Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Epigenetic modulation of intestinal Na + /H + exchanger-3 expression

doi: 10.1152/ajpgi.00293.2017

Figure Lengend Snippet: Effect of DNA methylation on Na+/H+ exchanger-3 (NHE3) promoter activity in Caco-2 cells. A: human (h) NHE3 promoter region flanking the area between −1509 and +127 (+1 is the transcription initiation site) was amplified by PCR and cloned in a promoterless cytosine guanine dinucleotide (CpG)-free vector upstream of the lucia gene (pCpG-EV). B: CpG-free EF1 promoter lucia vector (pCpG- hEF1) was used as control for methylation. The NHE3 promoter construct (pCpG-NHE3) and the control vector (pCpG- hEF1) were subjected to in vitro DNA methylation. The methylated and unmethylated control vector and NHE3 promoter construct were transiently transfected in Caco-2 cells along with the mammalian expression vector of β-galactosidase. Posttransfection (48 h), cells were harvested, and lucia activity and β-galactosidase activity were measured. The relative promoter activity was determined as a ratio of lucia to β-galactosidase. Results represent means ± SE of 3–4 separate experiments and are expressed as % of control comparing methylated control vector (pCpG-hEF1 methylated) or methylated NHE3 promoter construct (pCpG-NHE3 methylated) with unmethylated control vector (pCpG-hEF1) or unmethylated NHE3 promoter construct (pCpG-NHE3). **P < 0.01 compared with unmethylated vector/construct as assessed by Student's t-test. C: effect of 5-azacytidine on DNA methylation in the promoter region of NHE3 in Caco-2 cells: DNA was extracted from Caco-2 cells treated with or without 5-azacytidine (10 μM) for 48 h. DNA methylation was determined by qPCR after MethyMiner precipitation using specific primers described in materials and methods, normalized to DNA input, and expressed as fold change. Results represent means ± SE of 3–4 separate experiments. **P < 0.01 compared with untreated cells (control) as assessed by Student's t-test.

Article Snippet: A pCpG-free lucia vector with EF1 promoter (Invivogen) was used as a control for the methylation experiment.

Techniques: DNA Methylation Assay, Activity Assay, Amplification, Clone Assay, Plasmid Preparation, Methylation, Construct, In Vitro, Transfection, Expressing

Journal: International Journal of Neuropsychopharmacology

Article Title: Plasticity of Functional MAOA Gene Methylation in Acrophobia

doi: 10.1093/ijnp/pyy050

Figure Lengend Snippet: Functional analysis of MAOA promoter/exon I/intron I DNA methylation using luciferase-based reporter gene assays. Left: Normalized reporter gene activity was significantly decreased in the presence of pCpGfree-promoter Lucia_MAOA vectors containing the methylated insert spanning CpGs 1–13 compared with those carrying a nonmethylated insert; *** P < .001. Right: No significant difference in normalized reporter gene activity was discerned between methylated or non-methylated pCpGfree-promoter Lucia control vectors lacking the insert of the sequence spanning CpGs 1–13.

Article Snippet: Functional analysis was accomplished using the pCpGfree-promoter- Lucia vector (InvivoGen) expressing a Lucia luciferase under a human elongation factor-1 (hEF1) promoter.

Techniques: Functional Assay, DNA Methylation Assay, Luciferase, Activity Assay, Methylation, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *

doi: 10.1074/jbc.M113.546283

Figure Lengend Snippet: Effect of DNA methylation on NPC1L1 promoter activity. Human NPC1L1 promoter region flanking the area between −1741 and +56 (+1 is the transcription initiation site) was amplified by PCR and cloned into a promoterless CpG free vector (pCpG free basic lucia) upstream of the lucia gene. The NPC1L1 promoter construct as well as the empty vector were then subjected to in vitro methylation. The methylated and unmethylated empty vector (pEV) and NPC1L1 promoter construct (L1p) were transiently transfected into Caco2 cells along with the mammalian expression vector of β-galactosidase. 48 h post-transfection, cells were harvested, and lucia activity and β-galactosidase activity were measured. The relative promoter activity was determined as a ratio of lucia to β-galactosidase (A). Data are presented as fold increase compared with unmethylated empty vector and represent mean ± S.E. of three independent experiments performed on separate occasions. *, p < 0.05 compared with empty vector pEV. CpG free EF1 promoter lucia vector (pCpG free lucia) was used as control for methylation as described under “Results” (B). Data are presented as fold change relative to unmethylated vector.

Article Snippet: For the construction of pCpG free L1 vector, the DNA fragment containing −1741/+56 region of the human NPC1L1 promoter was amplified using forward 5-ATCGATGCGAGTACTTGGACTCTATCTCTCTGTGG-3′ and reverse 5-ATCGATGCGAAGCTTCCCAGGTCTGGGAAGGGGTCA-3′ primers, and the amplified promoter fragments were then inserted into a promoterless CpG free vector (pCpG free basic lucia, InvivoGen, San Diego) upstream of the lucia reporter gene.

Techniques: DNA Methylation Assay, Activity Assay, Amplification, Clone Assay, Plasmid Preparation, Construct, In Vitro, Methylation, Transfection, Expressing, Control