The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

The GCN4 bZIP can bind to noncognate gene regulatory sequences

We show that a minimalist basic region/leucine zipper (bZIP) hybrid, comprising the yeast GCN4 basic region and C/EBP leucine zipper, can target mammalian and other gene regulatory sequences naturally targeted by other bZIP and basic/helix-loop-helix (bHLH) proteins. We previously reported that this hybrid, wt bZIP, is capable of sequence-specific, high-affinity binding of DNA comparable to that of native GCN4 to the cognate AP-1 and CRE DNA sites. In this work, we used DNase I footprinting and electrophoretic mobility shift assay to show that wt bZIP can also specifically target noncognate gene regulatory sequences: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (Xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and the E-box (Enhancer box, 5'-CACGTG). Although wt bZIP still targets AP-1 with strongest affinity, both DNA-binding specificity and affinity are maintained with wt bZIP binding to noncognate gene regulatory sequences: the dissociation constant for wt bZIP in complex with AP-1 is 13 nM, while that for C/EBP is 120 nM, XRE1 240 nM, and E-box and HRE are in the microM range. These results demonstrate that the bZIP possesses the versatility to bind various sequences with varying affinities, illustrating the potential to fine-tune a designed protein's affinity for its DNA target. Thus, the bZIP scaffold may be a powerful tool in design of small, alpha-helical proteins with desired DNA recognition properties.     

Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.     

Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein–DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein–DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein–DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein–DNA binding.     

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k_cat ≈ 8 x 10⁻³ s⁻¹ and K_M < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower k_cat and a K_M ≈ 300 nM. The rate of ligation increased with addition of Mn²⁺, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.     

The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study

The structural competition between the G-quadruplex and Watson–Crick duplex has been implicated for the repetitive DNA sequences, but the factors influencing this competitive equilibrium in the natural and pharmacological context need to be elucidated. Using a 21mer 5′-Fluorescein-d[(G₃TTA)₃G₃]-TAMRA-3′ as a model system, extensive fluorescence resonance energy transfer analysis was carried out to investigate sensitivity of this equilibrium to osmotic stress and quadruplex selective small molecule. The binding affinities and kinetics involved in the hybridization of quadruplex to its complementary strand in the absence and presence of different concentrations of osmolytes (ethylene glycol and glycerol) and a quadruplex selective ligand (cationic porphyrin-TMPyP4) were determined. The presence of osmolytes and cationic porphyrin decreased the binding affinity of quadruplex to its complementary strand and slowed the kinetics of the reaction by delaying the hybridization process. Our binding data analysis indicates that the presence of either osmolytes or porphyrin increase the amount of quadruplex in the equilibrium. In 100 mM KCl solution, when 30 nM of each of the components, i.e. quadruplex and the complementary strand, were mixed together, the amount of quadruplex present in the system under equilibrium were 17.6, 23.4, 23.1 and 19.6 nM in the absence and presence of 10% ethylene glycol, 10% glycerol and 150 nM TMPyP4, respectively. Fluorescence melting profile of quadruplex in the absence and presence of these perturbants confirm the findings that osmolytes and cationic porphyrin stabilize quadruplex, and thus, shift the equilibrium to quadruplex formation.     

Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein–RNA interactions in the 3′-UTR of the collagen α1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is αCP₂. Recombinant αCP₂ is sufficient for binding to the 3′-UTR of collagen α1(I). To characterize the binding affinity of and specificity for αCP₂, we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3′-UTR of collagen α1(I) as probe. The binding affinity of αCP₂ for the 3′-UTR sequence is ~2 nM in vitro and the wild-type 3′ sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein–nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA–DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant αCP₂ to the wild-type 3′ sequence, although the kinetics of binding were slower.     

Vanadate binding to the (Na + K)-ATPase

A particulate (Na + K)-ATPase preparation from dog kidney bound [⁴⁸V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl₂ theK _d was 96 nM; substituting MnCl₂ decreased theK _d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl₂ increased binding, with aK _0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK _d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl₂ decreased binding markedly, with anI ₅₀ for NaCl of 7 mM. However, in the presence of MnCl₂ neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl₂, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K⁺ (corresponding to intracellular K⁺) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K⁺ (corresponding to extracellular K⁺) both stimulated catalysis and augmented vanadate binding.     

The bZIP mutant CEBPB (V285A) has sequence specific DNA binding propensities similar to CREB1

The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides containing 5-methylcytosine (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB, with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.