Alterations in Nucleosome Core Structure in Linker Histone-depleted Chromatin

Abstract

We have previously shown that the sequential arrangement of histone-DNA contacts is essentially the same in the nucleosomal core of sea urchin sperm nuclei, where chromatin is highly condensed and repressed, and in nuclei from lily bud sepals or yeast, where chromatin is highly active in transcription and replication and is significantly or completely depleted of histone H1. However, the difference in the strength of some histone-DNA contacts has not been understood or discussed. In this work, we demonstrate that some of these differences are due to a conformational change in the nucleosomal core. We show that the nucleosomal core in linker histone-depleted chromatin is in a different conformational state compared with the nucleosomal core in folded chromatin or in isolated core nucleosomes. This conformational state is characterized by altered strengths in the histone H4 and H2A/H2B contacts with the regions of sharply bent nucleosomal DNA around sites +/-1 and +/-4 and site +/-5, respectively. We demonstrate that this conformation, which we call the "stretched nucleosome," is a general feature of unfolded linker histone-depleted chromatin and may occur during chromatin activation. Our results suggest that this nucleosome structural alteration does not depend on chromatin sources and histone variants studied in this work. In addition, we show that this alteration is reversible and is caused by the stretching of linker DNA during chromatin unfolding.

Nucleosome Structural Transition during Chromatin Unfolding Is Caused by Conformational Changes in Nucleosomal DNA

“…This increase in the stiffness may cause stretching of the nucleosomal DNA that in turn may change the conformation of the nucleosome (27) and affect histone-DNA contacts. In the present paper, we show that the alterations in histone-DNA contacts in isolated core particles induced at low ionic strength are caused by a decrease in the neutralization of negative DNA charges and are identical to those observed in nucleosomes during chromatin unfolding (19). This suggests that the conformational changes in nucleosome core particles at low ionic strength and in nucleosomes in chromatin during chromatin unfolding are due to the stretching of the nucleosome core DNA.…”

“…Micrococcal Nuclease Digestion of Linker Histone-depleted Chromatin-Linker histone-depleted chromatin prepared from chicken erythrocyte nuclei as described earlier (19) was digested with micrococcal nuclease (3 g/1 mg of DNA) in 10 mM Tris-Cl, pH 8.0, and either 0.3 mM CaCl 2 or 2 mM CaCl 2 for the time course of 20, 40, and 80 min at 37°C. The reaction was stopped by adding EDTA to a final concentration of 4 mM.…”

“…In this work, using densitometric analysis, we qualitatively assessed the variation in histone H2B and H4 signal intensities representing the extent to which a particular histone is crosslinked to a particular site of the nucleosomal DNA. The main cross-linking sites on the nucleosomal DNA for histone H2B are around nucleotides 109, 119, and 129 and for histone H4 are around nucleotides 57, 66, and 93 from the 5Ј-end of the nucleosomal DNA (19,29,30). To estimate the extent of variation in these contacts, we compared their relative intensity within the same histone line and within the same gel because, as shown previously, such a comparison prevents ambiguity caused by choosing the reference spot at different exposures of different gels (19,30).…”

“…Transcriptionally active chromatin is unfolded and significantly depleted of linker histones (16), which play a key role in chromatin compaction (17,18). Recently, we have reported that certain histone-DNA contacts in the nucleosome core are heavily attenuated during chromatin unfolding (19), indicating that the nucleosome structure is changed at the first step of chromatin activation, chromatin decondensation. These changes may facilitate the second step, which involves either remodeling or removal of the nucleosome.…”

“…In contrast, in linker histone-depleted chromatin, the distance between the points where the linker DNA enters and leaves the nucleosome core may be increased by repulsion between adjacent linker DNA segments (17,(21)(22)(23)(24) and thereby can affect the conformation of the nucleosome core DNA. To address the question of whether or not the alterations in histone-DNA contacts during chromatin unfolding, which we observed earlier (19), are caused by the changes in the nucleosome core DNA conformation, we have analyzed the histone-DNA contacts in isolated core particles under ionic conditions affecting DNA stiffness.…”