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: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: GRN expression in human lymphoblast cell lines is inversely correlated to its promoter methylation. ( a ) GRN net secretion was measured by ELISA in LCLs derived from neurologically healthy individuals (LCL #1-13), unaffected relatives of FTLD patients (LCL #14, 15, highlighted in blue) and FTLD-patients (LCL#16, 17, highlighted in red). n = 3, mean ± SEM. ( b ) Scheme of GRN promoter region. Red bars depict PCR-amplicons analyzed for DNA methylation levels by MassARRAY. Blue bars indicate full length and short promoter region that was cloned into the pCpGL vector for luciferase assays (compare Figure ). White circles display CpG units in amplicons A-1 to A-5 and A-DAC quantified by MassARRAY. CpG units that were not analyzed are not shown. Asterisks indicate significant correlation between GRN mRNA expression or GRN secretion and GRN methylation at respective CpG unit (*p < 0.05, linear regression analysis, Benjamini Hochberg multiple testing and FDR correction, compare Additional file : Table S3). ( c ) Average DNA methylation levels in amplicons A-1 to A-5 for individual LCLs are plotted. Mean ± SD. Color code as in (a). ( d ) Correlation between GRN mRNA expression and average DNA methylation at CpG units 1, 2, 6, 8 and 11 is shown. GRN mRNA expression was quantified by qPCR and normalized to PGK1 and GAPDH. Relative mRNA expression levels were plotted against average DNA methylation levels. Correlation between parameters was quantified by linear regression analysis, r 2 and p-values are given. Color code as in (a). ( e ) Correlation between GRN secretion and average DNA methylation at CpG units 1, 2, 6, 8 and 11. GRN secretion was determined by ELISA and relative units (R.U.) were plotted against average DNA methylation levels. Correlation between parameters was quantified by linear regression analysis, r 2 and p-values are given. Color code as in (a).

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: Expressing, Methylation, Enzyme-linked Immunosorbent Assay, Derivative Assay, DNA Methylation Assay, Clone Assay, Plasmid Preparation, Luciferase

Journal: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: DNA methylation inhibits GRN promoter activity at distinct CpG units. In vitro methylated and unmethylated pCpGL plasmids containing the GRN core promoter region driving expression of firefly luciferase were transiently cotransfected into ( a ) HEK 293FT cells and ( b ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. The full length GRN promoter pCpGL plasmid, a GRN promoter construct with site specific mutations of the significant CpG units in amplicons A-1 and A-2, and a short GRN promoter construct lacking amplicons A-1 and A-2 were transiently cotransfected into ( c ) HEK 293FT cells and ( d ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. Luciferase reporter activity was measured 48 h (a + c, HEK 293FT) or 72 h (b + d, neurons) after transfection. Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. The empty vector was used as negative control. Mean ± SEM, n ≥ 3. ***p < 0.001, Student’s t-test, sign . significant.

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: DNA Methylation Assay, Activity Assay, In Vitro, Methylation, Expressing, Luciferase, Plasmid Preparation, Construct, Transfection, Negative Control

Journal: Acta Neuropathologica Communications

Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

doi: 10.1186/2051-5960-1-16

Figure Lengend Snippet: Overexpression of DNMT3a alters GRN promoter activity in primary cortical neurons and reduces GRN mRNA expression in LCLs. ( a ) pCpGL plasmid containing the GRN core promoter and a DNMT3a overexpression construct were transiently transfected in HEK 293FT cells (left panel) and in rat primary cortical neurons (right panel). Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. Empty vectors were used as negative control. Firefly luciferase expression was significantly reduced upon DNMT3a overexpression. Mean ± SEM, n ≥ 3. ***p < 0.001, ANOVA with Tukey’s Multiple Comparison test. ( b ) Lentiviral expression of DNMT3a in LCLs #3 and #14. Overexpression was verified by qPCR five days after viral transduction. n = 5, mean ± SEM, *p < 0.05, Student’s t-test. ( c ) GRN mRNA expression levels were significantly reduced in DNMT3a overexpressing LCLs as quantified by qPCR and normalized to PGK1 expression levels. n = 5, mean ± SEM, *p < 0.05, Student’s t-test.

Article Snippet: The GRN promoter region (−2423 to +207 bp relative to transcriptional start site, for primers see Additional file : Table S1) was cloned directly into the CpG-free vector pCpGL and in vitro methylated using recombinant DNA methyltransferase M.SssI (New England Biolabs).

Techniques: Over Expression, Activity Assay, Expressing, Plasmid Preparation, Construct, Transfection, Luciferase, Negative Control, Comparison, Transduction

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

Journal: Molecular and Cellular Biology

Article Title: Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding

doi: 10.1128/MCB.00436-19

Figure Lengend Snippet: Estrogen inducibility of VTG-CpGL luciferase reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S-adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The enhancer/promoter region of the chicken vitellogenin II gene, spanning nucleotides −638 to −1, was cloned into a CpG-free firefly luciferase plasmid (pCpGL-basic; InvivoGen) using HindIII and NcoI.

Techniques: Luciferase, Plasmid Preparation, Transfection, Expressing, In Vitro, Methylation, Sequencing, Purification, Quantitative RT-PCR, Isolation, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: MBD2 Directly Binds to CpG Islands of Promoter of PKCη and Positively Activates Transcription of Them by Hypomethylation of the Promoter (A) The patterns of CpG islands of the PKCη promoter and five pairs of the primer were predicted by the software of MethPrimer Promoter2.0. (B) ChIP assays represent the binding sites of MBD2 interaction with CpG islands of the promoter of PKCη. (C) Relative luciferase activity of MBD2 or MBD2 mutation plasmids cotransfected with the methylated PKCη pCpGI plasmid in BUMPT cells. (D) CpG-DNA methylation of the PKCη promoter region. (E) Immunoblot analysis of PKCη, cleaved PKCη, and GAPDH expression after LPS treatment with or without transfection of MBD2. (F) Grayscale image analysis between them after LPS treatment with or without transfection of MBD2. (G) Immunoblot analysis of PKCη, cleaved PKCη, and GAPDH expression after LPS treatment with or without transfection of MBD2 siRNA. (H) Grayscale image analysis between them after LPS treatment with or without transfection of MBD2 siRNA. These data are representative of at least four separate experiments shown as means ± SD (n = 6). #p < 0.05 versus the control group; ∗p < 0.05 versus the MBD2 plasmid or LPS group.

Article Snippet: The methylation promoter PKCη was subcloned into a CpG-free pCpGI luciferase reporter vector by Invitrogen Biotechnology (Shanghai, China).

Techniques: Software, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Methylation, Plasmid Preparation, DNA Methylation Assay, Western Blot, Expressing, Transfection