artificial or synthetic transcription factor for regulation of gene expression
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The document discusses synthetic transcription factors, including artificial transcription factors (ATFs) based on zinc fingers and transcription activator-like effectors (TALs), and their roles in regulating gene expression. It explains how these factors can be constructed and applied in functional genomics and biotechnology, as well as the use of CRISPR/Cas9 technology in developing tailored transcriptional activators and repressors. The conclusion highlights the potential of modular DNA binding domains for artificial gene control.
artificial or synthetic transcription factor for regulation of gene expression
- 1. Synthetic transcription factors for regulation of gene expression Balaji Rathod PGS16AGR7165
- 2. Outline • Introduction about transcription factor • artificial TFs: those based on • zinc fingers, • TALEs, and on • the CRISPR/Cas9 technology • conclusion
- 4. What is transcription factor • Transcription factors are proteins that help turn specific genes "on" or "off" by binding to nearby DNA. • Transcription factors that are activators boost a gene's transcription. Repressors decrease transcription. • Groups of transcription factor binding sites called enhancers and silencers can turn a gene on/off in specific parts of the body. • Transcription factors allow cells to perform logic operations and combine different sources of information to "decide" whether to express a gene.
- 5. what is synthetic transcription factor • An artificial transcription factor (ATF) is an example of a chimeral protein, designed to target and modulate gene transcription • They are generally composed of a DNA-binding domain (specific to a certain sequence) coupled to a modulatory domain (which acts upon other transcription factors) in order to alter the expression of a particular gene. • artificial TF-mediated regulation of gene expression, it would be better to employ the term “gene control region” rather than “promoter”. This control region is usually defined as the portion of a eukaryotic gene containing the core promoter as well as any other regulatory sequences that control or influence transcription of that gene
- 8. Zinc finger domains ZF domain useful for construction of DBP because of two properties 1) Sequence specificity 2) Modularity (Nicoletta et al 2000)
- 11. Construction of ZF ATFs (Takasi sera 2009)
- 12. (Sabine et .al. 2012)
- 13. • A) Schematic representation of a 6 ZF ATF bound to DNA with the orientation of the domains depicted • B) Schematic illustration of the SOX2 promoter outlining the ZF-552SKD, ZF- 598SKD, ZF-619SKD and ZF-4203SKD targeted sequences and their location relative to the transcription start site (TSS). Highlighted are the core promoter (red), regulatory region 1 (green), and regulatory region 2 (blue). Arrows show the orientation of the 18-bp binding site in the promoter (from 5′ to 3′).
- 14. • (C) Alpha-helical ZF amino acid sequences chosen to construct the ATFs. Residues at position –1, +3 and +6 making specific contacts with the recognition triplets are indicated in color (red refers to position –1, blue to position +3 and green to +6 of the ZF recognition helix. • (D) Quantification of SOX2 expression in 12 breast cancer cell lines by western blot
- 16. Applications of artificial transcription factors possible applications of designed TF and ZF technology in areas of functional genomics, molecular therapeutics, and biotechnology.
- 18. transcription activator-like effectors (TALEs) • The TALE DNA-binding domain consists of multiple repeats of 34 amino acids where variability in positions 12 and 13, referred to as the repeat-variable di-residues (RVDs), confer binding specificity for one specific DNA base The TALE DNA-binding domain consists of multiple repeats of 34 amino acids where variability in positions 12 and 13, referred to as the repeat-variable di-residues (RVDs), confer binding specificity for one specific DNA base
- 19. fusion of TALEs to transcriptional activator,or repressor domains, such as VP16 or KRAB could generate TALE-TFs that can target selected promoter regions and modulate expression of corresponding genes The array of TALE domains is then fused to an effector domain to induce a specific action at a user- determined genomic locus
- 20. Construction of TALES • There are multiple platforms available for constructing TALEs. Golden Gate Assembly Golden Gate cloning is a method of assembling multiple DNA fragments in a single reaction. The protocol utilizes type IIS restriction enzymes that cleave sequences adjacent to their recognition sequence In this manner, one restriction enzyme creates multiple unique overhangs that can only ligate into the destination vector in the correct orientation The advantage of this platform is that it does not require a PCR step Fast ligation-based automatable solid-phase high throughput (FLASH) developed by the Joung Lab Iterative Capped Assembly (ICA) developed by the Church group
- 21. • TALE repressors have been used to successfully suppress multiple endogenous genes in human cells ,model organism and plant (Cong et al 2012) • TALE-repressors have not yet been widely used, which may be in part due to their recent development • This may also be due to competition with other technologies, including creating knockouts via genome editing with engineered nucleases and gene silencing by RNA interference (RNAi). TALE repressors
- 22. • Synthetic TALE activators have been used to successfully induce expression of many endogenous genes • Certain tightly regulated genes, targeting the proximal promoter alone induces very low expression levels that may not be sufficient for functional effects • One example is activation of the Oct4 locus. Targeting the human Oct4 proximal promoter with a single TALE activator led to modest changes in gene expression (Zhang et al 2011) TALE transcriptional activators
- 23. Human Gene Expression by Ligand-Inducible TALE Transcription Factors Andrew et al 2014
- 24. Activation of gene expression by ligand-responsive TALE transcription factors the luciferase reporter system used to evaluate ligand-inducible TALE-TF gene activation. Avr15 target site repeats indicated. (b−d) Fold-activation of luciferase expression observed with the (b) PR/RU486, (c) ER/4-OHT, and (d) RXE/PonA regulatory systems. (e) Fold activation of luciferase expression observed by RXE-TFs with increasing concentrations of PonA. Error bars indicate standard deviation (n = 3; p-value < 0.0005; paired t-test).
- 25. (H) Analysis of various combinations of TALE-VP64 hOs for activating the endogenous human OCT4 gene in HEK293T cells. Significant transcriptional synergy was observed with multiple combinations. (I) OCT4 proteins were detected in HEK293T cells at 48 h after transfection with single or combinations of TALE-VP64 hOs. Western blot was performed with anti-OCT4. GAPDH was included as control for equal loading Activation of silenced Oct4 genes in human somatic cells by TALE-VP64s.
- 28. • With only two amino acid substitutions (D10A and H840A), Cas9 endonuclease activity can been eliminated while simultaneously maintaining its RNA-guided DNA-binding activity. This deactivated Cas9 (dCas9) functions as a modular DNA-binding domain, similar to TALEs. RNA-guided transcriptional activators and repressors have been created by fusing dCas9 with different effector domains
- 29. • Figure 1: Schematic illustration of transcriptional regulation via effector domains: A). Repression of transcription initiation via repressor domain (KRAB) pairing with dCas9 to block gene expression (heterochromatinization) B). Activation of transcription via activation domain (VP16) pairing with dCas9 to increase gene expression
- 30. A Modular CRISPR Fusion System for Efficiently Repressing and Activating Transcription in Human Cells (A) dCas9 fused to effector domains can serve as an RNA-guided DNA-binding protein to target any protein to any DNA sequence. (B) A minimal CRISPRi system in human cells contains an sgRNA expression plasmid and dCas9 or dCas9 fused to the repressive KRAB effector domain. Both dCas9 constructs are fused to two copies of a nuclear localization sequence and a blue fluorescent protein.
- 31. • (C) A dCas9-KRAB fusion protein efficiently silences GFP expression. Eight sgRNAs targeting GFP are transfected into GFP+HEK293 stably expressing either dCas9 (light gray) or dCas9-KRAB (dark gray). GFP fluorescence is quantified by flow cytometry 6 days following transfection and is displayed as a signal normalized to a vector control. The data are displayed as mean ± SD for three independent experiments. See also Figure S1. (D) CRISPRi gene repression is stable over time. GFP+HEK293 cells were infected with lentivirus constructs expressing a negative control sgRNA or a sgRNA targeting GFP and either dCas9 or dCas9-KRAB. Cells were grown for 14 days and then analyzed for GFP expression. A histogram displays GFP fluorescence for each sample and a control population of HEK293 cells that do not express GFP. Data are representative of three independent experiments.
- 32. (E) Two dCas9 fusion proteins were constructed with VP64 or p65AD. The sgRNA is expressed as before. Shown is a diagram of the Gal4 UAS-GFP reporter and data showing transient transfection of either dCas9-VP64 or dCas9-p65AD and sgGAL4-1 can activate gene expression in HEK293 cells. Cells were transfected with the indicated plasmids and 48 hr later were analyzed by flow cytometry for GFP expression. The data are displayed as mean ± SD for two independent experiments
- 33. (G) Activation of endogenous human OCT4 gene by sgRNA-guided dCas9-VP64s. Individual or combined sgRNAs were transfected with dCas9-VP64s into HEK293T cells. Up-regulation of endogenous OCT4 gene was examined using qRT-PCR analysis at 48 h after transfection. (H) OCT4 proteins were detected in HEK293T cells at 48 h after transfection with dCas9- VP64 and combinations of sgRNAs (H3 and H4, or H1–H4). Western blot was performed with anti-OCT4. GAPDH was included as control for equal loading. (I) Combinatory effect of sgRNA/dCas9-VP64s and hO TALEVP64s. sgRNAs H3 and H4 and TALE-VP64 (hO1, 3*, 4 and 6) were transfected together with dCas9-VP64 into HEK293T cells. Expression of endogenous OCT4 gene was examined using qRT-PCR at 48 h after transfection. Activation of human Oct4 promoters by sgRNA-guided dCas9-VP64s.
- 37. conclusion • The availability of modular DNA binding domains allows for a wealth of possibilities for artificial regulation of gene expression.
- 38. THANK YOU