Journal: Genome Medicine

Article Title: Potential contribution of the uterine microbiome in the development of endometrial cancer

doi: 10.1186/s13073-016-0368-y

Figure Lengend Snippet: Maximum likelihood phylogenetic tree of the V3-V5 16S rDNA region of the recovered Porphyromonas sp. a Recovered from children with atopic dermatitis. b Recovered from buffaloes with postpartum endometritis. c Recovered from Holstein dairy cows with postpartum metritis. Produced with FASTTREE

Article Snippet: In samples that failed 16S rDNA amplification, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs Inc., Ipswitch, MA, USA) was used to separate the microbiome from the human DNA to increase the odds of a successful amplification from samples naturally enriched with human DNA (mostly tissue samples).

Techniques: Produced

Journal: Nature structural & molecular biology

Article Title: BRD4 is a Histone Acetyltransferase that Evicts Nucleosomes from Chromatin

doi: 10.1038/nsmb.3228

Figure Lengend Snippet: BRD4 has intrinsic histone acetyltransferase activity (a) HAT assays of H3 and H4 (1μg) with radiolabeled AcCoA with recombinant BRD4 (500 ng), mTAF1 (250 ng) or mock. (b) HAT assays of rHis-mBRD4 (500 μg) from insect sf9 cells, GST-hBRD4 from E.coli , Flag-hBRD4 from HeLa cells and rTAF1 with histone H3 (1μg) and acetyl CoA. Immunoblotting was with anti-AcH3, anti-H3 or anti-BRD4 antibodies. Bottom panel, silver staining as a loading control. (c) Recombinant mBRD4 from sf9 cells, hBRD4 (3.5 μg) and control Sf9 extract resolved on a denaturing gel (left panel), immunoblotted with anti-BRD4 (middle panel) or subjected to an in-gel HAT assay; acetylated H4 peptide visualized with anti-AcH4 (right hand panel). Faster mobility band in hBRD4 lane is a degradation product. (d) Histones H2A, H2B, H3, H4 (1μg) or all four (1μg each) incubated with or without 500ng BRD4 in a HAT assay followed by autoradiography or coomassie staining. (e) Mononucleosomes (equivalent to ~700bp) (10pmol) assembled on 5S rDNA (208 bp), visualized in ethidium bromide stained gels (left panel), subjected to a HAT assay with or without recombinant p300 (500 ng) or BRD4 (500 ng) with radiolabeled AcCoA and visualized by autoradiography or coomassie staining. (f) Histone H3, H4ac and H3ac immunoblots of whole cell extracts (WCE) from HeLa cells grown 14, 16 and 18hr following transfection with 3μg hBRD4 expression plasmid or control. AU: arbitrary units. Data are representative of 3 independent experiments. Uncropped images of gels and immunoblots are shown in Supplementary Data Set 1

Article Snippet: Purified 5S rDNA (208bp) was from New England Biolabs (NEB).

Techniques: Activity Assay, HAT Assay, Recombinant, Silver Staining, Incubation, Autoradiography, Staining, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Nature structural & molecular biology

Article Title: BRD4 is a Histone Acetyltransferase that Evicts Nucleosomes from Chromatin

doi: 10.1038/nsmb.3228

Figure Lengend Snippet: BRD4 acetylation of H3K122 mediates histone eviction and nucleosome clearance a) Autoradiogram showing mononucleosomes (10 pmol) assembled in vitro with radiolabeled 5S rDNA and subjected to HAT assays for 15 and 30 minutes with or without 500ng BRD4 or TAF1 in the presence or absence of acetyl CoA or 500 μM JQ1. Free DNA resolved from mononucleosomes by electrophoresis (upper panel) . Immunoblots of identical HAT assays done in parallel with non-radiolabeled mononucleosomes, showing extent of acetylation of H3K122 and H3K9; histone H3 immunoblot served as control (lower panels) . (b) HAT assays and immunoblots as in (a) with or without BRD4, BRD4 HAT mutants or TAF1 (500ng). (c) Ethidium Bromide stained agarose gels showing native chromatin isolated from U2OS cells that was incubated in HAT assays with or without increasing amounts of BRD4 (Lane 3: 1 μg; Lane 4, 5, 6: 0.2, 0.5, 1 μg respectively; Lane 7: 1μg), Acetyl CoA and 500 μM JQ1 (lane 7) and then digested with 0.1U MNase for 3 min (left panel). Quantification of digested bands showing increased MNase digestion correlated with increasing BRD4 (right panel) . (d) Ethidium Bromide stained agarose gels showing native chromatin isolated from U2OS cells transfected with 3μg hBRD4 (BRD4 WT), hBRD4 HAT mutant (Mt BRD4) or pCMV2 vector (control) in the presence of sodium butyrate and incubated with or without 0.1U MNase for 3 min (middle panel). Quantification of digested bands from cells transfected with vector (1), BRD4 WT (2) and Mt BRD4 (3) (right panel) . Immunoblots of WCEs from the transfected cells showing H3K122ac, H3K9ac and BRD4 levels (left panel). Uncropped images of gels and immunoblots are shown in Supplementary Data Set 1

Article Snippet: Purified 5S rDNA (208bp) was from New England Biolabs (NEB).

Techniques: In Vitro, HAT Assay, Electrophoresis, Western Blot, Staining, Isolation, Incubation, Transfection, Mutagenesis, Plasmid Preparation