Optical mapping discerns genome wide DNA methylation profiles

BACKGROUND: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification. RESULTS: A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping. CONCLUSION: The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function

Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery

The salt dependence of the interaction of F-σ70 or F-σ32 with 10 nM Tb-core.

<p>To determine the effect of NaCl and KGlu on the interaction strength, binding curves were generated for the interaction of either F-σ70 or F-σ32 with 10 nM Tb-core at 100 mM, 250 mM, or 500 mM NaCl or KGlu. Shown is the average acceptor/donor signal (A₅₂₀/A₄₉₀) for each sample. The error bars represent the standard deviation for a sample size of 4. The curves were fit with Origin 7 and the K_D values are listed for each curve. A) The effect of NaCl on the interaction of F-σ70 with 10 nM Tb-core. B) The effect of KGlu on the interaction of F-σ70 with 10 nM Tb-core. C) The effect of NaCl on the interaction of F-σ32 with 10 nM Tb-core. D) The effect of KGlu on the interaction of F-σ32 with 10 nM Tb-core.</p

Electrophoretic mobility shift assay to determine quality of labeled proteins.

<p>An electrophoretic mobility shift assay was used to determine if all of the fluorescein-labeled sigma factors were able to form holoenzyme and if the terbium-labeled core could bind fluorescein-sigma as well as unlabeled core. Proteins were incubated for 1 hr at 22°C and run on a native 4-12% Tris-glycine gel. The F-sigma factors were visualized using the Typhoon Imager to produce a fluorescent scan of the gel (right). The total protein was visualized by staining the gel with Coomassie stain (left). The molar ratio (MR) of core to sigma is indicated. A) F –σ70 (780 nM final concentration) was incubated with increasing concentrations of Tb-core or unlabeled core (90–900 nM final concentration). B) F –σ32 (780 nM final concentration) was incubated with increasing concentrations of Tb-core or unlabeled core (90–900 nM final concentration).</p