Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters

Abstract

Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter ‘601’ arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and ‘601’ DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers’ global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains.

The shelterin component TRF2 mediates columnar stacking of human telomeric chromatin

“…The force-extension curves (Fig. 4A ) of #Telo-18# and 601-20 arrays display similar features to previously studied nucleosome arrays (Brouwer et al, 2021 ; Kaczmarczyk, 2019 ; Korolev et al, 2022 ; Kruithof et al, 2009 ; Meng et al, 2015 ) with parameters in agreement with results for the identical arrays published earlier (Soman et al, 2022b ). Increasing the stretching force leads to a series of transitions reflecting the unfolding of the compact nucleosome array and gradual DNA unpeeling from the histone core.…”

“…To this end, we used single molecule force spectroscopy measurements using multiplexed magnetic tweezers (MMT). This method enables the analysis of folded single chromatin fibres without staining, fixation or surface deposition and to characterise the force-extension curve from sub-pN to tens of pN force, following the nucleosome unstacking to complete DNA unwrapping (Brouwer, 2020 ; Kaczmarczyk et al, 2018 ; Korolev et al, 2022 ; Kruithof et al, 2009 ; Meng et al, 2015 ; Soman et al, 2022b ). Subsequent data analysis yields mechanical (stiffness) and thermodynamic (free energies of nucleosome unstacking and DNA unwrapping) parameters specific to the chromatin fibre under investigation.…”

“…Most MMT data can be reliably fitted to the current statistical mechanics model used previously to describe nucleosome array stretching (Brouwer et al, 2021 ; Kaczmarczyk, 2019 ; Korolev et al, 2022 ; Meng et al, 2015 ). In the absence of any nucleosome positioning signal in the telomeric DNA sequence, independent methods are necessary to determine an NRL value required to fit MMT data for the #Telo-18# arrays.…”

“…The homemade MMT setup and flow cells used in this work were assembled at Nanyang Technological University using the design developed in the laboratory of Chromatin Dynamics of Leiden Institute of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University (Brouwer et al, 2018 ; Kaczmarczyk et al, 2018 ). A detailed description of the apparatus is given in (Korolev et al, 2022 ).…”

Genome organization across scales: mechanistic insights from in vitro reconstitution studies

“…For example, integrating histone modifications in reconstituted chromatin could allow for detailed investigation of the cause-consequence relationship between the ‘histone code’, chromatin organization, and transcriptional regulation [ 82 , 83 ]. In addition, the development of a system in which reconstitution of larger regions of chromatin [ 84 ] with in vivo -like nucleosome positioning is combined with single-molecule analysis of SMC complexes and/or in vitro transcription experiments could resolve important open questions about the interplay between nucleosome positioning, loop extrusion and transcription in the context of higher-order chromatin structures.…”