Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Studying the effects of the TEN1-ICD on transcriptional regulation

ABSTRACT: Teneurins are large type II transmembrane proteins that are necessary for the normal development of the central nervous system (CNS). While many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus where it can influence gene transcription. Target genes as well as mechanisms have yet to be elucidated, and thus we are investigating the transcriptional activity of the human teneurin-1 ICD in this study. For the whole transcriptome analysis of TEN1-ICD overexpression, we used a modified and improved tet-system. Two separate vectors are required to make the cell line stable. One contains the tet-activating domain fused to a glucocorticoid binding domain (GBD). The other contains the tetO operator sequences directly upstream of a CMV promoter and the gene to be overexpressed. BS149 cells were first transfected with pirtetR-GBD and made stable by Puromycin selection, and then after further transfection with either ptetO-eGFP-His (negative control) or ptetO-TEN1-ICD-eGFP-His by Hygromycin selection. The stable BS149 cell lines were split into three 10 cm Petri dishes each. The triplicate cell lines were cultured once before induction with Dexamethasone and Doxycycline. The overexpressing cells were then FACS-sorted directly into RLT lysis buffer (Qiagen) at a 3:1 volume ratio of lysis buffer to cells in PBS, 24 h post-induction.

ORGANISM(S): Homo sapiens

SUBMITTER: Tim Roloff

PROVIDER: E-GEOD-61704 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor (MITF) by binding to transcriptional repressor HINT1.

Schöler Jonas J Ferralli Jacqueline J Thiry Stéphane S Chiquet-Ehrismann Ruth R

The Journal of biological chemistry 20150203 13

Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Becau ...[more]

PMID: 25648896

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Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

Dataset Information

Studying the effects of the TEN1-ICD on transcriptional regulation

Publications

The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor (MITF) by binding to transcriptional repressor HINT1.

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