An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.     

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies

Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f=formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5'-ACGCGT-3' through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6x10(8)M(-1) and 2.0x10(10)M(-1) for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (DeltaH of -3.3 and -1.0 kcal mol(-1) for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (DeltaC(p)) were determined to be -116 and -499 cal mol(-1)K(-1), respectively. These results are in general agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5'-CCACGCGTGG)(2). According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.     

Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides

Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.