Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Preparation of DNL and its use in in vitro ChIP-Seq experiments. ( a ) Modified histone variants prepared by protein semi-synthesis are assembled with the respective barcoded DNA into a barcoded nucleosome (‘NUC’) library (‘DNL’). After biochemical assays with a writer, reader or nuclear extract, the binders and reaction products are isolated by affinity- or immunoprecipitation, followed by DNA experiment multiplexing. NUC identity and abundance is analyzed by next generation sequencing (NGS). ( b ) Combinations of histone modifications (‘mod’) selected for the first version of the library (‘DNL-1’). Unmodified (‘–mod’) or modified H3 proteins (vertical axis) were combined with otherwise unmodified histones (‘–mod’), H2AK119ub, H2BK120ub, or mono-/hyperacetylated H4 (horizontal axis). Additionally, a NUC bearing H2BK120ub and H4Kac 5 was prepared. Asterisk: this variant was employed in the Brd4 experiment . ( c ) Analysis of the combined DNL-1 by native gel electrophoresis and ethidium bromide (EtBr) DNA staining. The bands from NUCs containing combinations of unmodified, Kac or Kme3 histones overlap, whereas the shifted fainter upper band represents NUCs containing ubiquitylated H2A or H2B. For details on modified histones, DNA preparation, NUC assembly and NGS, see .

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: In Vitro, ChIP-sequencing, Modification, Isolation, Immunoprecipitation, Multiplexing, Next-Generation Sequencing, Variant Assay, Nucleic Acid Electrophoresis, Staining

Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Profiling the substrate specificity of histone mark readers. ( a ) Left: Model showing bivalent nucleosomal recognition behavior of BPTF. Right: Immobilized GST-tagged BPTF-PHD (gray) and BPTF-PHD-BD (blue) were incubated with DNL-1, followed by DNA isolation and experiment multiplexing. The resulting DNA was analyzed by next generation sequencing, and the raw sequencing reads were normalized against input and the H3K4me3-containing variant, set as 1 (red asterisks). Values (mean ± SD, n=3) for the respective NUC variants are plotted as a 3D bar graph using the same grid as shown in . ( b ) Left: Model depicting potential binding modes of p300. Right: Library incubation of resin-bound GST-tagged p300-BD (gray) and p300-BD-PHD (blue). All subsequent steps were performed as in ( a ). Internal normalization: H4Kac 5 variant, set as 1 (red asterisks). Values are shown as (mean ± SD, n=3). ( c ) Left: Model depicting potential interaction between Brd4 and acetylated NUC. Right: Library incubation of resin-bound Flag-Brd4-BD1-BD2. All subsequent steps were performed as in ( a ) except for pulldown with an anti-Flag antibody. Internal normalization: H4Kac 5 variant, set as 1 (red asterisk). Experiment was performed in duplicate (replicate shown in ). See Source Data Table 1 for input-normalized sequencing reads.

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: Incubation, DNA Extraction, Multiplexing, Next-Generation Sequencing, Sequencing, Variant Assay, Binding Assay

Journal: PLoS pathogens

Article Title: X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

doi: 10.1371/journal.ppat.1003576

Figure Lengend Snippet: Figure 2. Absence of the XBP1s transcription factor is responsible for reduced C12 cytotoxicity. A. Fluorescence images of calcein AM stained wt MEFs treated with inhibitors of IRE1a RNase activity (STF-080310) and JNK (SP600125) in control conditions (top) and with C12 (25 mM, 4 hours, bottom). B. Normalized caspase 3/7 activation in wt MEFs in control conditions (black) and after treatment with STF-083010 (grey). C. Assessment of IRE1a activation in C12-treated cells. (left) Schematic of luciferase-based IRE1a activity reporter. Luciferase (luc) expression is prevented under control conditions by an ‘in-frame’ stop codon (top); however, IRE1a activation results in non-conventional splicing and removal of 26 nucleotides (26 nt) from the reporter pre-mRNA which shunts the stop codon ‘out-of-frame’ and the luciferase ‘in-frame’ (bottom). (right) IRE1a activation in wt MEFs in control conditions (black) and after 2 hours treatment with 25 mM C12 (grey) or 250 nM thapsigargin (blue). Statistical analysis was by ANOVA with Dunnett post hoc test; ** p,0.0001 versus control cells. D. Deletion of Xbp1 prevents C12-cytotoxicity assessed by calcein AM labelling (left) and caspase 3/7 activation (right). (bottom) Analysis of Xbp12/2 MEF by RT-PCR and western blot. E. Analysis of time- (left) and dose- dependent (right) C12-mediated normalized caspase 3/7 activation in wt (black) and Xbp12/2 (grey) MEFs. Cells were treated with 25 mM C12 over 0– 8 hours and for 4 hours with 0–100 mM C12. Scale bar in panel A refers to all images. doi:10.1371/journal.ppat.1003576.g002

Article Snippet: Plasmids, molecular biology and luciferase-based reporter assays Plasmids containing FLAG-tagged murine XBP1u and XBP1s cDNA were obtained from Addgene (plasmids 21832 and 21833; [63]).

Techniques: Fluorescence, Staining, Activity Assay, Control, Activation Assay, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS pathogens

Article Title: X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

doi: 10.1371/journal.ppat.1003576

Figure Lengend Snippet: Figure 3. Transient expression of XBP1s restores caspase 3 cleavage in Xbp12/2 and Ire1a2/2 MEFs. A. Caspase activation in C12-treated wt MEFs assessed by cleaved caspase 3 immunocytochemistry. Images are shown in the absence (top) and presence (bottom) of C12 (25 mM, 2 hours) with DAPI (magenta) and cleaved caspase 3 (red) staining. B. Cleaved caspase 3 immunostaining in C12-treated Xbp12/2 cells. Untransfected cells (left panels) show limited cleaved caspase 3 immunostaining in the presence of C12 although transient expression of cDNA encoding XBP1u (middle panels) or XBP1s (right panels) restores caspase 3 cleavage upon C12 treatment. C. Cleaved caspase 3 immunostaining in C12-treated Ire1a2/2 cells. Untransfected cells (left panels) and cells transfected with cDNA encoding XBP1u (middle panels) show limited cleaved caspase 3 immunostaining in the presence of C12 although expression of cDNA encoding XBP1s (right panels) restores caspase 3 cleavage upon C12 treatment. D. Number of cells showing cleaved caspase 3 immunostaining after C12 treatments. Data is shown for wt MEFs (white bars) and Xbp12/2 and Ire1a2/2 MEFs transiently transfected with cDNA encoding XBP1u (black) or XBP1s (grey) in the presence and absence of C12 (25 mM, 2 hours). Scale bar in panel A refers to all images. doi:10.1371/journal.ppat.1003576.g003

Article Snippet: Plasmids, molecular biology and luciferase-based reporter assays Plasmids containing FLAG-tagged murine XBP1u and XBP1s cDNA were obtained from Addgene (plasmids 21832 and 21833; [63]).

Techniques: Expressing, Activation Assay, Immunocytochemistry, Staining, Immunostaining, Transfection

Journal: PLoS pathogens

Article Title: X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

doi: 10.1371/journal.ppat.1003576

Figure Lengend Snippet: Figure 4. Leucine zipper and transcriptional activation domains of XBP1s are sufficient for C12-mediated caspase activation. A. Acute inhibition of RNA or protein synthesis with actinomycin (actin.) or cycloheximide (cyclo.) does not inhibit C12-induced caspase activation in wt MEFs. B. Domain structure of XBP1s and XBP1s truncations. XBP1s contains a polybasic domain (grey), leucine zipper domain (black) and transcriptional activation domain (blue). The location of the splice site that converts XBP1u pre-mRNA to XBP1s mRNA is also shown (red). The FLAG- tag on all constructs is located at the amino-terminus. C. Analysis of cellular localization of XBP1s and XBP1s truncations by immunocytochemistry. Panels are shown for anti-FLAG immunostaining (top), DAPI staining (middle), and merged images (bottom). D. Expression of XBP1s truncations that contain the leucine zipper and transcriptional activation domains (XBP1D1 and XBP1D2) is sufficient to restore C12-mediated caspase activation in Ire1a2/2 (top panels) and Xbp12/2 (bottom panels) MEFs. Expression of XBP1N failed to restore C12-mediated caspase activation. Images are shown for DAPI staining (magenta) and caspase immunostaining (red) in the absence and presence of C12. E. XBP1s truncations that restore C12-mediated caspase activation (XBP1D1 and XBP1D2) are transcriptionally inactive. Normalized XBP1s transcriptional activity was measured in wt MEFs using an XBP1s-responsive luciferase-based reporter and co-expression of control plasmid, XBP1s, or XBP1 truncations. Individual scale bars are shown for panels C and D. doi:10.1371/journal.ppat.1003576.g004

Article Snippet: Plasmids, molecular biology and luciferase-based reporter assays Plasmids containing FLAG-tagged murine XBP1u and XBP1s cDNA were obtained from Addgene (plasmids 21832 and 21833; [63]).

Techniques: Activation Assay, Inhibition, FLAG-tag, Construct, Immunocytochemistry, Immunostaining, Staining, Expressing, Activity Assay, Luciferase, Control, Plasmid Preparation

Journal: PLoS pathogens

Article Title: X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

doi: 10.1371/journal.ppat.1003576

Figure Lengend Snippet: Figure 5. XBP1s mediates cellular responses to the C14 homoserine lactone and in different cell types. A. Structure of N-(3-oxo- tetradecanoyl) homoserine lactone (C14). B. Ire1a2/2 MEFs are protected from C14-mediate cell death as assessed by calcein AM staining. C. Normalized caspase 3/7 activity in wt (black) and Ire1a2/2 (grey) MEFs in control conditions and after treatment with C12 or C14 (25 mM, 4 h). D. Caspase activation in C14-treated MEFs assessed by cleaved caspase 3 immunocytochemistry. Images are shown in the absence (top) and presence (bottom) of C14 (25 mM, 2 hours) with DAPI (magenta) and cleaved caspase 3 staining (red). Wildtype MEFs (first panels) and Ire1a2/2 MEFs that are either untransfected (second panels) or transfected to express XBP1s (third panels) or XBP1D2 (fourth panels) are shown. E. Over-expression of XBP1D2 enhances executioner caspase activation when heterologously expressed in FRT cells. (left) Fluorescence images of cell enriched for expression of XBP1D2 (FRT-XBP1D2), anti-FLAG immunostaining (top), DAPI fluorescence (middle) and a merged image (bottom) are shown. (right) Caspase 3/7 activation is enhanced in FRT-XBP1D2 cells (grey bars) relative to control cells (black bars), by C12 (25 mM) at 2 and 4 hours and by C14 (25 mM) at 4 hours. F. Over-expression of XBP1D2 enhances C12- and C14- (25 mM) mediated caspase 3/7 activation when heterologously expressed in Hela cells. Statistical analysis was by t-test and for pairwise comparison of control and XBP1D2 overexpressing cells, * p,0.001 and ** p,0.0001. doi:10.1371/journal.ppat.1003576.g005

Article Snippet: Plasmids, molecular biology and luciferase-based reporter assays Plasmids containing FLAG-tagged murine XBP1u and XBP1s cDNA were obtained from Addgene (plasmids 21832 and 21833; [63]).

Techniques: Staining, Activity Assay, Control, Activation Assay, Immunocytochemistry, Transfection, Over Expression, Fluorescence, Expressing, Immunostaining, Comparison

Journal: PLoS pathogens

Article Title: X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

doi: 10.1371/journal.ppat.1003576

Figure Lengend Snippet: Figure 7. Role of XBP1s in C12-mediated cellular responses. Activation of caspases is dependent upon XBP1s, albeit by a mechanism that does not involve transcription. Phosphorylation of p38 MAPK and eIF2a is XBP1s-independent. Stimulation of cells with C12 does not activate IRE1a and presumably does not activate other ER stress response pathways. No inferences about c-Jun synthesis can be drawn from the current study. doi:10.1371/journal.ppat.1003576.g007

Article Snippet: Plasmids, molecular biology and luciferase-based reporter assays Plasmids containing FLAG-tagged murine XBP1u and XBP1s cDNA were obtained from Addgene (plasmids 21832 and 21833; [63]).

Techniques: Activation Assay, Phospho-proteomics