Journal: PLOS Biology

Article Title: H2A.Z deposition by the SWR complex is stimulated by polyadenine DNA sequences in nucleosomes

doi: 10.1371/journal.pbio.3003059

Figure Lengend Snippet: (A) The histone exchange assay. Blue flag indicates the 3xFLAG tag on Htb1. The S-S-Biotin label indicates the cleavable biotin tag on Htz1(V126C). Red sectors: A-B dimers. Green sectors: Z-B dimers. Grey sectors: H3-H4 dimers. (B) Native SWR complex purified by the ASAP technique was separated on an 8-16% polyacrylamide gel and analyzed by SYPRO orange staining. (C) Histone exchange reactions analyzed by 6% polyacrylamide / 0.5x TBE electrophoresis. Recombinant nucleosomes were used in lanes 1 and 2, native nucleosomes in lanes 3 and 4. (D) Strategy for isolating SWR-remodeled nucleosomes. Input: nucleosomes partially remodeled by SWR in the presence of Z-B dimers that were biotinylated on Htz1 and FLAG-tagged on Htb1. (E) Recombinant nucleosomes and native nucleosomes partially remodeled by SWR (input) were pulled down with streptavidin-coated beads and eluted with DTT (eluate). FT fractions represent the unbound materials of the streptavidin pulldown. Cell equivalent amounts of input, FT, and eluate were analyzed by 6% PAGE and SYBR green staining. (F) Same as E except that the remodeling reactions were quenched by EDTA at the indicated times before subjected to streptavidin pulldown.

Article Snippet: The FLAG beads were washed with Buffer O [25 mM HEPES (pH 7.6), 1 mM EDTA, 0.01% NP-40, 0.3 M KCl, 1x PI] and eluted with 0.5 μ g/ μ L 3xFLAG peptides (Biopeptide) in Buffer P [25mM HEPES (pH 7.6), 1mM EDTA, 0.1% NP-40, 0.3M KCl, 0.1% DOC, 1x PI] overnight at 4 ∘ C. Nucleosomal DNA was extracted by incubating with 0.34 μ g/ μ L Proteinase K (Invitrogen; Cat.# 25530049) in the Buffer Q [340 mM NaCl, 8.5 mM EDTA, 0.43% SDS, 0.1 ug/uL Glycogen] at 55 ∘ C for 1 hr followed by extraction with phenol:chloroform:IAA (25:24:1) and ethanol/sodium acetate precipitation.

Techniques: Purification, Staining, Electrophoresis, Recombinant, SYBR Green Assay

Journal: PLOS Biology

Article Title: H2A.Z deposition by the SWR complex is stimulated by polyadenine DNA sequences in nucleosomes

doi: 10.1371/journal.pbio.3003059

Figure Lengend Snippet: (A) Schematic representation of the asymmetric nucleosomal substrates used in the assay. These substrates feature a a 50-bp linker on one side and a 7-bp linker on the other. Where indicated (dA 10 ), a 10-bp poly(dA:dT) tract was inserted between SHL-4 and -5 on the side closer to the long linker. Red: A-B dimers. Green: Z-B dimers, which contain a 3xFLAG tag on H2B (Htb1). Grey: H3-H4 dimers. Yellow: dA 10 tract. (B–C) The AZ nucleosome has the Z-B dimer inserted on the side distal to the long linker, whereas the ZA nucleosome has the Z-B dimer positioned proximal to the long linker. (D–E) Histone exchange reactions were allowed to proceed for 2 hours in D and for the indicated times in E. The resulting nucleosomes were resolved on a 6% polyacrylamide gel with 0.5x TBE at 110V for 2 hours, followed by SYBR Gold staining.

Article Snippet: The FLAG beads were washed with Buffer O [25 mM HEPES (pH 7.6), 1 mM EDTA, 0.01% NP-40, 0.3 M KCl, 1x PI] and eluted with 0.5 μ g/ μ L 3xFLAG peptides (Biopeptide) in Buffer P [25mM HEPES (pH 7.6), 1mM EDTA, 0.1% NP-40, 0.3M KCl, 0.1% DOC, 1x PI] overnight at 4 ∘ C. Nucleosomal DNA was extracted by incubating with 0.34 μ g/ μ L Proteinase K (Invitrogen; Cat.# 25530049) in the Buffer Q [340 mM NaCl, 8.5 mM EDTA, 0.43% SDS, 0.1 ug/uL Glycogen] at 55 ∘ C for 1 hr followed by extraction with phenol:chloroform:IAA (25:24:1) and ethanol/sodium acetate precipitation.

Techniques: Staining

Journal: Nature Communications

Article Title: The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas

doi: 10.1038/s41467-019-11989-x

Figure Lengend Snippet: CrCO and NF-YB are crucial for photoprotection. a The bleaching phenotypes of the reference strain ( LHCSR1 – Luc717 ) and the DSR mutants visualized in multiwell plates. Representative cell cultures treated with low light (LL; left wells) or high light (HL; right wells). Concentrations of the cultures were adjusted to 1.0 × 10 7 cells/mL. b Chlorophyll content per cell after LL (closed bar) or HL (open bar) treatment of the cells shown in a . c Maximum quantum yield of photosystem II (Fv/Fm) during HL treatment. d qE quenching capability during HL treatment. e Immunoblot analysis of 3xFLAG-fused proteins (CrCO–FLAG and NF-YB–FLAG in crco-2 / CrCO and nfyb-1 / NFYB , respectively), LHCSR1, LHCSR3, and PSBS during HL treatment. ATPB protein levels are shown as the loading control. The experiments were performed three times with different biological samples ( n = 3 biological replicates; mean ± S.E.M); a representative experiment is shown in e

Article Snippet: The Venus–3xFLAG-fused proteins were immunoprecipitated using 100 μL of SURE-beads (Bio-Rad Laboratories) conjugated with 5 μg of FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich) at 4 °C for 1 h. The immunoprecipitates were then eluted using 3xFLAG peptide (ProteinArk, Sheffield, UK) at 500 ng/μL with shaking at room temperature for 15 min, and the eluted polypeptides were separated on a 12.5% PAGEL-HR gel (ATTO) at a constant current 10 mA for 90 min and in-gel trypsin digested according to Shevchenko et al. .

Techniques: Western Blot, Control

Journal: Nature Communications

Article Title: The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas

doi: 10.1038/s41467-019-11989-x

Figure Lengend Snippet: CrCO interacts with NF-Y isoforms to form a transcriptional complex that associates with the promoter regions of the photoprotective genes. a Confocal live-cell imaging of the complemented strains, crco-2 / CrCO (labelled as CrCO) and nfyb-1 / NFYB (labelled as NF-YB), to visualize the localization of CrCO and NF-YB fused with Venus–3xFLAG in C. reinhardtii cells. Scale bars, 5 µm. b GAL4-based yeast two-hybrid (Y2H) assays. CrCO was fused to the GAL4-activation domain (AD–CrCO), whose ability to form heterodimers was tested by cotransformation of the GAL4-binding domain fused with NF–YB or NF-YC (BD–NF-YB or BD–NF-YC). Autoactivation was tested using empty vectors (AD or BD). The cells were plated on permissive (+His) or selective (−His) media and grown at 30 °C for 40 h after being spotted on the plates. c Chromatin immunoprecipitation (ChIP)-PCR assay of CrCO and NF-YB. Agarose gel electrophoresis showing the strength of the association of CrCO or NF-YB with the promoter regions of the photoprotective genes under different light treatments. ChIP was performed using 2 × 10 8 cells/mL of crco-2 / CrCO (labelled as CrCO) or nfyb-1 / NFYB (labelled as NF-YB) cells cross-linked with 0.35% (v/v) formaldehyde after a 1-h light treatment

Article Snippet: The Venus–3xFLAG-fused proteins were immunoprecipitated using 100 μL of SURE-beads (Bio-Rad Laboratories) conjugated with 5 μg of FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich) at 4 °C for 1 h. The immunoprecipitates were then eluted using 3xFLAG peptide (ProteinArk, Sheffield, UK) at 500 ng/μL with shaking at room temperature for 15 min, and the eluted polypeptides were separated on a 12.5% PAGEL-HR gel (ATTO) at a constant current 10 mA for 90 min and in-gel trypsin digested according to Shevchenko et al. .

Techniques: Live Cell Imaging, Activation Assay, Binding Assay, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis

Journal: Nature Communications

Article Title: The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas

doi: 10.1038/s41467-019-11989-x

Figure Lengend Snippet: UVR8 interacts with COP1 and SPA1 to activate the CrCO-based transcriptional complex under UV irradiation. a Confocal images of UVR8–Venus–3xFLAG proteins in the DSR1 – comp15 ( uvr8 / UVR8 ) strain. The cells were treated with UV for 30 min. Scale bars, 10 µm. b After 1 h of UV treatment, the cells were harvested and UVR8–Venus–3xFLAG proteins were immunoprecipitated by FLAG (M2) antibody with SURE-beads. The coimmunoprecipitated proteins were identified by LC-MS/MS analysis of the Coomassie Brilliant Blue-stained polypeptide bands obtained via SDS-PAGE separation after in-gel trypsin digestion. M, molecular mass standard. c Interaction profiles among COP1, SPA1, and CrCO visualized with Y2H assays, which were performed using COP1, SPA1, and CrCO fused with the AD and/or BD domains of GAL4. The culture conditions were as described for Fig. . d Immunoblot analysis of LHCSRs, PSBS, and 3xFLAG-fused CrCO to visualize protein accumulation in the wild-type control (WT), crco-2 , crco-2 / CrCO , spa1 , spa1 crco-2 , and spa1 crco-2 / CrCO strains before and after UV treatment. ATPB proteins were included as the loading controls

Article Snippet: The Venus–3xFLAG-fused proteins were immunoprecipitated using 100 μL of SURE-beads (Bio-Rad Laboratories) conjugated with 5 μg of FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich) at 4 °C for 1 h. The immunoprecipitates were then eluted using 3xFLAG peptide (ProteinArk, Sheffield, UK) at 500 ng/μL with shaking at room temperature for 15 min, and the eluted polypeptides were separated on a 12.5% PAGEL-HR gel (ATTO) at a constant current 10 mA for 90 min and in-gel trypsin digested according to Shevchenko et al. .

Techniques: Irradiation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Staining, SDS Page, Western Blot, Control