Journal: bioRxiv

Article Title: Depolymerized lamins link nuclear envelope breakdown to mitotic transcriptional quiescence

doi: 10.1101/334110

Figure Lengend Snippet: a. pS22-LMNA and UnP-LMNA ChIP-seq signals at LADs. LADs are defined by UnP-LMNA ChIP-seq ( Methods ). A subset of LADs (885 of total 2,178) that do not have adjacent LADs within 250 kb downstream of LADs are shown. Inset shows location of strong ChIP-seq signals (marked in red). FE, fold enrichment. b. pS22-LMNA and UnP-LMNA ChIP-seq signals at 34,489 dLASs. c. Feature size of dLASs and LADs. d. Location of dLASs relative to LADs. e. Ty1 ChIP-seq signals at dLASs. Ty1 ChIP-seq was performed in BJ-5ta cells overexpressing Ty1-LMNA, Ty1-LMNC (Lamin C), phospho-deficient Ty1-LMNA-S22A/S392, or phospho-mimetic Ty1-LMNA-S22D/S392D. f. (Left) Fraction of dLASs overlapping anti-Ty1 ChIP-seq peaks in the overexpression cells shown in e . (Right) Fraction of anti-Ty1 ChIP-seq peaks overlapping dLASs. g. ATAC-seq, K27ac ChIP-seq, K4me3 ChIP-seq, and pS22-LMNA ChIP-seq signals at 73,933 ATAC-defined accessible sites in wild-type BJ-5ta cells.

Article Snippet: Antibodies used in ChIP are: rabbit monoclonal anti-phospho-Ser22-LMNA antibody D2B2E (Cell Signaling 13448S, Lot # 1; 5 µL per IP); mouse monoclonal anti-unphospho-Ser22-LMNA antibody E1 (Santa Cruz Biotechnology sc-376248, Lot # H2812; 10 µL per IP); mouse monoclonal anti-acetyl-Lys27 histone H3 antibody (Wako MABI0309, Lot # 14007; 2 µL per IP); mouse monoclonal anti-trimethyl-Lys4 histone H3 antibody (Wako MABI14004, Lot # 14004; 2 µL per IP); mouse monoclonal anti-Ty1 antibody (Diagenode C15200054, Lot # 005; 1 µL per IP).

Techniques: ChIP-sequencing, Over Expression

Journal: PLoS Pathogens

Article Title: Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

doi: 10.1371/journal.ppat.1000322

Figure Lengend Snippet: (A) Schematic representation of episomally expressed AMA-1 GFP fusion proteins. Signal peptide: blue; transmembrane domain: grey; pro-domain (P); domain I-III (DI-DIII) and cytoplasmic domain (CPD): brown; GFP: green. 3D7-derived AMA-1 is represented in lighter brown colour. Cleavage sites of the signal peptide and the pro-domain are marked with arrows. Sizes of the processed proteins are indicated either in green (GFP fusion) or in brown (endogenous protein). (B) Western blot analysis of transgenic parasites using anti-GFP antibodies recognize 3D7- and W2mef-derived AMA-1-GFP proteins (the 110 kDa and the processed 92 kDa AMA-1-GFP fusion protein) in the transgenic parasite lines but not in wild type (WT) parasites. (C) Western blot analysis of transgenic AMA-1 3D7 -GFP and AMA-1 W2mef -GFP expression using an AMA-1 specific polyclonal antibody. In wild type parasites, AMA-1 is recognized as an 83 kDa and a 66 kDa fragment (with and without pro peptide). In the transgenic cell lines, two additional proteins are apparent, representing the 110 kDa and the processed 92 kDa AMA-1-GFP fusion. (D) The monoclonal 1F9 antibody recognizes only the 3D7-derived but not the W2mef-derived AMA-1-GFP fusion protein. (E,F) Full-length AMA-1-GFP (green) localizes in schizonts (s) at the apical end of merozoites (m) and is distributed over the surface of merozoites after schizont rupture. This is shown in both unfixed (E) and fixed parasites (F). The distribution of the endogenous protein visualized with the 3D7-specific 1F9 antibody (red) is identical to the localization of the fusion protein (merge). (G,H) AMA-1-GFP dynamics during schizont rupture. (G) Time lapse microscopy of live AMA1-GFP–expressing parasites before and after schizont rupture. Strong apical GFP fluorescence with some surface staining was observed during schizont rupture and immediately after merozoite release (time frames from ). (H) This distribution is indistinguishable from AMA-1 in wild type parasites visualized by a polyclonal AMA1 antibody (green).

Article Snippet: Antibodies used in immunodetection were rabbit polyclonal anti AMA-1 anti AMA-1 monoclonal 1F9 , monoclonal anti TY1 (Diagenode) and monoclonal anti-GFP (Roche).

Techniques: Derivative Assay, Western Blot, Transgenic Assay, Expressing, Time-lapse Microscopy, Fluorescence, Staining

Journal: PLoS Pathogens

Article Title: Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

doi: 10.1371/journal.ppat.1000322

Figure Lengend Snippet: (A) Schematic representation of episomally expressed mutant AMA-1-GFP fusion proteins without cytoplasmic domain (AMA-1 Δtail -GFP). (B) Expression of mutant AMA-1-GFP was verified using anti-GFP specific antibodies. (C,D) The deletion of the cytoplasmic domain does not affect the localization of the AMA-1 Δtail -GFP fusion protein. This is shown in unfixed (C) and fixed (D) parasites. Mutant protein (green) colocalizes with the endogenous protein (red) as shown in the merge pictures. (E) Immunoprecipitation and immunoblot of AMA-1-GFP– interacting proteins. Coloured marker lines show the predicted size of the endogenous (brown), or the GFP-tagged AMA-1 (green). Supernatant (SN) of extract of AMA-1-GFP–expressing segmented schizonts was used on an AMA-1-GFP column. Supernatant (SN), flow through (FT), washing (W1-3), and the elution fraction (E) were separated on an SDS-PAGE gel and probed with polyclonal anti-PfAMA1 (PR) and anti-RON4.

Article Snippet: Antibodies used in immunodetection were rabbit polyclonal anti AMA-1 anti AMA-1 monoclonal 1F9 , monoclonal anti TY1 (Diagenode) and monoclonal anti-GFP (Roche).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Western Blot, Marker, SDS Page

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: Histone acetylation prevents c-Myb binding to native K562 histones. a 18% SDS-PAGE of native histones isolated from K562 cells treated with DMSO (lane 1–3) or TSA (lane 4–6) and recombinant human histones loaded as reference (lane 7–9). b Acetylation of K562 histones was validated by western blotting using anti-H3K27ac primary antibody (ab177178, Abcam) and anti-H3 primary antibody (ab1791, Abcam) as reference. c GST pull-down assay performed with GST-fused c-Myb DBD (NR123) and 6 µg native K562 histone octamers. The western blot was analysed by using anti-H3 and anti-H3K27ac primary antibodies. About 10% (0.6 µg) of the K562 histones was loaded as reference

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Binding Assay, SDS Page, Isolation, Recombinant, Western Blot, Pull Down Assay

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: ChIP analysis of c-Myb occupancy and H3K27ac levels at the mim - 1 locus. a Map of the mim - 1 locus ( LECT2 leucocyte cell derived chemotaxin 2 ( Gallus gallus (Chicken) ). The enhancer mapped in is indicated. Myb recognition elements (MREs) are indicated by colour-coded asterisk (red: the strong MRE = YAACGG , green: MRE = YAACNG and blue: double MREs with palindromic orientation). The amplicons used for qRT-PCR quantification are indicated. b , c Occupancy of c-Myb wild-type (hcM) and D152V as measured by ChIP and qRT-PCR at the indicated regions. IgG IP controls are included. Bar graphs represent the mean ± SEM of three independent biological replicates each measured in triplicate. d Fragmentation of chromosomal DNA of one replicate. e – g Levels of H3K27ac as measured by ChIP and qRT-PCR at the indicated regions. Bar graphs represent the mean ± SEM of two independent biological replicates each measured in triplicate. ChIP was performed as described on HD11 cells 24 h after transfection

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Derivative Assay, Quantitative RT-PCR, Transfection

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: c-Myb-dependent transcriptional activation of a chromatin-embedded gene involves increased histone acetylation. HD-11 cells were transfected with plasmids encoding full-length HA-tagged c-Myb or c-Myb D152V. The cells were treated with 15 ng/ml TSA or DMSO 8 h after transfection. Total RNA was isolated 24 h after transfection, and mim - 1 and MYB expression was measured by qRT-PCR. The values of RNA expression were normalized to the relative amount of the reference gene hprt . The western blots were analysed using anti-c-Myb (ab45150, Abcam), anti-GAPDH (AM4300, Invitrogen) and anti-H3K27ac primary antibodies. The qRT-PCR results are presented as mean ± SEM of three independent biological replicates. Significance was evaluated by unpaired, two-tailed t tests on selected pairs and indicated with p values (* p < 0.05; ns p > 0.05). No p values were calculated for the MYB expression since only two biological replicates were used for this measurement

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Activation Assay, Transfection, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Western Blot, Two Tailed Test

Journal: Epigenetics & Chromatin

Article Title: The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

doi: 10.1186/s13072-018-0208-y

Figure Lengend Snippet: Pioneer factor model. We propose a model for the pioneer factor function of c-Myb (left panel) where c-Myb binds to closed chromatin, possibly marked by H3K4me1, then recruits H3K27 acetyltransferases such as p300/CBP, facilitating histone acetylation. Due to the reduced nucleosomal stability caused by histone acetylation, chromatin accessibility increases and c-Myb detaches from chromatin because of the lost interaction with acetylated histones. The pioneer-defect D152V mutant (right panel) binds to chromatin and recruits H3K27 acetyltransferases, but is unable to facilitate H3K27ac due to the weakened histone interaction. Therefore, the D152V mutant is unable to increase chromatin accessibility, and the chromatin-embedded target gene remains silent

Article Snippet: The ChIP analysis was performed essentially as previously described [ , ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac.

Techniques: Mutagenesis