New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens
The tetracycline repressor (tetR)‐regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA‐binding protein 43 (TDP‐4...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-10-01
|
| Series: | FEBS Open Bio |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/2211-5463.13482 |
| _version_ | 1811202417568514048 |
|---|---|
| author | Valeria Colicchia Maria Häggblad Oleksandra Sirozh Bartlomiej Porebski Mirela Balan Xuexin Li Louise Lidemalm Jordi Carreras‐Puigvert Daniela Hühn Oscar Fernandez‐Capetillo |
| author_facet | Valeria Colicchia Maria Häggblad Oleksandra Sirozh Bartlomiej Porebski Mirela Balan Xuexin Li Louise Lidemalm Jordi Carreras‐Puigvert Daniela Hühn Oscar Fernandez‐Capetillo |
| author_sort | Valeria Colicchia |
| collection | DOAJ |
| description | The tetracycline repressor (tetR)‐regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA‐binding protein 43 (TDP‐43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP‐43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA‐approved drugs. While the primary screen identified several compounds that prevented TDP‐43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline‐dependent TDP‐43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet‐On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome‐wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP‐43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline‐dependent expression of TDP‐43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells. |
| first_indexed | 2024-04-12T02:39:19Z |
| format | Article |
| id | doaj.art-ec127910a33a46728af06e40f73ca107 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-04-12T02:39:19Z |
| publishDate | 2022-10-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-ec127910a33a46728af06e40f73ca1072022-12-22T03:51:23ZengWileyFEBS Open Bio2211-54632022-10-0112101896190810.1002/2211-5463.13482New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screensValeria Colicchia0Maria Häggblad1Oleksandra Sirozh2Bartlomiej Porebski3Mirela Balan4Xuexin Li5Louise Lidemalm6Jordi Carreras‐PuigvertDaniela Hühn7Oscar Fernandez‐Capetillo8Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenGenomic Instability Group Spanish National Cancer Research Centre (CNIO) Madrid SpainScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenScience for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics Karolinska Institute Stockholm SwedenThe tetracycline repressor (tetR)‐regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA‐binding protein 43 (TDP‐43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP‐43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA‐approved drugs. While the primary screen identified several compounds that prevented TDP‐43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline‐dependent TDP‐43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet‐On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome‐wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP‐43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline‐dependent expression of TDP‐43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells.https://doi.org/10.1002/2211-5463.13482ALSchemical screendoxycyclineTDP‐43tetR |
| spellingShingle | Valeria Colicchia Maria Häggblad Oleksandra Sirozh Bartlomiej Porebski Mirela Balan Xuexin Li Louise Lidemalm Jordi Carreras‐Puigvert Daniela Hühn Oscar Fernandez‐Capetillo New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens FEBS Open Bio ALS chemical screen doxycycline TDP‐43 tetR |
| title | New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens |
| title_full | New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens |
| title_fullStr | New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens |
| title_full_unstemmed | New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens |
| title_short | New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens |
| title_sort | new regulators of the tetracycline inducible gene expression system identified by chemical and genetic screens |
| topic | ALS chemical screen doxycycline TDP‐43 tetR |
| url | https://doi.org/10.1002/2211-5463.13482 |
| work_keys_str_mv | AT valeriacolicchia newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT mariahaggblad newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT oleksandrasirozh newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT bartlomiejporebski newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT mirelabalan newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT xuexinli newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT louiselidemalm newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT jordicarreraspuigvert newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT danielahuhn newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens AT oscarfernandezcapetillo newregulatorsofthetetracyclineinduciblegeneexpressionsystemidentifiedbychemicalandgeneticscreens |