High-resolution nucleosome mapping of targeted regions using BAC-based enrichment.

We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. We achieved ≈ 10 000-fold enrichment of specific loci, which enabled sequencing nucleosomes at up to ≈ 500-fold higher coverage than has been reported in a mammalian genome. We demonstrate the advantages of generating high-sequencing coverage for mapping the center of discrete nucleosomes, and we show the use of the method by mapping nucleosomes during T cell differentiation using nuclei from effector T-cells differentiated from clonal, isogenic, naïve, primary murine CD4 and CD8 T lymphocytes. The analysis reveals that discrete nucleosomes exhibit cell type-specific occupancy and positioning depending on differentiation status and transcription. This method is widely applicable to mapping many features of chromatin and discerning its landscape in large genomes at unprecedented resolution.

Direct quantitation of Mg2+-RNA interactions by use of a fluorescent dye.

The ionic composition of a solution strongly influences the folding of an RNA into its native structure; of particular importance, the stabilities of RNA tertiary structures are sharply dependent on the concentration of Mg2+. Most measurements of the extent of Mg2+ interaction with an RNA have relied on equilibrium dialysis or indirect measurements. Here we describe an approach, based on titrations in the presence of a fluorescent indicator dye, that accurately measures the excess Mg2+ ion neutralizing the charge of an RNA (the interaction or Donnan coefficient, Gamma2+) and the total free energy of Mg2+-RNA interactions (DeltaG(RNA-2+)). Automated data collection with computer-controlled titrators enables the collection of much larger data sets in a short time, compared to equilibrium dialysis. Gamma2+ and DeltaG(RNA-2+) are thermodynamically rigorous quantities that are directly comparable with the results of theoretical calculations and simulations. In the event that RNA folding is coupled to the addition of MgCl2, the method directly monitors the uptake of Mg2+ associated with the folding transition.

Importance of partially unfolded conformations for Mg(2+)-induced folding of RNA tertiary structure: structural models and free energies of Mg2+ interactions.

RNA molecules in monovalent salt solutions generally adopt a set of partially folded conformations containing only secondary structure, the intermediate or I state. Addition of Mg2+ strongly stabilizes the native tertiary structure (N state) relative to the I state. In this paper, a combination of experimental and computational approaches is used to estimate the free energy of the interaction of Mg2+ with partially folded I state RNAs and to consider the possibility that Mg2+ favors "compaction" of the I state to a set of conformations with a higher average charge density. A sequence variant with a drastically destabilized tertiary structure was used as a mimic of I state RNA; as measured by small-angle X-ray scattering, it adopted a progressively more compact conformation over a wide Mg2+ concentration range. Average free energies of the interaction of Mg2+ with the I state mimic were obtained by a fluorescence titration method. To interpret these experimental data further, we generated molecular models of the I state and used them in calculations with the nonlinear Poisson-Boltzmann equation to estimate the change in Mg2+-RNA interaction free energy as the average I state dimensions decrease from expanded to compact. The same models were also used to reproduce quantitatively the experimental difference in excess Mg2+ between N and I states. On the basis of these experiments and calculations, I state compaction appears to enhance Mg2+-I state interaction free energies by 10-20%, but this enhancement is at most 5% of the overall Mg2+-associated stabilization free energy for this rRNA fragment.

Mg2+-RNA interaction free energies and their relationship to the folding of RNA tertiary structures.

Mg2+ ions are very effective at stabilizing tertiary structures in RNAs. In most cases, folding of an RNA is so strongly coupled to its interactions with Mg2+ that it is difficult to separate free energies of Mg2+-RNA interactions from the intrinsic free energy of RNA folding. To devise quantitative models accounting for this phenomenon of Mg2+-induced RNA folding, it is necessary to independently determine Mg2+-RNA interaction free energies for folded and unfolded RNA forms. In this work, the energetics of Mg2+-RNA interactions are derived from an assay that measures the effective concentration of Mg2+ in the presence of RNA. These measurements are used with other measures of RNA stability to develop an overall picture of the energetics of Mg2+-induced RNA folding. Two different RNAs are discussed, a pseudoknot and an rRNA fragment. Both RNAs interact strongly with Mg2+ when partially unfolded, but the two folded RNAs differ dramatically in their inherent stability in the absence of Mg2+ and in the free energy of their interactions with Mg2+. From these results, it appears that any comprehensive framework for understanding Mg2+-induced stabilization of RNA will have to (i) take into account the interactions of ions with the partially unfolded RNAs and (ii) identify factors responsible for the widely different strengths with which folded tertiary structures interact with Mg2+.

Ions and RNA folding.

The problem of how ions influence the folding of RNA into specific tertiary structures is being addressed from both thermodynamic (by how much do different salts affect the free energy change of folding) and structural (how are ions arranged on or near an RNA and what kinds of environments do they occupy) points of view. The challenge is to link these different approaches in a theoretical framework that relates the energetics of ion-RNA interactions to the spatial distribution of ions. This review distinguishes three different kinds of ion environments that differ in the extent of direct ion-RNA contacts and the degree to which the ion hydration is perturbed, and summarizes the current understanding of the way each environment relates to the overall energetics of RNA folding.