Regulation of gene expression in a spatiotemporal manner specifies cellular identity. Transcription factors (TFs) bind to DNA regulatory elements to remodel chromosome structure, to recruit transcription machinery to initiate gene transcription or to prevent the assembly of such machinery to repress gene transcription, thus they lie at the heart of gene regulation. Given important roles of TFs in gene regulation, numerous attentions have been attracted for engineered transcription factors (eTFs). The recent advance of generating customized DNA-sequence specific binding domains, including transcription activator-like effectors (TALEs) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene Cas9, has greatly accelerated the study and application of eTFs. The eTFs with these new binding domains offer a powerful and precise approach for modulating gene expression.
Oct4 is an important TF and it plays essential roles in the formation of inner cell mass during embryogenesis, and the maintenance of embryonic stem cells in culture as well as the reinstatement of cellular pluripotency from somatic cells.
In this study, we systematically investigated the potential of TALE-TFs and CRISPR /Cas9-TFs in activating Oct4. We designed a number of TALEs and small guide RNAs (sgRNAs) targeting various regions in the mouse and human Oct4 promoters. Using luciferase assays, we found that the most efficient TALE-VP64s bound on the region -120 to -80 bp upstream of transcription start site (TSS), while highly effective sgRNAs targeted -147 to -89 bp upstream of TSS to induce high activity of luciferase reporters. This positional effect can serve as a simple guideline for designing eTFs for activating transcription from a reporter system. Next, we examined the potential of TALE-VP64 and sgRNAs to activate endogenous Oct4 transcription. We found that the positional effect was less obvious as individual eTFs exhibited marginal activity to up-regulate endogenous gene expression. Interestingly, we found that when multiple eTFs were applied simultaneously, Oct4 could be induced significantly and synergistically. This phenomenon was well supported by activation of human SOX2, KLF4, cMYC, CDH1 and NANOG by TALE-VP64s.
Using optimized combinations of TALE-VP64s, we successfully enhanced endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64- and sgRNA/dCas9-VP64-induced transcription of endogenous OCT4. Taken together, this study demonstrated that engineered TALE-TFs and dCas9-TFs are useful tools for modulating gene expression in mammalian cells.
|Advisor:||Yee, Chan Wood|
|School:||The Chinese University of Hong Kong (Hong Kong)|
|School Location:||Hong Kong|
|Source:||DAI-B 76/08(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Systematic, School administration|
|Keywords:||Cas9, Oct4, P300, TALE, Transcription factor, Vp64|
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