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 currently 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. Non-peptide, oligonucleotide, and polyamide DNA-binding domains have recently been explored which permit rational design. The type of DNA binding domain chosen depends on the desired application of the ATF, common DNA-binding domains are presented in Types of ATF DNA-Binding Domains section below.Regulatory Domain
The regulatory domain is responsible for activating or repressing the bound gene and accomplishes this regulation by either directly regulating gene expression or recruiting other proteins and transcription factors to change transcription levels. One route to upregulate a gene is for the ATF to recruit proteins that loosen the DNA wrapping around histones allowing RNA polymerase to bind and transcribe the gene; likewise, compacting the DNA would downregulate gene expression by inhibiting RNA polymerase from binding. 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. Factors influencing the effect the ATF has on transcription include 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. 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. 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.Linkers
Linkers covalently or non-covalently link the DNA-binding domain and regulatory domain. Frequently, peptide linkers are used, butHistory
Most ATFs have been constructed by exchanging existing DNA-binding domains and regulatory domains to generate ATFs with new targeting sites and transcription regulation consequences. Designed DNA-binding domains, such as CRISPR-Cas, with new targeting capabilities are being explored to engineer higher specificity and control potential side effects. In the future, ATFs which can respond to physiological cues, only change transcription levels in a specific cell type, and can easily be delivered without the use of electroporation are of great interest.Types of ATF DNA-Binding Domains
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). 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. 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.TALEs
Transcription activator-like effectors (TALEs) are peptide structures composed of repeating 34 amino acids long segments forming a peptide ranging in total length from 340 to 510 amino acids. Each repeating segment folds into two alpha helices and amino acids at residue positions 12 and 13 in the repeating segment determines the DNA binding sequence. The TALEs peptide has 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.Zinc Fingers
ATF Applications
Reprogramming Cell State
Directing cell differentiation and reprogramming cell fate have traditionally been achieved via a mixture of transcription factors. The field gained significant interest once four transcription factors Oct4/Sox2/cMyc/Klf4 were found to reprogram cells from a differentiated state into anAngelman Syndrome
Angelman syndrome is a neurological development disorder caused by the deactivation of the maternal UBE3A gene. Two potential treatment strategies using ATFs are to upregulate the expression of the maternal UBE3A gene or downregulate the expression of UBE3A-AS gene, the gene that causes repression of the paternal UBE3A gene. Zinc finger ATF TAT-S1 acts as a strong repressor against the UBE3A-AS gene, and when administered to mice, resulted in increased Ube3a in the brain.Cancer
Abnormal gene expression is regularly associated with cancer and uncontrolled tumor growth, making ATFs a promising therapeutic for cancer treatment. By linking 6 zinc fingers together in an ATF, the ATF only binds to an 18 base pair sequence containing smaller subsequences complementary to each zinc finger in the ATF, so the ATF is more specific than one zinc finger which only targets a specific 3 to 4 base pair sequence. ATFs linked to the KRAB repressor regulatory domain decreases cancer cells' drug resistance to chemotherapy, and ATFs linked to activator domains can upregulate Bax gene expression causing cell apoptosis; however, these treatments remain in the early stages because of inadequate delivery methods.See also
*References
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