Glutaraldehyde Modified Mica: A New Surface for Atomic Force Microscopy of Chromatin

Abstract

We have found that mica surfaces functionalized with aminopropyltriethoxysilane and aldehydes bind chromatin strongly enough to permit stable and reliable solution imaging by atomic force microscopy. The method is highly reproducible, uses very small amounts of material, and is successful even with very light degrees of surface modification. This surface is far superior to the widely used aminopropyltriethoxysilane-derivatized mica surface and permits resolution of structure on the nanometer-scale in an aqueous environment, conditions that are particularly important for chromatin studies. For example, bound nucleosomal arrays demonstrate major structural changes in response to changes in solution conditions, despite their prior fixation (to maintain nucleosome loading) and tethering to the surface with glutaraldehyde. By following individual molecules through a salt titration in a flow-through cell, one can observe significant changes in apparent nucleosome size at lower [salt] and complete loss of DNA from the polynucleosomal array at high salt. The latter result demonstrates that the DNA component in these arrays is not constrained by the tethering. The former result is consistent with the salt-induced loss of histones observed in bulk solution studies of chromatin and demonstrates that even histone components of the nucleosome are somewhat labile in these fixed and tethered arrays. We foresee many important applications for this surface in future atomic force microscopy studies of chromatin.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner

“…Atomic Force Microscopy-To minimize the surface influence on the structure of TrmBL2-and histone-DNA complexes in our AFM experiments, we used glutaraldehyde-coated mica, which better preserves the native conformation of protein-DNA samples compared with freshly cleaved or (3-aminopropyl)triethoxysilane-modified mica (27,28). To prepare it, freshly cleaved mica was treated with 0.1% (3-aminopropyl)triethoxysilane solution for 15 min, then rinsed with deionized water, dried with nitrogen gas, and incubated in a desiccator overnight.…”

NAP1 Modulates Binding of Linker Histone H1 to Chromatin and Induces an Extended Chromatin Fiber Conformation

“…AUC experiments describe the unperturbed, global conformation of chromatin fibers under physiological conditions (37)(38)(39)(40)(41)(42)(43)(44)(45)(46), and SFM allows it to identify changes of the fiber geometry at the single nucleosomal level (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57). By using a combination of these two biophysical techniques in conjunction with biochemical characterizations, it is shown here that NAP1 mediates the reversible removal of the linker histones from chromatin fibers.…”

Micro- and nanofluidic technologies for epigenetic profiling

“…27 Since the structure of chromatin itself likely regulates aspects of gene transcription, 28 understanding these factors is of critical importance in probing epigenetics. Intensive research has been conducted to characterize the structural and functional properties of chromatin using diverse experimental and modeling techniques including atomic force microscopy (AFM), [29][30][31] optical/magnetic tweezers, 32,33 electron microscopy, 34,35 and X-ray scattering methods. 36,37 DNA is coiled around a nucleosome core comprising four pairs of histone proteins, and nucleosomes are connected to each other by linker DNA (often represented by a "beads-on-astring" model) ( Fig.…”