Linker histones play essential roles in the chromatin structure of higher eukaryotes. While binding to the surface of nucleosomes is directed by an ϳ80-amino-acid-residue globular domain, the structure and interactions of the lysine-rich ϳ100-residue C-terminal domain (CTD), primarily responsible for the chromatincondensing functions of linker histones, are poorly understood. By quantitatively analyzing binding of a set of H1 CTD deletion mutants to nucleosomes containing various lengths of linker DNA, we have identified interactions between distinct regions of the CTD and nucleosome linker DNA at least 21 bp from the edge of the nucleosome core. Importantly, partial CTD truncations caused increases in H1 binding affinity, suggesting that significant entropic costs are incurred upon binding due to CTD folding. van't Hoff entropy/enthalpy analysis and intramolecular fluorescent resonance energy transfer (FRET) studies indicate that the CTD undergoes substantial nucleosome-directed folding, in a manner that is distinct from that which occurs upon H1 binding to naked DNA. In addition to defining critical interactions between the H1 CTD and linker DNA, our data indicate that the H1 CTD is an intrinsically disordered domain and provide important insights into the biological function of this protein.Linker histones are essential proteins in higher eukaryotes that play multiple critical roles in eukaryotic chromatin, including defining nucleosome spacing (5, 14, 21), stabilizing folding and condensation of chromatin (7,32,37), and directly regulating gene expression (14, 32). In higher eukaryotes, these small, basic proteins are composed of three distinct domains: a well-conserved ϳ80-amino-acid (aa)-residue globular domain, a long ϳ100-residue C-terminal domain (CTD), and a short ϳ25-residue N-terminal tail (41) (Fig. 1A). While linker histone binding to the surface of nucleosomes is directed by the 80-aa globular domain (1, 6, 12), the chromatin-condensing functions of the protein are primarily provided by the highly basic CTD (2, 25). Indeed, the linker histones of some protists consist of only regions resembling the CTD (42). While the structure of the globular domain and its location within nucleosomes have been elucidated (29, 36), the specific interactions and structure of the CTD remain poorly defined.Previous analysis of the electrostatic properties of the C-terminal tail of linker histone H1 concluded that all basic residues are fully charged under physiological conditions (35). Moreover, analyses of the ion dependence of chromatin folding and the extent of DNA charge neutralization by H1 as predicted by Manning polyelectrolyte theory indicate that nearly all negatively charged residues in the CTD are involved in the neutralization of the linker DNA backbone in condensed chromatin (9). Thus, a simple model predicts a roughly linear association of the CTD with the linker DNA. However, recent work by Lu et al. provided evidence that two discontinuous regions of approximately 24 aa residues played distinct roles...
The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC 5 AC and MUC 5 B, but not MUC 2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the delta isoform.
We previously documented condensation of the H1 CTD consistent with adoption of a defined structure upon nucleosome binding using a bulk FRET assay, supporting proposals that the CTD behaves as an intrinsically disordered domain. In the present study, by determining the distances between two different pairs of sites in the C-terminal domain of full length H1 by FRET, we confirm that nucleosome binding directs folding of the disordered H1 C-terminal domain and provide additional distance constraints for the condensed state. In contrast to nucleosomes, FRET observed upon H1 binding to naked DNA fragments includes both intra- and inter-molecular resonance energy transfer. By eliminating inter-molecular transfer, we find that CTD condensation induced upon H1-binding naked DNA is distinct from that induced by nucleosomes. Moreover, analysis of fluorescence quenching indicates that H1 residues at either end of the CTD experience distinct environments when bound to nucleosomes, and suggest that the penultimate residue in the CTD (K195) is juxtaposed between the two linker DNA helices, proposed to form a stem structure in the H1-bound nucleosome.
Optogenetics provides promising tools for the precise control of receptor-mediated cell behaviors in a spatiotemporal manner. Most photoreceptors, yet, require extensive genetic manipulation and respond only to ultraviolet or visible light, which are suboptimal for in vivo applications because they do not penetrate thick tissues. Here we report a novel near-infrared light-activated DNA agonist (NIR-DA) nanodevice for nongenetic manipulation of cell signaling and phenotype in deep tissues. This nanodevice is prepared by conjugating a pre-inactivated DNA agonist onto the gold nanorods (AuNRs). Upon NIR light treatment, the DNA agonist is released through the localized surface plasmon resonance (LSPR)-based photothermal effect of AuNRs and becomes active. The active DNA agonist dimerizes the DNA-modified chimeric or native receptor tyrosine kinase (RTK) on cell surfaces and activates downstream signal transduction in live cells. Such NIR-DA activation of RTK signaling enables the control of cytoskeletal remodeling, cell polarization, and directional migration. Furthermore, we demonstrate that the NIR-DA system can be used in vivo to mediate RTK signaling and skeletal muscle satellite cell migration and myogenesis, which are critical cellular behaviors in the process of skeletal muscle regeneration. Thus, the NIR-DA system offers a powerful and versatile platform for exogenous modulation of deep tissues for purposes such as regenerative medicine.
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