Artificial transcription factor
Artificial transcription factors (ATFs) are engineered individual or multi molecule transcription factors that either activate or repress gene transcription.[1]
ATFs often contain two main components linked together, a DNA-binding domain and a regulatory domain, also known as an effector domain or modulatory domain.[1] The DNA-binding domain targets a specific DNA sequence with high affinity, and the regulatory domain is responsible for activating or repressing the bound gene.[1] The ATF can modify the DNA structure directly, recruit proteins to compact or loosen the DNA wrapping around histones, which inhibits or allows RNA polymerase from binding and transcribing the DNA, or affect other transcription factors.[1][2] Because ATFs are composed of two separable components, the DNA-binding domain and the regulatory domain, the two domains are interchangeable permitting the design of new ATFs from existing natural transcription factors.[1]
It is also possible to downregulate expression of a gene by targeting the 5' untranslated region with a DNA-binding domain that lacks a regulatory domain; this will reduce transcription simply by blocking RNA polymerase progression along the DNA template.
A library of ATFs has been created and used to induce pluripotency in mouse embryonic fibroblasts.[3]
ATF Design
DNA-Binding Domain
The DNA-binding domain routes the ATF to a specific gene sequence. Natural DNA binding proteins are commonly used because of their high affinity for their DNA target sequence, however no algorithm that matches the protein amino-acid sequence to the complementary DNA binding sequence exists, limiting the rational design of new DNA-binding proteins.[4] Non-peptide, oligonucleotide, and polyamide DNA-binding domains have recently been explored which permit rational design.[4]
Regulatory Domain
The regulatory domain is responsible for activating or repressing the bound gene and accomplishes this regulation by directly altering the DNA structure, recruiting proteins to compact or loosen the DNA wrapping around histones, which inhibits or allows RNA polymerase from binding and transcribing the DNA, or affecting other transcription factors.[5][6] Regulatory domains promoting gene transcription are usually acidic activators, composed of acidic and hydrophobic amino acids, and regulatory domains repressing gene transcription usually contain more basic amino acids.[5] Factors influencing the effect the ATF has on transcription includes the distance the regulatory domain is from the transcription site, the cell type, and the number of activating or repressing sequences present in the regulatory domain.[5] Activating domains, regulatory domains that promote gene transcription, are often capable of upregulating transcription by 5 to 40-fold and RNA regulatory domains have been shown to result in 100 fold transcription levels.[5] An alternative strategy for repressing genes is for the ATF to out compete natural transcriptions factors and physically block transcription by RNA polymerase; however, creating ATFs with higher affinity for the DNA sequence than the natural transcription factors remains a challenge.[5]
Linkers
Linkers covalently or non-covalently link the DNA-binding domain and regulatory domain.[7] Frequently, peptide linkers are used, but Polyethylene glycol and small molecules linkers also exist.[7] The linkers enable the DNA-binding domains and regulatory domains to be interchangeable allowing the design of new ATFs from natural transcription factor components.[7] The linker length is important because it alters the extent of impact the regulatory domain has on gene expression.[7]
ATF DNA-Binding Domain
CRISPR-Cas
The clustered regularly interspaced short palindromic repeats - Cas (CRISPR-Cas) system has been extensively studied to target a specific DNA sequence using a single guide RNA (sgRNA).[8] For ATF applications the CRISPR-Cas system is modified to inactivate the Cas enzyme's natural function and link a regulatory domain to the Cas enzyme.[9] The CRISPR-Cas system benefits from high specificity between the sgRNA and the target DNA sequence and the simplicity of designing new sgRNAs; however, the CRISPR-Cas system requires a PAM sequence directly upstream of the target DNA site and the large size of the Cas protein hinders delivery into the cell.[9]
TALEs
Transcription activator-like effectors (TALEs) are peptide structures ranging in length from 340 to 510 amino acids and are composed of repeating length segments of 34 amino acids.[10] The TALEs fold into a super helical structure with high specificity to the target DNA preventing secondary side effects, but this high specificity prevents the ATF from binding to multiple sites and requires a different ATF for each desired effect.[10]
Zinc Fingers
ATF Applications
Reprogramming Cell State
Angelman Syndrome
See Also
References
- ^ a b c d e Ansari, Aseem Z; Mapp, Anna K (2002-12-01). "Modular design of artificial transcription factors". Current Opinion in Chemical Biology. 6 (6): 765–772. doi:10.1016/S1367-5931(02)00377-0. ISSN 1367-5931.
- ^ Heiderscheit, Evan A.; Eguchi, Asuka; Spurgat, Mackenzie C.; Ansari, Aseem Z. (2018). "Reprogramming cell fate with artificial transcription factors". FEBS Letters. 592 (6): 888–900. doi:10.1002/1873-3468.12993. ISSN 1873-3468. PMC 5869137. PMID 29389011.
{{cite journal}}: CS1 maint: PMC format (link) - ^ Eguchi, Asuka; Wleklinski, Matthew J.; et al. (2016). "Reprogramming cell fate with a genome-scale library of artificial transcription factors". Proceedings of the National Academy of Sciences. 113 (51): E8257 – E8266. doi:10.1073/pnas.1611142114. ISSN 0027-8424. PMC 5187731. PMID 27930301.
- ^ a b Ansari, Aseem Z; Mapp, Anna K (2002-12-01). "Modular design of artificial transcription factors". Current Opinion in Chemical Biology. 6 (6): 765–772. doi:10.1016/S1367-5931(02)00377-0. ISSN 1367-5931.
- ^ a b c d e Ansari, Aseem Z; Mapp, Anna K (2002-12-01). "Modular design of artificial transcription factors". Current Opinion in Chemical Biology. 6 (6): 765–772. doi:10.1016/S1367-5931(02)00377-0. ISSN 1367-5931.
- ^ Heiderscheit, Evan A.; Eguchi, Asuka; Spurgat, Mackenzie C.; Ansari, Aseem Z. (2018). "Reprogramming cell fate with artificial transcription factors". FEBS Letters. 592 (6): 888–900. doi:10.1002/1873-3468.12993. ISSN 1873-3468. PMC 5869137. PMID 29389011.
{{cite journal}}: CS1 maint: PMC format (link) - ^ a b c d Ansari, Aseem Z; Mapp, Anna K (2002-12-01). "Modular design of artificial transcription factors". Current Opinion in Chemical Biology. 6 (6): 765–772. doi:10.1016/S1367-5931(02)00377-0. ISSN 1367-5931.
- ^ Nidhi, Sweta; Anand, Uttpal; Oleksak, Patrik; Tripathi, Pooja; Lal, Jonathan A.; Thomas, George; Kuca, Kamil; Tripathi, Vijay (2021-03-24). "Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives". International Journal of Molecular Sciences. 22 (7): 3327. doi:10.3390/ijms22073327. ISSN 1422-0067. PMC 8036902. PMID 33805113.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ a b Heiderscheit, Evan A.; Eguchi, Asuka; Spurgat, Mackenzie C.; Ansari, Aseem Z. (2018). "Reprogramming cell fate with artificial transcription factors". FEBS Letters. 592 (6): 888–900. doi:10.1002/1873-3468.12993. ISSN 1873-3468. PMC 5869137. PMID 29389011.
{{cite journal}}: CS1 maint: PMC format (link) - ^ a b Heiderscheit, Evan A.; Eguchi, Asuka; Spurgat, Mackenzie C.; Ansari, Aseem Z. (2018). "Reprogramming cell fate with artificial transcription factors". FEBS Letters. 592 (6): 888–900. doi:10.1002/1873-3468.12993. ISSN 1873-3468. PMC 5869137. PMID 29389011.
{{cite journal}}: CS1 maint: PMC format (link)
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