Journal: Human Molecular Genetics

Article Title: Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers

doi: 10.1093/hmg/ddx188

Figure Lengend Snippet: CSC-induced changes in enhancer marks in A459 cells. (A) Each diagram displays RefSeq genes at top (if present; hg19 coordinates) and from top to bottom, ENCODE data in A549 cells: Illumina Infinium HumanMethylation450 BeadChip CpG methylation status (blue = unmethylated; orange/purple = partially methylated; the CpGs of interest are marked by black arrowheads at bottom); DNase hypersensitive sites (DHSS) as black boxes; H3K27ac and H3K4me1 profiles with called peaks underlined and peak height scale indicated. Locations of PCR primers used for ChIP assays of H3K27ac and H3K4me1 panel B are indicated by purple and green vertical lines respectively. The region highlighted in pale yellow was cloned into a basal promoter-containing luciferase gene reporter vector (pGL4.26, panel C). The positions of AHR binding sites are indicated by gray arrowheads at the bottom. (B) ChIP assays of H3K27ac and H3K4me1 in vehicle- or CSC-treated A549 cells expressed as % input. * indicates luciferase activity statistically significantly different from vehicle (P < 0.05 paired Student's t test). Bars represent mean ± SEM of samples assayed in three or more independent trials of A549 cells exposed to CSC for 48 h. (C) Luciferase reporter assays to test for CSC-regulated enhancer activity. The region highlighted in pale yellow in panel A was cloned into the basal promoter-containing luciferase vector pLG4.26. In each case we ensured that the DNase hypersensitive site closest to the hypomethylated CpG was included. The plasmids were transfected into A549 cells that were treated with vehicle or CSC at the indicated doses 24 h after transfection. A renilla expression plasmid was cotransfected for DNA quantity normalization. Luciferase activity was measured at 24 h after CSC exposure, and is indicated relative to empty vector. All expression levels were statistically significantly elevated relative to pGL4.26. Significant induction relative to vehicle is indicated by an asterisk and the fold induction is noted for 20 µg/ml CSC. Bars represent mean ± SEM of three or more independent experiments.

Article Snippet: Luciferase reporter gene assays Putative enhancer regions spanning each hypomethylated CpG and closest A549 DNase HSS were PCR amplified from normal human gDNA (Promega) and inserted upstream of the minimal promoter contained within the pGL4.26-luciferase construct (Promega).

Techniques: CpG Methylation Assay, Methylation, Clone Assay, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Transfection, Expressing

Journal:

Article Title: Molecular Characterization and Placental Expression of HERV-W, a New Human Endogenous Retrovirus Family

doi:

Figure Lengend Snippet: Definition of a putative LTR. (A) The 2,364- to 2,720-nt region of the cl.PH74 clone, identified as a putative promoter region with Signalscan 4.05, was PCR amplified and subcloned into pCAT3 reporter vector. The resulting pCAT3-3′LTR plasmid and pCAT3 control plasmid were used to transfect HeLa cells prior to CAT activity determination. Cells, HeLa cell extract; pCAT3 and pCAT3-3′LTR, extract of HeLa cells transfected with control plasmid and promoter plasmid, respectively. (B) Alignment of 5′ and 3′ UTRs of placental experimental clones with the 5′ (5-RG-28000-28872) and 3′ (3-RG-37500-38314) repeated sequences of the 28,000- to 38,314-nt fragment of human DNA sequence RG083M05. The end of the 3′-most ORF is indicated (env orf). The tandemly repeated CAAC flanking sequences are doubly underlined on DNA sequences. The 783-bp LTR consensus sequence is positioned at the bottom. The polypurine tract (PPT) upstream from the 5′ end of the LTR and the tRNATrp PBS downstream from the 3′ end of LTR are indicated. U3, R, and U5 subparts are indicated, except for the U3-R junction. Transcription factor sites determined with Signalscan 4.05 are underlined and labelled from I to VI. The sites V and VI may correspond to CCAAT box and TATA box, respectively. The 3′ region of clone cl.PH74 used in the CAT assay is underlined on the cl.PH74 sequence. ∗ and ▴, potential cap sites obtained in 5′ RACE experiments with human placental poly(A)+ RNA and total RNA extracted from pCAT-3′LTR-transfected HeLa cells, respectively. [polyA], polyadenylation signal.

Article Snippet: The putative promoter region was cloned into pCAT3 Enhancer reporter vector purchased from Promega Biotec (Madison, Wis.).

Techniques: Amplification, Plasmid Preparation, Activity Assay, Transfection, Clone Assay, Sequencing