypes and siRNAs. Raw MFI values of at least 300 cells per cell line/siRNA were collected from digital images and normalized as described in Materials and Methods. The left panel includes all cells, EdU negative and EdU positive, and the right panel shows only EdU-positive cells. Vertical marks are distribution means. (C) Summary of data from multiple independent experiments similar to the one shown in panel B. For each experiment, EdU incorporation value data sets were processed as follows. The K-S test D statistic was calculated as a metric of the difference between EdU incorporation of siControl and siATAD2 cells for each genotype. The results were grouped by genotype as described above for panel A and plotted. For visualization purposes, in cases where siATAD2 cells showed reduced EdU incorporation compared to siControl, the value of the D statistic was assigned a negative sign. Black circles denote highly significant differences (P < 0.01), and white circles denote differences with P values of >0.01. Lines indicate means of the D statistic distributions. (D) Experimental design to measure fork progression rates by maRTA and data from a representative maRTA experiment. Cells were labeled sequentially with CldU and IdU for 30 min, harvested, and processed as described in Materials and Methods. Lengths of CldU and IdU segments in 100 to 300 two-segment tracks corresponding to ongoing forks were measured and plotted. P values were calculated by using K-S tests. (E) Summary of data from several independent maRTA experiments. For each experiment, differences between lengths of the 1st-label (CldU) segments in ongoing forks of siControl and siATAD2 cells were expressed as a K-S test D statistic, and the D statistic values were plotted as described above for panel C for each genotype. Black circles are the values that correspond to statistically significant differences (P < 0.01) (GM639-Cas9-EV for the WT; hdac1-50, hdac1-47, hdac1-12, and hdac1-5 for hdac1; and hdac2-21 and hdac2-36 for hdac2).

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: Labeling, Immunofluorescence, In Situ, Transfection, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: ATAD2 depletion reduces RNA synthesis levels. (A) IF in situ tests for specificity of BrU and EdU detection. GM639-Cas9-EV cells were labeled for 30 min with 5 mM BrU only (left), 10 μM EdU only (middle), and both labels (right). All samples were clicked to biotin-azide and then incubated with both an antibody to BrdU (without denaturation) and an antibody to biotin. (B) GM639-Cas9-EV cells were labeled with EdU and BrU, as described above for panel A, in the absence or presence of 50 μM cordycepin added concurrently with the labels, and subjected to IF in situ as described above. BrU MFI data were plotted separately for EdU− and EdU+ cells. (C) Data from a representative experiment showing distributions of BrU incorporation in cells with the indicated genotypes and siRNAs. Cells were labeled simultaneously with 5 μM EdU and 5 mM BrU for 1 h, harvested, stained for IF in situ, and imaged. The acquired data were subsetted by EdU incorporation status (EdU positive [S phase] and EdU negative [non-S phase]). Mean BrU incorporation levels in 200 to 300 S-phase nuclei per genotype/siRNA are shown. Vertical marks are distribution means. (D) Statistics summary for independent experiments similar to the ones for panel C. For each experiment, differences between BrU incorporation in siControl and siATAD2 cells were expressed as a K-S test D statistic and plotted (GM639-Cas9-EV for the WT [3 independent experiments with biological replicates] and hdac2-21, hdac2-27, and hdac2-51 for hdac2 [4 independent experiments with biological replicates]). Black circles are the values that correspond to statistically significant differences (P < 0.01). (E) Levels of BrU incorporation in EdU-positive (S-phase) primary fibroblasts upon ATAD2 depletion. Cells were labeled simultaneously with 5 mM BrU and 10 μM EdU, fixed, and analyzed by IF in situ, similarly to panel C.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: In Situ, Labeling, Incubation, Staining

Journal: Molecular and Cellular Biology

Article Title: Mutual Balance of Histone Deacetylases 1 and 2 and the Acetyl Reader ATAD2 Regulates the Level of Acetylation of Histone H4 on Nascent Chromatin of Human Cells

doi: 10.1128/MCB.00421-19

Figure Lengend Snippet: Working model of the relationship between HDAC1, HDAC2, ATAD2, maturation of the nascent chromatin, and DNA metabolism. The open (hyperacetylated)-to-closed (hypoacetylated) dynamic in chromatin, which takes place during replication, may affect replication itself as well as local transcription. Activities and levels of HDAC1, HDAC2, and ATAD2 may delay (e.g., when the ATAD2 level is high) or speed up (e.g., when there is no ATAD2 and no HDAC2) chromatin maturation. See Discussion for more details. RNApol, RNA polymerase.

Article Snippet: The C-terminally tagged ATAD2 ORF (ATAD2-V5) was derived from GFP-ATAD2 (a gift from Kyle Miller) (Addgene plasmid 65370 [ http://n2t.net/addgene:65370 ]; RRID, Addgene_65370) and transferred into the pLenti-EFS-T2A-BSD lentiviral expression vector by using a Gibson assembly kit (NEB).

Techniques: