Klenow DNA Research Articles

Convenient synthesis of nucleoside 5′-(α-P-thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA)

Modified deoxy- and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S -modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy- and ribo-nucleoside 5′-(α-P-thio)triphosphates (dNTPαS and NTPαS) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5′-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTPαS and NTPαS analogs (mixtures of S p and R p diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPαS and NTPαS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the S p diastereomers of dNTPαS and NTPαS analogs, respectively, while the R p diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.

Intracellular Detection of Cytosine Incorporation in Genomic DNA by Using 5‐Ethynyl‐2′‐Deoxycytidine

5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase β. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.

Interactions of replication versus repair DNA substrates with the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3′ overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5′- or 3′-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

BackgroundBacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found.Methodology/Principal FindingsWe herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species.Conclusions/SignificanceBroad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.

Screening for additives that stabilize enzymes at higher temperatures

In Molecular Biology, enzymes are often required that function at elevated temperatures. Enzymes produced by mesophilic organisms usually denature at elevated temperatures, followed by aggregate formation. We developed a rapid fluorescence‐based assay for assessing a range of parameters impacting the thermal stability of an enzyme. Overall, protein stability is monitored by a fluorogenic dye that selectively detects aggregated protein. Stability can be measured in the presence of different buffers, cryoprotectants and excipients. By systematically raising the temperature of the protein in solution, the temperature at which the protein aggregates (Tagg) can be determined. Using this method with Klenow DNA polymerase, we determined that trehalose significantly increases Tagg. The DNA polymerase activity of the enzyme is significantly enhanced at 50°C in the presence of trehalose under the same conditions. Low amounts of the detergents, Tween20® and Triton X‐100® (0.05%) decreased Tagg and also compromised enzyme activity, especially at elevated temperatures. T7 RNA polymerase was tested in a similar manner. Trehalose less significantly increased Tagg, and had a more modest effect on increasing enzyme activity at 42°C. The described assay facilitates screening for buffers and additives that structurally stabilize a protein of interest at elevated temperatures.This work was supported by ENZO Life Sciences.

Origins for the Thermostability of Taq DNA Polymerase: Entropy Versus Heat Capacity

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. The free energy versus temperature stability curves of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases have been compared, and by utilizing the ΔG and ΔCp information obtained from the stability curves, the ΔH and TΔS of folding of each protein as a function of temperature can be calculated directly from the Gibbs-Helmholtz equation. This analysis reveals that Klentaq's extreme stability originates from a significantly decreased entropic folding penalty (TΔS of folding). In contrast, Klentaq's native state enthalpic stabilization (ΔH of folding) is actually considerably less favorable than Klenow's at all temperatures. We have also examined published data on 17 other mesophilic-thermophilic protein pairs using the same approach to calculate ΔH and TΔS as a function of temperature from the stability curves. There are 6 other data sets (7 total including Klenow/Klentaq) with the same thermodynamic pattern, where a reduced entropic penalty is the primary basis for thermostabilization. In contrast, 8 of the 18 total data sets exhibit a highly popularized thermodynamic pattern where a significantly reduced ΔCp of folding is correlated with the enhanced stability of the thermophile. ΔCp has been correlated with changes in accessible surface area during protein folding, so a reduced ΔCp of folding for a thermophilic protein has been suggested to reflect a more compact denatured state of the thermophilic protein, although direct empirical evidence of this is still lacking for any mesophilic-thermophilic protein pair. It is notable that there is minimal overlap between the 8 systems exhibiting a lowered ΔCp of folding and the 7 systems showing a lowered entropic folding penalty.

The Interleukin 13 (IL-13) Pathway in Human Macrophages Is Modulated by MicroRNA-155 via Direct Targeting of Interleukin 13 Receptor α1 (IL13Rα1)

Macrophages play a central role in the balance and efficiency of the immune response and are at the interface between innate and adaptive immunity. Their phenotype is a delicate equilibrium between the M1 (classical, pro-Th(1)) and M2 (alternative, pro-Th(2)) profiles. This balance is regulated by cytokines such as interleukin 13 (IL-13), a typical pro-M2-Th(2) cytokine that has been related to allergic disease and asthma. IL-13 binds to IL-13 receptor α1 (IL13Rα1), a component of the Type II IL-4 receptor, and exerts its effects by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) through phosphorylation. MicroRNAs are short (∼22 nucleotide) inhibitory non-coding RNAs that block the translation or promote the degradation of their specific mRNA targets. By bioinformatics analysis, we found that microRNA-155 (miR-155) is predicted to target IL13Rα1. This suggested that miR-155 might be involved in the regulation of the M1/M2 balance in macrophages by modulating IL-13 effects. miR-155 has been implicated in the development of a healthy immune system and function as well as in the inflammatory pro-Th(1)/M1 immune profile. Here we have shown that in human macrophages, miR-155 directly targets IL13Rα1 and reduces the levels of IL13Rα1 protein, leading to diminished activation of STAT6. Finally we also demonstrate that miR-155 affects the IL-13-dependent regulation of several genes (SOCS1, DC-SIGN, CCL18, CD23, and SERPINE) involved in the establishment of an M2/pro-Th(2) phenotype in macrophages. Our work shows a central role for miR-155 in determining the M2 phenotype in human macrophages.

Quantitation of cellular deoxynucleoside triphosphates

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5' --> 3' DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3' --> 5' exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3' terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.

Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases

MicroRNAs (miRNAs) as endogenous regulators of gene expression have spurred a surge of interest for their quantification and expression analysis. High-sensitivity and high-specificity miRNA detection techniques, such as real-time polymerase chain reaction and recently introduced bioluminescent miRNA detection, require systematic study of DNA polymerases for use with miRNAs. In this study, a variety of DNA polymerases were studied to assess their capabilities of using miRNA as a primer and incorporating 2′-deoxyadenosine-5′- O-(1-thiotriphosphate) as a dATP alternative during DNA strand extension. Five DNA polymerases were investigated: mesophilic DNA polymerase I large (Klenow) fragment, 3′ → 5′ exo − Klenow DNA polymerase, thermophilic Bst DNA polymerase large fragment, Therminator DNA polymerase, and Taq DNA polymerase. The experimental results show that, except for Taq DNA polymerase, the polymerases can use miRNA as a primer and have both common and divergent properties of the nucleotide analog incorporation and miRNA discrimination. DNA polymerase I large (Klenow) fragment showed no detectable polymerization product with the thio-modified dATP as a substrate. Thermophilic Bst DNA polymerase had the highest specificity for miRNA recognition on a DNA template. The study provides a novel method for miRNA detection without reverse transcription to complementary DNA that is faster, simpler, and less prone to biases and errors.

Osmotic enhancement of DNA binding by glutamate: Klenow and Klentaq DNA binding studies

Glutamate (Glu−) is the major osmolyte accumulated in E.coli in response to external salt. In vitro, the 'glutamate effect' is primarily characterized by enhanced DNA binding when Glu− replaces Cl−. Some proteins also exhibit reduced ion release upon DNA binding in Glu−. Klenow polymerase (E.coli) exhibits both aspects of the 'glutamate effect'. Upon replacement of Cl− by Glu−, the ion release upon DNA binding is reduced by >50%. In addition, the apparent water release upon DNA binding is reduced by ~70% (150 waters) and proton linkage increases 5 fold. The decrease in apparent water release highlights the osmotic nature of the glutamate effect: Glu− and Cl− salts behave as ionic inhibitors of DNA binding but Glu− salts also behave as osmotic enhancers. While Klentaq's (T.aquaticus) DNA binding is enhanced in Glu−, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq polymerase but it is not as significant in the salt concentration range at which nanomolar Klentaq‐DNA binding occurs. E.coli DNA‐binding proteins might have evolved to bind tightly at higher salt concentrations in order to use osmotic enhancement of Glu− upon its intracellular accumulation. NSF.

Water Release upon DNA Binding by E. coli and T. aquaticus Pol I DNA Polymerases

Klentaq (T. aquaticus) and Klenow (E. coli) are a thermophilic‐mesophilic pair of Type I DNA polymerases with similar morphologies. The thermodynamic linkage between binding affinity and the osmotic pressure induced by non‐interacting osmolytes can be used to calculate the number of water molecules bound or released during a macromolecular process such as DNA binding. Approximately 600 water molecules are released when Klentaq polymerase binds to either primed‐template or blunt‐end DNA. In contrast, only approximately 500 waters are released when Klenow binds primed‐template DNA, however, the necessity of measuring Klenow DNA binding at significantly higher salt concentrations may be the source of this difference. For both proteins, the measured water release is significantly higher than that predicted from the burial of surface area upon complex formation calculated from structural data (approximately 300 waters). Steric displacement of non‐surface associated water within the DNA binding cleft of the proteins may account for this difference, and indicates that these binding reactions will be more enhanced at intracellular osmotic pressures than would be predicted from surface area calculations.

Klenow and Klentaq-DNA Binding: the ‘Glutamate Effect’ is Primarily an Osmotic Effect

DNA binding by Klenow (E.coli) and Klentaq (T.aquaticus) DNA polymerases has been studied as a function of monovalent salt concentration, pH and osmotic stress. We previously showed that DNA binding resulted in the net release of 4.5∼5 ions from Klenow and 3∼3.5 ions from Klentaq. Here, we report that Klenow and Klentaq have minimal sensitivity to pH changes, with proton linkages of ∼0.06 and ∼0.3 respectively in KCl. Furthermore, osmotic stress data in KCl indicates 500∼600 waters are released upon binding by both polymerases.Glutamate is the major intracellular anion accumulated in E.coli in the presence of KCl in the external environment. The ‘glutamate effect’ is primarily characterized by an increase in DNA binding affinity when chloride is replaced by glutamate. Some proteins also exhibit decreased ionic linkage in glutamate.Klenow exhibits both aspects of the ‘glutamate effect’. Substituting glutamate for chloride reduces the ionic linkage for Klenow by >50%. The presence of glutamate also increases the proton linkage of Klenow five fold and decreases water release by ∼70% to approximately 150 waters. The dramatic decrease in water release highlights the osmotic nature of the glutamate effect. Glutamate and chloride salts behave as ionic inhibitors of DNA binding but glutamate salts also exhibit an osmotic enhancement effect.While Klentaq's DNA binding affinity is also enhanced by glutamate, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq but it is not as significant in the salt concentration range at which nanomolar Klentaq-DNA binding occurs. E.coli DNA-binding proteins might have evolved to bind tightly at higher salt concentration to utilize the glutamate effect while accumulating intracellular glutamate.

2Am-DNT Induces Cell Death and Apoptosis in Human Cells

During microbial or mammalian cell metabolism, TNT (2,4,6-tinitrotoluene) is reduced to 2Am-DNT (2-amino-4,6-dinitrotoluene), 4Am-DNT, or 2,4-diamino-NT (2,4-diaminonitrotoluelne) depending on the specific organism. The metabolite 2Am-DNT is the most common of the TBT biotransformation pathways in bacterial and fungal species studied to date. in the mammalian liver cells, TNT is metabolized to 2Am-DNT by the P450 enzyme system. Apoptosis is rapidly emerging as a relevant endpoint for detecting low-dose toxin exposure. We report in this study that 2Am-DNT treatment of mammalian cells causes cell death by apoptosis. Cell death was assayed by the Trypan Blue method. Apoptotic changes, such as DNA break down, were detected in treated cells by the production of a dark-brown DAB (diaminobenzidine) signal using the Fragel Klenow DNA fragment detection system, by immunohistochemical techniques with fluorescence microscopy, and by using a microplate reader for a single-stranded DNA binding assay. All of these results showed that 2am-DNT is toxic to mammalian cells and induces apoptosis.

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.

An Iron Enhancer Element in the FTN-1 Gene Directs Iron-dependent Expression in Caenorhabditis elegans Intestine

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5 '-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATA-binding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

An Antiproliferative Genetic Screening Identifies a Peptide Aptamer That Targets Calcineurin and Up-regulates Its Activity

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G SO3) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G SO3 is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G SO3 does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Control of MyoD Function during Initiation of Muscle Differentiation by an Autocrine Signaling Pathway Activated by Insulin-like Growth Factor-II

The insulin-like growth factors (IGFs) play key roles in muscle development, maintenance, and repair, but their mechanisms of action are incompletely defined. We previously identified an autocrine pathway involving production of IGF-II and activation of the IGF-I receptor, phosphatidylinositol 3-kinase, and Akt in myoblast differentiation induced by MyoD in 10T1/2 mesenchymal stem cells and found that blocking this pathway prevented differentiation (Wilson, E. M., Hsieh, M. M., and Rotwein, P. (2003) J. Biol. Chem. 278, 41109-41113). We now have analyzed regulation of MyoD function in this model system. Inhibition of IGF-II production impaired the transcriptional actions of MyoD, as seen by a 70-80% decline in activity of transfected reporter genes, including the myogenin and creatine kinase promoters, and by complete inhibition of transcription of the endogenous myogenin gene but had no effect on MyoD protein levels, post-translational modifications, or nuclear localization, and neither blocked the rapid disappearance of the inhibitory molecule Id1 nor altered the nuclear expression or abundance of the MyoD binding partner E12/E47. Impaired signaling through the IGF-I receptor also did not decrease the ability of MyoD or E12/E47 to bind to target DNA sites at the proximal myogenin promoter, as assessed by chromatin immunoprecipitation assay but, rather, blocked chromatin remodeling at this site, as indicated by reduced recruitment of co-activators p300 and P/CAF and diminished acetylation of histones H3 and H4. Taken together, these results show that IGF-II-initiated signaling through the insulin-like growth factor-I receptor targets transcriptional co-regulators that are essential co-factors for MyoD and suggests that the phosphatidylinositol 3-kinase-Akt pathway plays a key role in establishing an amplification cascade that is essential for sustaining the earliest events in muscle differentiation.