Journal: Molecular Cancer

Article Title: RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

doi: 10.1186/1476-4598-13-27

Figure Lengend Snippet: SiRNA knockdown of MLL-AF9 in THP1 cells. (A) Alignments of siRNAs to MLL-AF9 mRNA. Bold sequence represents the MLL part of the fusion transcript. (B) Confirmation of MLL-AF9 knockdown via RT-qPCR. Mean relative MLL-AF9 and HOXA9 transcript levels of knockdown (siRNA-A, siRNA-B) as compared to control treatments (control siRNA-1, -2) during the time course of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. (C) Immunoblot of MLL-AF9 from total protein extracted from day 8 experimental samples. Normalized MLL-AF9 levels are indicated at the bottom.

Article Snippet: DNA new synthesis rate was measured by flow cytometry after pulse labeling THP1 cells with 25 μM EdU (5-ethynyl-2’-deoxyuridine) for 2 hours and staining with AF488-azide and SYTOX AADvanced nuclear stain (Invitrogen, Life Technologies) according to supplier’s protocol.

Techniques: Knockdown, Sequencing, Quantitative RT-PCR, Control, Standard Deviation, Western Blot

Journal: Molecular Cancer

Article Title: RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

doi: 10.1186/1476-4598-13-27

Figure Lengend Snippet: Gene set enrichment plots for our gene expression profile after knockdown of MLL-AF9 in THP1 cells. (A) Gene set enrichment plot of direct MLL-AF9 targets previously detected by Bernt et al. in a mouse model. (B) Gene set enrichment plot of the leukemia gene set defined by FunDO. Because MLL-AF9 acts as a transcriptional activator, enrichment of direct targets was analyzed for the set of up- or down-regulated genes (A) , while the gene set altered in leukemia was analyzed for deregulated genes in general (B) . FDR, false discovery rate q-value; NES, normalized enrichment score.

Article Snippet: DNA new synthesis rate was measured by flow cytometry after pulse labeling THP1 cells with 25 μM EdU (5-ethynyl-2’-deoxyuridine) for 2 hours and staining with AF488-azide and SYTOX AADvanced nuclear stain (Invitrogen, Life Technologies) according to supplier’s protocol.

Techniques: Gene Expression, Knockdown

Journal: Molecular Cancer

Article Title: RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

doi: 10.1186/1476-4598-13-27

Figure Lengend Snippet: Effects of MLL-AF9 knockdown in THP1 cells related to cell size. (A) Mean cell diameter between MLL-AF9 knockdown and non-targeting siRNA control cells was significantly altered on day 7 and 8 of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. ** p < 0.005. (B) Gene set enrichment plot of genes from the gene ontology term “structural constituent of ribosome” (GO:0003735) for our gene expression profile after MLL-AF9 knockdown in THP1 cells. FDR, false discovery rate; NES, normalized enrichment score.

Article Snippet: DNA new synthesis rate was measured by flow cytometry after pulse labeling THP1 cells with 25 μM EdU (5-ethynyl-2’-deoxyuridine) for 2 hours and staining with AF488-azide and SYTOX AADvanced nuclear stain (Invitrogen, Life Technologies) according to supplier’s protocol.

Techniques: Knockdown, Control, Standard Deviation, Gene Expression

Journal: Molecular Cancer

Article Title: RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

doi: 10.1186/1476-4598-13-27

Figure Lengend Snippet: Strategy to prioritize candidate genes concerning mediation of MLL-AF9 leukemogenic effects. Four approaches (second line) were used to select 70 genes from the set of 425 differentially expressed genes after MLL-AF9 knockdown in THP1 cells. Each gene’s likelihood to play a role in leukemogenesis was rated via a structured rating strategy based on data obtained through a literature search.

Article Snippet: DNA new synthesis rate was measured by flow cytometry after pulse labeling THP1 cells with 25 μM EdU (5-ethynyl-2’-deoxyuridine) for 2 hours and staining with AF488-azide and SYTOX AADvanced nuclear stain (Invitrogen, Life Technologies) according to supplier’s protocol.

Techniques: Knockdown

Journal: Molecular Cancer

Article Title: RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

doi: 10.1186/1476-4598-13-27

Figure Lengend Snippet: THP1 cell characteristics influenced by 10 μM of the dopamine receptor antagonist SCH39166 compared to DMSO control. (A) Proliferation in serum reduced conditions. (B) Colony formation in methyl cellulose. Images show representative wells of DMSO control (left) and 10 μM SCH39166 treatment (right). (C) Cell cycle distribution in serum reduced conditions. (D) Cell migration in 5 μm transwells. (E) DNA new synthesis rate measured via EdU incorporation and SYTOX AADvanced nuclear stain. Ratio of SCH39166 compared to DMSO control treatment is shown. Mean over three (A-C) or two (D-E) independent experiments are shown. Bars indicate standard error of the mean; *p < 0.05. † Non significant differences because of interexperimental fluorescence intensity standard deviation, although the ratio between the two treatments remains similar.

Article Snippet: DNA new synthesis rate was measured by flow cytometry after pulse labeling THP1 cells with 25 μM EdU (5-ethynyl-2’-deoxyuridine) for 2 hours and staining with AF488-azide and SYTOX AADvanced nuclear stain (Invitrogen, Life Technologies) according to supplier’s protocol.

Techniques: Control, Migration, Staining, Fluorescence, Standard Deviation

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: The POLE3-POLE4 Complex Is a Bona Fide H2A-H2B Histone Fold Complex (A) Cartoon depicting human POLE3 and POLE4 proteins and their predicted structural motifs. α helices are represented as squared boxes, while histone fold motifs are indicated by brackets. (B) Modeled POLE3-POLE4 dimer with conserved Phe44 and Phe74 in stick format. Consurf was used to color residues by conservation from blue (most conserved) to red (least conserved). (C) In vitro GST pull-downs of the indicated GST-tagged proteins in the presence of His-POLE4 (left) or His-POLE3 (right). (D) Western blot analysis of FLAG IPs from whole-cell extracts of HeLa TRex-expressing empty FLAG, FLAG-POLE3 WT, or F44D mutant. (E) Western blot analysis of FLAG IPs from whole-cell extracts of HeLa TRex-expressing empty FLAG, FLAG-POLE4 WT, or F74D mutant.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: In Vitro, Western Blot, Expressing, Mutagenesis

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: The POLE3-POLE4 Complex Interacts In Vitro with H3-H4 (A) In vitro GST pull-down of the GST-POLE4/His-POLE3 complex in the presence of H3-H4 at the described NaCl concentrations. (B) In vitro GST pull-down of the indicated GST-tagged proteins in the presence of H3-H4. Experiments were performed in 150 mM NaCl. (C) In vitro GST pull-down of the indicated GST-tagged proteins in the presence of H2A-H2B dimers. Experiments were performed in 150 mM NaCl. (D) Limited trypsin digestion of recombinant POLE3-POLE4 complex in the presence of the indicated trypsin concentrations. (E) Analytical gel filtration of POLE3-POLE4, H3-H4, and POLE3-POLE4-H3-H4 complexes in 300 mM NaCl concentrations. (F) SDS-PAGE analysis of gel filtration fractions from (E).

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: In Vitro, Recombinant, Filtration, SDS Page

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: H/D Exchange Mass Spectrometry and Protein Pull-Downs Identify Interaction Domains between POLE3-POLE4 and H3-H4 Complexes (A) Difference plot of hydrogen/deuterium (H/D) exchange data from POLE3 (upper left), POLE4 (upper right), H3 (lower left), and H4 (lower right). A cartoon depicting the domains of the analyzed proteins is shown on the top of the graph. Experimental error is reported in gray, while different colors represent different D 2 O incubation times. More exposed and protected regions are located, respectively, on the higher and lower part of the graph. Peptide aa numbers from highly protected or exposed regions of POLE3 and H4 are shown. ±0.5 Dalton difference is indicated with red/blue lines, which represents 98% confidence limit, so that peptides above or below these lines can be considered significantly changed. Difference heatmaps are provided in Figure S3 A. (B) Analytical gel filtration of the described protein complexes in 300 mM NaCl concentrations. (C) SDS-PAGE analysis of gel filtration fractions from the described protein complexes.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: Mass Spectrometry, Incubation, Filtration, SDS Page

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: The POLE3-POLE4 Complex Binds to H3-H4 Dimers and Tetramers and Promotes Tetrasome Formation and Supercoiling In Vitro (A) DSS crosslinking experiments performed in 150 mM NaCl. Lane 4 shows control without crosslinking; lanes 1, 2, and 5 show samples incubated with 1 mM DSS for 30 min at 23°C and resolved by SDS-PAGE and Coomassie staining. Lane 3 was left empty. Two black arrows indicate the molecular weight species identified upon crosslinking of POLE3-POLE4/H3-H4 co-complex. T (tetramers) indicates POLE3-POLE4 binding to H3-H4 tetramers, while D (dimers) indicates POLE3-POLE4 binding to dimers. (B) Western blot analysis of DSS crosslinking experiments performed in 150 mM NaCl. Lane 1 shows control without crosslinking; lane 2 shows sample incubated with 1 mM DSS for 30 min at 23°C. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with α-H3, α-H4, α-POLE3, and α-POLE4 antibodies, from left to right. (C) Analytical gel filtration of POLE3-POLE4 complex alone or in combination with H3-H4 or H3(EE)-H4. Experiments were performed in 300 mM NaCl to prevent histone aggregation. (D) SDS-PAGE analysis and Coomassie staining of gel filtration fractions from (C). (E) Tetrasome assembly on linear DNA (Widom 601 sequence) monitored by native PAGE. Lane 2 shows tetrasome preassembled by salt dilution method; lanes 3–5 show linear DNA incubated with the indicated proteins. (F and G) Tetrasome assembly on linear DNA, performed as in (E), with the indicated proteins. (H) Plasmid supercoiling assay resolved by native agarose gel electrophoresis. Lane 1 shows supercoiled control phix174 RF1 DNA; lane 2, phix174 RF1 DNA relaxed by TOPO I; and lanes 3–7, phix174 RF1 DNA incubated, in the presence of TOPO I, with histones and increasing concentrations of POLE3-POLE4. (I) Plasmid supercoiling assay performed as described in (H) using increasing concentrations of POLE3ΔC-POLE4 or POLE3-POLE4.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: In Vitro, Control, Incubation, SDS Page, Staining, Molecular Weight, Binding Assay, Western Blot, Filtration, Sequencing, Clear Native PAGE, Plasmid Preparation, Agarose Gel Electrophoresis

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: POLE3 and POLE4 Interact with H3-H4 In Vivo (A) IP of endogenous POLE3 from human HeLa cells performed after CSK-Triton (0.5%) extraction on soluble and DNaseI-digested chromatin fractions. After SDS-PAGE, western blotting was performed using antibodies against the indicated proteins. (B) FLAG IP experiments from HeLa TRex-expressing empty FLAG or FLAG-POLE4 under tetracycline-regulated promoter. Cells were induced with doxycycline for 24 hr and lysed in CSK-Triton 0.5%. FLAG IPs were performed on soluble and DNaseI-digested chromatin fractions. After SDS-PAGE and nitrocellulose transfer, membranes were incubated with antibodies against the indicated proteins. (C) FLAG IP experiments from HeLa TRex-expressing empty FLAG, FLAG-POLE3 WT, or POLE3ΔC mutants under a tetracycline-regulated promoter. Cells induced with doxycycline for 24 hr were lysed in CSK-Triton 0.5%, and FLAG IP was performed on DNaseI-digested chromatin. After SDS-PAGE, western blotting was performed using antibodies against the indicated proteins. (D) HA tag IP experiments from HeLa S3 cells stably expressing HA-H3.1 and HA-H3.3 from soluble and DNaseI-digested chromatin fractions. After SDS-PAGE, western blotting was performed using antibodies against HA tag, POLE3, and POLE4. (E) FLAG IPs from HeLa TRex-expressing empty FLAG or POLE4-FLAG mutants under tetracycline-regulated promoter. Cells were induced with doxycycline for 24 hr and lysed in CSK-Triton 0.5%. FLAG IPs were performed on DNaseI-digested chromatin fractions; after SDS-PAGE and western blotting, membranes were incubated with antibodies against marks specific of newly synthesized or parental histones. (F) Sequential IP experiments performed on empty FLAG or FLAG-POLE3 HeLa SNAP-HA-H3.1-transfected cells. After cell lysis in CSK-Triton 0.5%, chromatin was solubilized with benzonase and incubated with anti-FLAG agarose beads. After subsequent FLAG bead elution in 3xFLAG peptides (1 mg/mL concentration), HA IPs were performed, followed by SDS-PAGE and western blotting using antibodies against the indicated proteins.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: In Vivo, Extraction, SDS Page, Western Blot, Expressing, Incubation, Stable Transfection, Synthesized, Transfection, Lysis, Concentration Assay

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: Constitutive or Transient Depletion of the POLE3-POLE4 Complex Affects Nucleosome Disruption/Maturation at the Replication Fork (A) FACS analysis of endogenous chromatin-bound RPA from POLE4 +/+ and POLE4 −/− MEFs treated or not with 2 mM hydroxyurea for 2 hr. (B) FACS analysis of chromatin from U2OS cells stably expressing GFP-RPA, subjected or not to HU treatment as described in (A). Cells were transfected with siRNAs against the indicated genes, and chromatin purification and FACS analysis were performed 48 hr later. (C) Bar graph showing percentage of chromatin RPA in cells transfected with the indicated siRNA and treated or not with 2 mM HU for 2 hr. Gates for quantification were selected as shown in (B). Biological triplicates (n = 3) are reported with mean, standard deviation, and p value ( ∗ p < 0.05, ∗∗ p < 0.01). (D) Bar graph showing the median of PCNA staining intensity (arbitrary units) from U2OS RFP-PCNA transiently transfected with siRNAs against the indicated genes. Biological triplicates (n = 3) are reported with mean, standard deviation, and p value ( ∗ p < 0.05, ∗∗ p < 0.01). (E) Western blot analysis of cell lysates from HeLa TRex cells expressing empty FLAG, FLAG-POLE3, or FLAG-POLE3ΔC and transfected with the indicated siRNAs in the presence of doxycycline. After SDS-PAGE and nitrocellulose transfer, membranes were incubated with antibodies against the indicated proteins. (F) FACS analysis of endogenous chromatin RPA from HeLa TRex FLAG-POLEΔC cells transfected with the indicated siRNAs and treated or not with 2 mM hydroxyurea for 2 hr.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: Disruption, Stable Transfection, Expressing, Transfection, Purification, Standard Deviation, Staining, Western Blot, SDS Page, Incubation

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet: Transient Depletion of the POLE3-POLE4 Complex Affects Histone Deposition in SNAP Tag H3.1-Expressing Cells (A) In HeLa H3.1-SNAP: at left, quench-chase-pulse experiment to follow new H3.1; at right, pulse-chase experiment to follow parental H3.1. A quenching step labels all pre-existing histones with a non-fluorescent dye. A chase step allows synthesis and deposition of new unlabeled H3.1-SNAP. A pulse using the fluorophore TMR (red) labels available H3.1-SNAP. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). A Triton extraction step is performed prior to fixation to eliminate soluble histones and analyze chromatin-bound H3.1. (B) Representative images of new (left) or parental (right) H3.1 (TMR, red) and replication sites (EdU, green) in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions. Scale bars, 10 μm. (C) Quantification of TMR fluorescence signal per nucleus normalized to control mean for new (left) or parental (right) H3.1 in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions (n = 3). For new H3.1 (left), only cells in S phase were quantified.

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: Expressing, Pulse Chase, Extraction, Control, Fluorescence

Journal: Molecular Cell

Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

doi: 10.1016/j.molcel.2018.08.043

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal Anti POLE3 , Bethyl , Cat#A301-245A; RRID: AB_890598.

Techniques: Produced, Recombinant, Protease Inhibitor, Blocking Assay, Mass Spectrometry, Staining, Flow Cytometry, Mutagenesis, Control, Synthesized, Sequencing, Software

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: Immunofluorescence of endogenous macroH2A1 and histone H3 trimethylation (H3K27me3). Enrichment of endogenous macroH2A1 co-localized with H3K27me3 in the two inactive X chromosomes was detected at interphase in a HEK 293T cell. Bar=10 µm. (B) Live cell imaging of mCherry-tagged macroH2A during mitosis. mCherry-tagged macroH2A1.2 was expressed under a CMV promoter. A mitotic HEK 293T cell was imaged using widefield microscopy. Time series images were acquired every 15 minutes. HEK 293T cells contain three X chromosomes, with two inactivated, apparent as the two bright subnuclear signals of mCherry-tagged macroH2A. Bar=10 µm.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Immunofluorescence, Live Cell Imaging, Microscopy

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: The timeline of synchronization and labeling of cells. Cells in prometaphase were collected by shake-off following nocodazole treatment for 12 hours. Three hours after spreading onto a MatTeck cell culture dish, the pre-existing SNAP-tagged macroH2A was labeled with SNAP-Oregon Green (OG) to cause the existing MacroH2A to fluoresce green. The cells were then pulsed with SNAP-Block to prevent any remaining SNAP-tagged macroH2A from being able to conjugate with fluorophores. Following further culture, the cells were arrested at the G2/M transition by RO-3306 treatment, allowing SNAP-macroH2A newly incorporated since the G2/M transition to be labeled with SNAP-TMR STAR (TMR) for a red fluorescence signal. Live cell imaging was then performed after releasing from the RO-3306 block. (B) Live cell imaging of pre-existing and newly incorporated SNAP-macroH2A distribution into daughter cells during mitosis. Pre-existing (OG, green) and newly incorporated (TMR, red) SNAP-macroH2A were imaged at 20 minute intervals. (C) In vivo imaging reveals equal distributions of pre-existing and newly incorporated SNAP-macroH2A to daughter cells (n=5).

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Labeling, Cell Culture, Blocking Assay, Fluorescence, Live Cell Imaging, In Vivo Imaging

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: (A) The time line of synchronization and harvesting of HEK 293T cells is shown. Equal numbers of cells were synchronized at the G1/S phase border by a double thymidine block. The cells were harvested (Pre-S) or released from thymidine block and synchronized prior to M phase with RO-3306 and harvested (Post-S). Chromatin fractions were isolated for Western blotting, testing endogenous macroH2A1, histone H3, and phosphorylation of H3 at serine 10 (H3S10P) as a marker of mitosis, with hnRNP k/j as a loading control. We show that H3 increase during mitosis, as expected, but there is no concurrent increase in macroH2A. n=3, Error bars = Standard deviation. (B) The time line of synchronization and labelling of cells at S/G2 and G1 phases for the analysis in (C). To label newly incorporated histones in S/G2 phase, HEK 293T cells stably expressing SNAP-tagged H3 or macroH2A were synchronized at the G1/S phase border by double thymidine block. Cells were treated with SNAP-Oregon Green to label pre-existing histones (green arrow), subsequently blocking non-labelled proteins using the non-fluorescent SNAP-Block reagent. The cells were allowed to progress to the G2/M transition until they were blocked using RO-3306 (a CDK1/cyclin B1 and CDK1/cyclin A inhibitor), labelling newly-incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). To label newly incorporated histones in G1 phase, mitotic cells were collected by shake-off following nocodazole treatment for 12 hours and spread onto coverslips. After two hours, cells were labelled with Oregon Green and treated with the blocking reagent. Cells were then allowed to progress to the G1/S transition, when they were synchronized by double thymidine block, then labelling newly-incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). After being released from the first synchronization, the cells were also incubated with EdU until the second synchronization, allowing cells that had undergone DNA synthesis to be identified. (C) An example of images showing the detection of pre-existing and newly-synthesized SNAP-tagged histones in S/G2 or G1 phases. Bar = 10 µm. (D) Image analysis measurements of red and green nuclear signals, representing the ratio of newly-incorporated to pre-existing histones H3 and macroH2A in the S/G2 and in G1 phases. The error bars represent one standard deviation. The P values were determined using two-tailed unpaired t-tests.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Blocking Assay, Isolation, Western Blot, Marker, Standard Deviation, Stable Transfection, Expressing, Incubation, DNA Synthesis, Synthesized, Two Tailed Test

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: (A) Image analysis of SNAP-tagged macroH2A incorporation on the two inactive X chromosomes (Xi) in HEK 293T cells. A line of HEK 293T cells stably expressing SNAP-tagged macroH2A was synchronized and pulse-chase labeled as shown in . The left panels show the merged images of pre-existing (Oregon Green: green) and newly incorporated (TMR: red) SNAP-macroH2A. In the right panels we show the signal intensities measured along the white dashed lines in the left panels. The green and red lines show the signal intensities of pre-existing and newly incorporated macroH2A per pixel, respectively. (B) We show the areas measured. NUC -Xi is the area of nucleus excluding the two inactive X chromosomes (Xi). (C) The signal intensities of pre-existing macroH2A in NUC -xi and Xi. (D) Comparison of signal intensities of newly incorporated macroH2A between the NUC -Xi and Xi subnuclear domains in S/G2 and in G1. (E) The signal intensities of EdU incorporation on NUC -Xi and Xi. No differences are apparent between the dynamics of incorporation of newly-incorporated macroH2A into the inactive X chromosome compared with the rest of the nucleus.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Stable Transfection, Expressing, Pulse Chase, Labeling

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: (A) The time line of synchronizing and labelling cells. Newly synthesized macroH2A was labelled with SNAP substrate JF646 at each 2 hour time point after release from thymidine block. (B) Flow cytometric analysis of pre-existing (Oregon Green) and newly incorporated macroH2A (JF646) and the Fucci cell cycle indicator. The stage of the cell cycle at each time point was determined by the levels of TagBFP (G1 marker) and mCherry (S/G2 marker) ( Figure S4 ), and indicated in the middle section. The black columns represent when pre-existing macroH2A (hours 2-12, S/G2) transitions to an enrichment for newly-incorporated macroH2A (hours 18-22, G1). (C) Examples of in vivo imaging of macroH2A dynamics in individual cells. Pre-existing macroH2A in non-synchronized cells was labelled with SNAP-Oregon Green and blocked with SNAP-Block. Newly-incorporated macroH2A was detected with JF646. Live cell images were acquired every 20 minutes for 18-20 hours. Signal intensity transitions were measured as shown in the left panels. Representative images from 4 hourly intervals are shown in the right. The upper cell is captured entering G1 (dashed arrow) and shows a substantial accumulation of new macroH2A, whereas the lower cell is captured during S/G2 and accumulates new macroH2A to a much lesser extent. Bar=10µm. (D) The summarized data from imaging of 22 cells. The cells were aligned temporally using the Fucci cell cycle images. The lines in lower panel represent the detection phase in each cell. The rate of change of signal of newly incorporated macroH2A was calculated as the difference of intensity of newly-incorporated macroH2A normalized to the signal in first time point of pre-existing macroH2A between two consecutive time points (Delta (X t -X (t- 1))/2). These delta values were then plotted through the cell cycle. Signal saturation at metaphase distorts the data, but the right panel allows comparison of temporal changes compared with the average delta (0.17) during S-G2 phase. The period of sustained accumulation of macroH2A is between hours ∼13-17 (red box). Error bars = Standard error of the mean.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Synthesized, Blocking Assay, Marker, In Vivo Imaging, Imaging

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: (A) The time line of synchronizing, blocking and harvesting of cells. (B) Using data from 500 kb genomic windows and identifying those with high confidence for macroH2A enrichment ( Figure S5 ), we find that these “peaks” overlap substantially between macroH2A newly incorporated in S/G2 and in G1. (C) MacroH2A is enriched before and after cell division in Giemsa positive bands, specifically those categorized as gpos100 in the UCSC Genome Browser. The results of permutation tests demonstrate enrichment only for the most heterochromatic cytogenetic bands (P<0.01). (D) ChIP-qPCR of loci predicted from the ChIP-seq results to be positive and negative validates these genome-wide studies.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Blocking Assay, ChIP-sequencing, Genome Wide

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: Sequencing results of the immunoprecipitated (IP) SNAP-macroH2A (G1 or S/G2) and control input DNA were normalized to the same number of aligned reads per sample (∼127 million), with calculation of IP/input ratios for windows of 1 – 1,000 kb genome-wide. The top and bottom 1% of values were excluded and density plots generated as shown. No bimodality to suggest enrichment and specificity of targeting is apparent except in the windows of 500 – 1,000 kb.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Sequencing, Immunoprecipitation, Genome Wide, Generated

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: (A) Mononucleosomes (red box) were purified using high density sucrose gradient ultracentrifugation of samples synchronized as in . (B) Western blotting before and after immunoprecipitation of pure mononucleosomes using anti-SNAP antibody or rabbit IgG as a negative control. Input samples (and 1:10 dilutions) were loaded in the four lanes on the left. The lower Western blot shows that histone H3 is present in the isolated mononucleosomes and that the loading of the pairs of samples prior to or after S phase (indicated as Pre-S or Post-S) lanes are balanced. In the upper blot, there is no evidence for nucleosomes containing SNAP-macroH2A also containing detectable levels of endogenous macroH2A, as the signal intensities in the anti-SNAP lanes do not exceed those of the non-specific IgG lanes. (C) The brightness of individual nucleosomes containing Oregon Green-labelled SNAP-macroH2A measured by fluorescence correlation spectroscopy (FCS) was indistinguishable from individual beads with single molecules of SNAP-Oregon Green, demonstrating that individual nucleosomes contain only single molecules of SNAP-macroH2A.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Purification, Western Blot, Immunoprecipitation, Negative Control, Isolation, Fluorescence, Spectroscopy

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: Mononucleosomes from HEK 293T cells stably expressing either SNAP-tagged macroH2A (left lanes) or histone H3 (right lanes) were loaded on a Western blot. Detection was performed using the antibodies shown under each image, and relative signal intensities measured by densitometry (relative values shown for each band). We assume that each mononucleosome contains a pair of histone H3 molecules, allowing the left panel to serve as a loading control. The right lane has a higher total signal intensity, (1.0 + 0.2 =) 1.2 / 0.8 = 1.5 fold that of the left lane, indicating the increased proportion of mononucleosomes loaded in the right lane. The middle panel reveals the differences in expression levels of the transgenes encoding the SNAP-tagged proteins, the macroH2A-SNAP expressed at (0.8 / (1.0 / 1.5) =) 1.2 fold the level of the H3-SNAP transgene. This allows us to test the proportion of mononucleosomes with endogeneous macroH2A in the H3-SNAP cell line ( right panel ). This endogeneous macroH2A occurs as a proportion of SNAP-macroH2A of (1.0 / 1.5) / 1.1, a ratio of 1:1.65. As macroH2A-SNAP is expressed at a ratio of 1.2: 1 of H3-SNAP, we calculate that endogeneous macroH2A is expressed at a ratio of 1: (1.65 / 1.20 =) 1.375 of H3-SNAP. From the left panel , we see that H3-SNAP is (0.2 / (0.2+1.0)=) 1/6 of all nucleosomes, allowing us to infer that endogeneous macroH2A is present in 1 in (1.375 x 6 =) 8.25, or 12.1%, of all nucleosomes.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Stable Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Re-Targeting of Macroh2A Following Mitosis to Cytogenetic-Scale Heterochromatic Domains

doi: 10.1101/333468

Figure Lengend Snippet: The relative expression of SNAP-macroH2A relative to endogenous macroH2A in the HEK293T cell lines that were used in this study. We used the low-expressing line for all studies except for that involving FCS, for which we wanted to maximize the chances of incorporating two SNAP-macroH2A molecules per nucleosome. Titration of labeling SNAP-macroH2A with SNAP-Oregon Green for FCS analysis. The same amount of nuclei were used in labelling with 1, 5, 10, and 25 nmol of SNAP-Oregon Green and mononucleosomes labeled with SNAP-Oregon Green were detected by FCS. Values above 5 nmol of SNAP-Oregon Green saturate the fluorescence intensity values per molecule, indicating that the 5 nmol we used was occupying all available SNAP-tags.

Article Snippet: In order to generate the plasmids expressing SNAP-H3 and SNAP-macroH2A, the pSNAP f vector produced from New England BioLabs was used.

Techniques: Expressing, Titration, Labeling, Fluorescence