|
| Status |
Public on Jun 09, 2016 |
| Title |
A positive regulatory loop between a Wnt-regulated non-coding RNA and ASCL2 controls intestinal stem cell fate |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. Despite significant recent inroads, the full complement of Wnt target genes and the mechanisms of regulation remain incompletely understood. Here we identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its close neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition/physical contact of its own promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 expression. This feedforward regulatory loop controls stem cell-related gene expression and is highly amplified in colorectal cancer.
|
| |
|
| Overall design |
Derivatives of Ls174T colon cancer cells, overexpressing the Tet repressor were used for the construction of inducible overexpressing a shRNA against the WiNTRLINC1 long non coding RNA upon treatment with doxyxycline. siRNAs against WiNTRLINC1 were designed with the siDesign center tool from Dharmacon and their sequences were used for the construction of the shRNA stem loop structure as described in EMBO Rep. 2003 Jun;4(6):609-15. The modified pTER vector was used as a backbone for constructing the shRNA cassette as described in EMBO Rep. 2003 Jun;4(6):609-15. Positive cell clones were screened with RT-PCR in order to validate the efficiency of the knockdown of WiNTRLINC1. The Ls174T derivative cell line inducibly overexpressing a shRNA against ASCL2 has been described previously in Cell. 2009 Mar 6;136(5):903-12. RNA deep sequencing was performed in the WiNTRLINC1 KD and ASCL2 KD cells compared to controls cells in order to detect changes in gene expression due to the loss of either WiNTRLINC1 or ASCL2.
|
| |
|
| Contributor(s) |
Giakountis A, Moulos P, Zarkou V, Oikonomou C, Harokopos V, Hatzigeorgiou AG, Reczko M, Hatzis P |
| Citation(s) |
27292638 |
| |
| Submission date |
May 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Pantelis Hatzis |
| E-mail(s) |
hatzis@fleming.gr
|
| Organization name |
Biomedical Sciences Research Center 'Alexander Fleming'
|
| Department |
Molecular Biology and Genetics
|
| Street address |
34 Fleming Str
|
| City |
Vari |
| State/province |
Attiki |
| ZIP/Postal code |
16672 |
| Country |
Greece |
| |
|
| Platforms (1) |
| GPL17303 |
Ion Torrent Proton (Homo sapiens) |
|
| Samples (8)
|
|
| Relations |
| BioProject |
PRJNA284373 |
| SRA |
SRP058490 |
Less...
More...