Characterization of the human promoter and its regulation by the transcription factor E2F1-1

Gether with either one of two reverse primers targeting exon 3 (upper panel). First-strand cDNA was subjected to RT-PCR analysis with the indicated primers, and reaction products were visualized by ethidium bromide staining (lower panel). The lengths of the marker bands are indicated in bp. Expected fragment lengths are 192 bp, 232 bp, 216 bp and 256 bp for lanes 1–4. Arrows point to PCR products that were cloned and sequenced.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-9

Gether with either one of two reverse primers targeting exon 3 (upper panel). First-strand cDNA was subjected to RT-PCR analysis with the indicated primers, and reaction products were visualized by ethidium bromide staining (lower panel). The lengths of the marker bands are indicated in bp. Expected fragment lengths are 192 bp, 232 bp, 216 bp and 256 bp for lanes 1–4. Arrows point to PCR products that were cloned and sequenced.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-7

Onserved in the mouse, dog, and platypus genomes and comprises a full CRE sequence (with one mismatch to the canonical octamer) and a CRE half-site (boxed). B) Forskolin stimulates phosphorylation of CREB on Ser133. PC12 cells were treated for 30 min with forskolin (10 μM) or DMSO (Ctrl) before nuclear extracts were prepared. Phospho-Ser133 in CREB was detected by Western blot analysis with the help of a phosphospecific antibody. Migration of molecular mass standards is indicated in kDa. C) Northern blot. PC12 cells were treated for 24 h with forskolin (10 μM) or DMSO as the vehicle (Ctrl) before total RNA was isolated and subjected to Northern blot analysis with probes for DYRK1A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and . Duplicate lanes contain RNA samples from parallel plates. Migration of the ribosomal RNAs is indicated in the right margin (28S, 18S). D) Luciferase assays. The indicated cell lines (PC12, PC3, Saos2) were transfected with the reporter gene constructs as schematically depicted. Luciferase activity was determined from cells treated with forskolin (10 μM) for 24 h and from untreated cells. Data were normalized to β-galactosidase activity and are presented as fold induction relative to untreated cells. Bars reflect the means +/- SD of 3 independent experiments (Saos2, PC3) or the means of duplicate wells for the experiment with PC12 cells.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-5

He human DYRK1A promoter as indicated. Five hours after transfection, the medium was changed and cells were treated with doxycycline or were not treated. Data were normalized to β-galactosidase activity and are expressed as fold stimulation relative to untreated cells. The diagram integrates results of 2–6 independent experiments, and bars reflect the means +/- SD. B) Luciferase constructs of the human and the murine promoter were analyzed for upregulation by doxycycline-induced overexpression of E2F1. Data were normalized to β-galactosidase activity and are presented as the ratio relative to the activity of the unstimulated murine -660 construct. Bars reflect the means +/- SD of 3 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-3

Iferase cDNA in the vector pGL3basic. The position of the promoter fragments relative to TSS B (+1) is indicated. Segments upstream of TSS A and TSS B are highlighted in red and blue, respectively. Saos2 cells were co-transfected with the indicated reporter gene constructs and a β-galactosidase reporter control plasmid. Cells were lysed 48 h after transfection, and luciferase activities were determined from duplicate wells. Data were normalized to β-galactosidase activities and are presented as the ratio relative to the activity of the -742 construct. The diagram integrates results of 2–6 independent experiments. Bars reflect means +/- SD. The inset shows a magnified representation of the weakest signals.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-0

Red (1M, 1A, 1B, 2 and 3) and drawn to scale. Open boxes are noncoding regions, filled boxes represent the coding sequence. Open boxes with dotted lines are facultative exonic sequences that have been described in previous reports. Numbering of nucleotides in the promoter region refers to the 5'-end of transcript MNBHa which has previously been defined as the transcription start of the human gene [19] and corresponds to the transcript variant 3 (NM_101395) in the NCBI database. Position +1 corresponds to hchr21:37,661,729 in the assembly at the UCSC genome browser (release hg18). A CpG island identified by Yamada . [33] is indicated, with the bar representing the region that was analyzed in this report and found to be unmethylated. transcript variants that were previously described by Guimera et al. 1999 [19] or Wang et al. 1998 [32](database accession numbers are given in brackets) or were identified by 5'-RACE in the present study () are depicted thereunder. Positions of the primers used for RACE are indicated by arrows.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p

Characterization of the human promoter and its regulation by the transcription factor E2F1-6

Le E2F1 binding sites. B) The inducible E2F1-expressing Saos2 cells were transfected with the -742 promoter construct or the mutated versions thereof as indicated. Five hours after transfection, the medium was changed and cells were treated with doxycycline or were not treated. Data were normalized to β-galactosidase activity and are presented as the ratio relative to the activity of the unstimulated wild type construct. Luciferase activity of induced cells was significantly different from that in untreated cells (two-sided t test, p < 0.05, except for the double mutant) but there was no significant difference between wild type and mutants. Bars reflect the means +/- SD of 3 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p