Core Histone Hyperacetylation Impacts Cooperative Behavior and High-Affinity Binding of Histone H1 to Chromatin

Abstract

Linker histones stabilize higher order chromatin structures and limit access to proteins involved in DNA-dependent processes. Core histone acetylation is thought to modulate H1 binding. In the current study, we employed kinetic modeling of H1 recovery curves obtained during fluorescence recovery after photobleaching (FRAP) experiments to determine the impact of core histone acetylation on the different variants of H1. Following brief treatments with histone deacetylase inhibitor, most variants showed no change in H1 dynamics. A change in mobility was detected only when longer treatments were used to induce high levels of histone acetylation. This hyperacetylation imparted marked changes in the dynamics of low-affinity H1 population, while conferring variant-specific changes in the mobility of H1 molecules that were strongly bound. Both the C-terminal domain (CTD) and globular domain were responsible for this differential response to TSA. Furthermore, we found that neither the CTD nor the globular domain, by themselves, undergoes a change in kinetics following hyperacetylation. This led us to conclude that hyperacetylation of core histones affects the cooperative nature of low-affinity H1 binding, with some variants undergoing a predicted decrease of almost 2 orders of magnitude.

Dynamic properties of independent chromatin domains measured by correlation spectroscopy in living cells

“…3g): One binding domain of H1.0 interacts with the entry–exit site of DNA at the nucleosome and either dissociates quickly or engages the second domain to form a longer-lived binding state, from which it dissociates again later. Deriving the rate equations for the different binding states allowed us to calculate the remaining parameters in differently treated cells [65] and in euchromatin and heterochromatin (Additional file 1: Eq. S95; Table 1): The residence time of H1.0 in the short-lived binding state was ~1 s, whereas the average residence time on chromatin was ~4 s. Thus, the fluctuations observed with FCS with relaxation times of ~100 ms did not result from association/dissociation events but rather from chromatin dynamics.…”

The PAX3 Paired Domain and Homeodomain Function as a Single Binding Module In Vivo to Regulate Subnuclear Localization and Mobility by a Mechanism That Requires Base-Specific Recognition

“…Tethering of the two domains leads to a significant decrease in the effective diffusion coefficient, which is reduced by 4.9-fold to 9-fold (or 4.1-fold to 7.6-fold when compared to full-length PAX3) ( Table 1). This reflects a greater level of lowaffinity binding [33][34][35] for the PDLHD and PAX3-GFP fusions in comparison to the isolated PD and HD. In addition, the lower overall slope of the PDLHD and PAX3-GFP recovery curves identifies a high-affinity binding component that is largely absent when analyzing the individual domains (Fig.…”

“…19 Kinetic modeling was carried out as previously described. [33][34][35] Briefly, the method involved fitting a reaction-diffusion equation to the FRAP data. The solution of this equation assumed rapid mobility of the nuclear protein and reversible binding events, and provided kinetic information relating to protein-protein and protein-DNA interactions.…”

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

“…A reliable indicator of chromatin remodeling is histone mobility as can be measured via fluorescence photoperturbation methods. Fluorescence recovery after photobleaching (FRAP), and, to a lesser extent, photoactivation, have yielded important insights into the dynamics and binding properties of histones in intact, native chromatin (Kimura, 2005; Beaudouin et al, 2006; Wiesmeijer et al, 2008; Martin and Cardoso, 2010; Raghuram et al, 2010; Stasevich et al, 2010). Mobility changes triggered by DNA damage can be investigated easily on a global scale, by treating cells with genotoxic agents such as chemicals or ionizing radiation prior to a FRAP or photoactivation experiment.…”