ICH guideline E2F on development safety update report. June 2008: Adoption by CHMP for release for consultation : June 2008. Template for the qualified person's declaration equivalence to EU GMP for Investigational Medicinal Products. The RIP140 Gene Is a Transcriptional Target of.
E2F-6 CRISPR Knockout and Activation Products (m) are gene editing systems designed to knockout or upregulate gene expression of mouse E2F-6. E2F-4 CRISPR Knockout and Activation Products (h) are ready to use gene editing systems designed to knockout or upregulate gene expression of human E2F-4. 4 Supported by a fellowship from the Fond de Recherche sur la Nature et les.
The RIP1. 40 Gene Is a Transcriptional Target of E2. F1. Abstract. RIP1.
Source of information about advances in the causes, diagnosis, treatment, and prevention of cancer.
We demonstrate here that RIP1. E2. F1 transcription factor. Bioinformatics analysis, gel shift assay, and chromatin immunoprecipitation demonstrate that the RIP1. E2. F response elements. In transiently transfected MCF- 7 breast cancer cells, the RIP1.
E2. F1/DP1. Interestingly, RIP1. RNA is finely regulated during cell cycle progression (5- fold increase at the G1/S and G2/M transitions).
The positive regulation by E2. F1 requires sequences located in the proximal region of the promoter (. Finally, we show that E2. F1 participates in the induction of RIP1. Altogether, this work identifies the RIP1. E2. F1 which may explain some of the effect of E2.
F1 in both cancer and metabolic diseases. Citation: Docquier A, Augereau P, Lapierre M, Harmand P- O, Badia E, Annicotte J- S, et al. PLo. S ONE 7(5). e. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Institut National de la Sant! The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
Introduction. RIP1. Receptor Interacting Protein of 1. Da also known as NRIP1) is a nuclear protein of 1. The molecular mechanisms involved in this transrepression implicate several repressive domains within the RIP1.
HDACs and Ct. BPs. Several post- translational modifications (such as acetylation, methylation and conjugation to SUMO peptides or vitamin B6) also play key roles in the regulation of RIP1. Interestingly, we previously demonstrated that RIP1. Cloning of the human . For instance, this gene is required for a proper oocyte release during ovulation and involved in the regulation of fat accumulation and energy homeostasis in metabolic tissues. We recently reported that its expression is significantly decreased in basal- like breast cancers as compared to luminal ones .
We also demonstrated that RIP1. E2. Fs . E2. F1, the founding member of the E2. F family, has been shown to possess oncogenic properties and numerous evidences show that deregulation of E2. F activity plays a key role in tumorigenesis . In addition to its effect on cell cycle progression, E2. F1 can also induce apoptosis through p.
More recently, a clear implication of E2. F1 has been demonstrated in different metabolic processes including lipid and adipocyte metabolism or glucose homeostasis (for a review see . Most of the E2. F family members exhibit structural conserved features such as a DNA- binding domain and hydrophobic heptad repeats which allow heterodimerization with DP proteins (DRTF1 polypeptides DP1, DP2 and DP3) through coil- coil interactions. In their C- terminus moiety, E2. F1 to 5 exhibit a transactivation domain whose activity is tightly regulated by different post- translational modifications (such as phosphorylation and acetylation) and through the binding of pocket proteins (p. RB, p. 10. 7 and p.
E2. F transcriptional activity is controlled by a plethora of factors . The association with pocket proteins allows active repression through the recruitment of histone deacetylases and methyltransferases and is finely regulated by various members of the cyclin/cdk family .
Our data identify the RIP1. E2. F1 that might be involved in a wide range of pathological processes regulated by this transcription factor such as cancer or metabolic diseases. Materials and Methods.
Plasmids and Reagents. The pc. DNA3- E2.
F1 and 4, p. CMV- DP1, p. CMV- Rb expression vectors were given by Dr C. Sardet (IGMM, Montpellier, France) and the p. CMV- E2. F2, 3, 5 and E2. F6 expression vectors by Dr K. Helin (European Institute of Oncology, Milan, Italia). The reporter plasmids (Cyclin.
E- luc and (E2. F)3- TK- Luc) were obtained from Dr L. Fajas (IRCM, Montpellier). The p. EFc- myc. RIP1.
RIP1. 40 promoter (RIP9. The p. RL- CMVBis plasmid expressing Renilla luciferase (Stephan Vagner, Toulouse, France) was used to normalize transfection efficiency. The deletion of the Sp. CDNA3 E2. F1 sequence (from residues 1. The same protocol was used to generate DP1 mutants in the p. CMV expression vector (DP1 .
Potential transcription factor binding elements in the promoter region of both promoters were searched using Genomatix Mat. Inspector Program (www. Alignment of human and mouse NRIP1 promoter sequences and identification of the evolutionary conserved transcription factor binding elements were performed using Genomatix Di. Align Program (www. Cell Culture. MCF- 7 and He. La human cancer cell lines were purchased from the American Type Culture Collection and routinely maintained in the laboratory as previously described . For synchronization, 2.
When cells were at 8. At each time point, cells were washed, trypsinized and total RNA was extracted. Cell cycle analysis was performed using a FACs- Vatange flow cytometer (Becton- Dickinson, San Jose, CA).
For each sample, 2. Cell. Quest software was used to analyze the list- mode data. Mod. Fit software was used to determine the percentage of each G0/G1, S and G2/M phase in the population. Gel- Shift Assay. Gel shift assays were performed as previously described .
Sequences of sense strand oligonucleotides are given in Table S1. Where indicated, anti- E2. F1 antibody (Ab) was added in the incubation mixtures, 5 min before the radioactive probe.
Complexes were separated 1. The Champion. Ch. IP One- Day Kit (SABiosciences) was then used according to the manufacturer’s recommendations.
Immunoprecipitations were performed using the KH9. Santa. Cruz), C- 2.
Santa- Cruz) or 2. C6a (sc- 8. 13. 70 Santa- Cruz) antibodies against E2. F1, E2. F4 and RIP1. It should be noted that although the anti- E2.
Fs antibody were given by the supplier as being specific, we could not eliminate with certainty the possibility that they may cross- react with other E2. Fs. As a negative control, we performed IP with no antibody (in our hands, such IP controls gave the same background as an isotype- matched m.
Ab). Quantitative PCR was then performed using the Power SYBR Green PCR master mix, on an Applied Biosystems 7. Primers flanking the E2.
F site of the RIP1. A2 promoters are given in Table S1. The input DNA fraction corresponded to 1% of the immunoprecipitation. Transient Transfection and Luciferase Assays.
MCF- 7 cells were plated in 9. RLCMVBis (2. 5 ng per well) used as internal standard, and 2. E2. F1, 2, 3, 4, 5, 6 and DP1) and 2. RIP1. 40, p. Rb, p.
Renilla luciferase activity; all triplicate point values were expressed as mean + SD. Western- blot Analysis. Expression plasmids for E2.
F1, 2, 3, 4 or DP1 were transfected in MCF- 7 cells using Jet. PEI. After cell lysis in 2. M Tris- HCl, p. H7. M EDTA, 2 m. M DTT, 1. Glycerol, 1% Triton, supplemented with protease inhibitors, whole cell extracts were diluted in Laemmli sample buffer 2. X and resolved by SDS–PAGE.
Western blotting detection was performed using primary antibodies against E2. Fs (sc- 2. 51, sc- 9. E2. F1, 2, 3, 4 respectively and sc- 5. DP1 from Santa- Cruz Biotechnology). To check for gel loading, we used the anti- actin (A2. Sigma) or anti- TBP (MA1- 2. Thermo. Scientific) antibodies.
RNA Extraction and Quantitative PCRTotal RNA was extracted from cells as previously described . PCR were carried out in a final volume of 1. After a 1. 0 min preincubation at 9. Melting- curves of the PCR products were analyzed using the Light. Cycler software system to exclude amplification of unspecific products. Results were corrected for RS9 m.
RNA levels (reference gene) and normalized to a calibrator sample. Adipocyte Differentiation Experiments. E2. F1. Three different MEF cultures were obtained from E2. F1 wild- type and knock- out embryos and grown in F1. DMEM supplemented with 1.
HEPES. For adipocyte differentiation experiments, MEFs (at passage 3) were seeded in 6- well plates. Two days after confluence, differentiation was induced by treating cells for 2 days with a cocktail containing 0. M IBMX (3- isobutyl- 1- methylxanthine), 1.
Every two days, medium was changed with F1. DMEM, 1. 0% serum, 1. HEPES, 1. 0 . Differentiated cells were visualized with Oil Red O staining (Sigma). Nuclear protein extracts were prepared using the NE- PER kit (Thermo Scientific) and 2.
Total RNA was extracted using the Quick- RNA Miniprep (Zymo Research) and 1 . Results were corrected for RS9 m. RNA levels used as a reference gene. Statistics. Statistical analysis was performed using the Student t test except for Figure S1 where we used the paired t- test (*p< 0. Results. Identification of Bona Fide E2.
F Binding Sites in the RIP1. Promoter. We previously reported the cloning and characterization of the human RIP1. Upon close inspection of the proximal promoter region, we mapped several putative E2. F binding sites resembling the consensus sequence TTTSGCGCS. In the human RIP1. These sites spread into two clusters with sites a and b being in the distal part of the promoter and sites c, d and e around the transcription start site, all being flanked by putative Sp. It should be noted that the E2.
Fa site exhibited the sequence which is the closest to the consensus motif with only one nucleotide change and that the E2. Fe site was in fact a composite site with three different possibilities to bind the E2. F/DP heterodimers (Figure 1. A). Interestingly, the murine RIP1. E2. F binding sites, the E2. Fd site being perfectly conserved in term of position and sequence as compared to the human promoter (Figure 1 B and C).
Figure 1. The human and mouse promoters exhibit 4 to 5 potential E2. F binding sites (grey square) with a conserved distribution. Bioinformatics analysis also identified Sp. Sp. 1 sites) and in the human promoters (8 sites in two clusters). Using gel shift experiments, we demonstrated that E2.