Activation of gene expression by small molecule transcription factors

Abstract

Eukaryotic transcriptional activators are minimally comprised of a DNA binding domain and a separable activation domain; most activator proteins also bear a dimerization module. We have replaced these protein modules with synthetic counterparts to create artificial transcription factors. One of these, at 4.2 kDa, mediates high levels of DNA site-specific transcriptional activation in vitro. This molecule contains a sequence-specific DNA binding polyamide in place of the typical DNA binding region and a nonprotein linker in place of the usual dimerization peptide. Thus our activating region, a designed peptide, functions outside of the archetypal protein context, as long as it is tethered to DNA. Because synthetic polyamides can, in principle, be designed to recognize any specific sequence, these results represent a key step toward the design of small molecules that can up-regulate any specified gene.

Figure 1

Design and synthesis of activators. (A) The synthetic activator consisting of an activation domain (AD), a dimerization or linker domain (LD), and a DNA binding domain (DBD) complexed with the cognate palindromic DNA site of the hairpin polyamide-peptide conjugate. (B) The synthesis of polyamide 1 was accomplished according to established protocols. (a) Treatment of 1 with thiolane-2,5-dione followed by benzyl bromide provided thioester 2 in good yield (53%). (b) Combination of thioester 2 with each peptide in denaturing buffer then provided the targeted conjugates via the native ligation reaction.

Figure 2

Conjugate 3 (PA-Gcn4-AH) binds to its cognate palindromic DNA site and activates transcription in vitro when its predetermined DNA binding sites are present. (A) (Upper) Storage phosphor autoradiogram of a quantitative DNase I footprinting titration of 3 on the 3′-³²P-labeled 271-bp pPT7 EcoRI/PvuII restriction fragment carried out according to established protocols (15, 21). Preequilibration of 3 with the DNA fragment was carried out for 75 min before initiation of the cleavage reactions. From left to right the lanes are: the A sequencing lane; DNase I digestion products in the presence of 3 at concentrations of 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, and 0.25 nM, respectively; DNase I digestion products with no 3 present; undigested DNA. (Lower) Data for 3 in complex with the 19-bp palindromic site. The curve through the data points is the best-fit cooperative Langmuir binding titration isotherm (n = 2) obtained from a nonlinear least-squares algorithm. (B) An in vitro transcription reaction containing PA-Gcn4-AH (3) at 200 nM shows enhanced expression of a 277-nt transcript relative to basal levels whereas a reaction containing conjugate 4, lacking the activating region, does not. Inclusion of the parent hairpin polyamide (1) (lane 2) in the reaction does not impair basal transcription (lane 1). The variation in transcript position for lane 4 is caused by curvature of the gel and was confirmed by additional experiments (data not shown). (C) In vitro transcription reactions containing 3 (PA-Gcn4-AH) with templates bearing either the cognate palindromic binding sites (match template) or palindromic sites in which a G⋅C base pair has replaced a T⋅A base pair in each half site (mismatch template) upstream of the core promoter. The concentrations of 3 used were 0 (basal), 10 nM, 100 nM, and 500 nM.

Figure 3

Dependence of activation level upon time and activating region. (A) (Upper) Storage phosphor autoradiogram showing an in vitro transcription time course experiment with conjugates 3 and 4 present at 300 nM concentration. Aliquots were processed at 10, 20, 30, 40, and 60 min. (Lower) Comparison of the amount of transcript obtained at each time point relative to basal transcription levels in which no conjugate was present (fold activation). (B) (Upper) Storage phosphor autoradiogram showing the effect of increasing concentrations of untethered 21-aa AH peptide on transcription reactions containing either conjugates 4 or 3 at 300 nM concentration. Transcription reactions were performed for 30 min in the presence of 0, 0.2 μM, 2 μM, and 10 μM concentrations of AH peptide. (Lower) For each reaction, the amount of transcript obtained was compared with basal transcription levels to give the respective fold activation value. These values are displayed as percent activation compared with the results from the reaction containing conjugate 3 (lane 5), which is defined as 100%.

Figure 4

Substitution of the dimerization module with a flexible ethylene glycol-derived linker. (A) The synthesis of hairpin polyamides 6 and 7 and conjugates 8 and 9 was carried out as described in Fig. 1B. (B) (Left) Storage phosphor autoradiogram showing in vitro transcription reactions containing parent hairpin polyamides 1 (lane 2), 6 (lane 4), and 7 (lane 6) or conjugates 5 (lane 3), 8 (lane 5), and 9 (lane 7), which have the AH peptide attached by flexible linkers of increasing length at 500 nM concentration. A higher conjugate concentration relative to the experiments presented in Figs. 2 and 3 was used to accommodate the slightly lower binding affinity (2- to 3-fold) of conjugates 5, 8, and 9 relative to conjugate 3 (PA-Gcn4-AH). (Right) The activation levels for conjugates 3, 5, 8, and 9 were determined by comparison with the amount of transcript obtained from reactions containing the relevant parent hairpin polyamides. The fold activation values thus obtained are displayed as percentages relative to the fold activation mediated by conjugate 3, defined as 100%. (C) Data from DNase I footprinting titrations with 5 and 8. The curve through each data set is the best-fit Langmuir binding titration isotherm (n = 1) obtained from a nonlinear least-squares algorithm.