Name: Video: Nucleosome Reconstitution for Single-Molecule Chromatin Studies
Uploaded: 2024-09-06T12:50:00
Description: 84 Views. Nucleosome Reconstitution for Single-Molecule Chromatin Studies

nucleosome reconstitution for single-molecule chromatin studies

In Situ Nucleosome Reconstitution for Single-Molecule Studies

The primary packaging unit of eukaryotic chromatin is the nucleosome, in which ~147 base pairs (bps) of DNA are wrapped around an octamer of core histones proteins1,2. In addition to genome packaging, the nucleosome architecture serves as another rich layer of biophysical regulation that can be harnessed by chromatin-binding proteins when performing their various functions3,4. Experimentally accessing and measuring the physical characteristics of nucleosomes has been technically challenging since these units perform at minuscule scales (e.g., nanometer lengths, piconewton forces), and thus, their probing requires sufficient sensitivity and precision to meaningfully inform function. Moreover, chromatin-binding proteins often engage with their substrates transiently, and most ensemble approaches lack appropriate temporal resolution to inform the kinetics of these interactions5. Fortunately, the advent of single-molecule techniques has made it possible to visualize and manipulate individual proteins and their interactions in real-time, revealing mechanistic information about these molecular events occurring at the nanoscale6. In particular, single-molecule correlative force and fluorescence microscopy (smCFFM) harnesses both high-resolution fluorescence detection and force manipulation tools to simultaneously resolve the mechanics, composition, and coordination of chromatin and chromatin-protein complexes7,8.
In smCFFM that specifically employs &#34;optical tweezers&#34; as the force manipulation method, a tightly focused laser is used to trap a dielectric object, typically a micron-sized plastic bead, in three dimensions9. A biopolymer such as a piece of DNA can be conjugated to the bead (via, e.g., streptavidin-biotin, digoxigenin-anti-digoxigenin antibody linkage), and the user can both apply a calibrated force and measure the force/displacement generated by the system10. The added fluorescence module enables simultaneous multicolor fluorescence imaging to track protein composition and behavior.
The mainstream method for reconstituting nucleosome templates for smCFFM involves assembling nucleosomes on custom DNA templates by salt dialysis11,12,13. In this procedure, purified histone octamers are incubated with DNA templates in a high-salt buffer (~1 M sodium chloride), and as the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA in a sequence-dependent positional manner14,15. As such, it is customary that strong nucleosome positioning sequences (e.g., Widom 60116, X. laevis 5S rDNA17) are incorporated within the DNA templates to generate custom nucleosome arrays. Although this well-established method offers precise control over the placement and number of nucleosomes across DNA, it suffers from several disadvantages: (1) incorporation of strong nucleosome positioning DNA sequences may generate artificially stable nucleosomes that bias the activity of chromatin-binding proteins, (2) the process of salt dialysis for nucleosome formation requires high amounts of DNA and octamers, and (3) the procedure takes one to several days of work as well as a considerable effort to titrate the correct DNA to octamer ratio for optimal nucleosome array density.
In this manuscript, we describe an alternative method to form nucleosomes along DNA in situ within a single-molecule correlative force and fluorescence microscope using recombinant histone chaperone Nap1, which resembles the physiological pathway of nucleosome formation in vivo18,19,20,21,22. This method allows users to assemble nucleosomes on non-specific DNA sequences, easily adjust nucleosome density, and utilize significantly fewer amounts of reagents, all within minutes. Additionally, the formation of nucleosomal DNA tethers in situ enables simpler experimental workflow and the convenience of built-in single-molecule visualization and manipulation. Thus, when uniform and specific nucleosome positioning is not a necessity, this protocol offers a useful option for the investigation of nucleosome mechanics or protein behavior on chromatin, for which we also describe example assays.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosome Reconstitution for Single-Molecule Chromatin Studies

nucleosome-reconstitution-for-single-molecule-chromatin-studies

Research

JoVE Journal

Biochemistry

84 Views. Nucleosome Reconstitution for Single-Molecule Chromatin Studies

Video: Nucleosome Reconstitution for Single-Molecule Chromatin Studies

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...