Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes
| Autor: | Reilly McCracken, Cassandra M Barrett, Karmella A. Haynes, Jacob Elmer |
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| Rok vydání: | 2019 |
| Předmět: |
Transcription
Genetic Polycomb-Group Proteins MYB Regulatory Sequences Nucleic Acid Epigenesis Genetic lcsh:Chemistry chemistry.chemical_compound 0302 clinical medicine Genes Reporter Heterochromatin Transcriptional regulation Transgenes lcsh:QH301-705.5 Spectroscopy 0303 health sciences food and beverages General Medicine epigenetic silencing Computer Science Applications 3. Good health Chromatin Cell biology 030220 oncology & carcinogenesis Transcription Initiation Site Protein Binding Transcriptional Activation animal structures Transgene Biology Models Biological Catalysis Article activator Inorganic Chemistry 03 medical and health sciences Proto-Oncogene Proteins c-myb c-myb Humans Gene Silencing Physical and Theoretical Chemistry Molecular Biology 030304 developmental biology Binding Sites Base Sequence Organic Chemistry HEK 293 cells fungi transgene Fusion protein DNA binding site lcsh:Biology (General) lcsh:QD1-999 chemistry Gene Expression Regulation Trans-Activators polycomb DNA |
| Zdroj: | International Journal of Molecular Sciences Volume 21 Issue 2 International Journal of Molecular Sciences, Vol 21, Iss 2, p 530 (2020) |
| ISSN: | 1422-0067 |
| Popis: | A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell&rsquo s genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines. |
| Databáze: | OpenAIRE |
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