Studies on the Role and Mode of Operation of the Very-Lysine-Rich Histone H1 (F1) in Eukaryote Chromatin. Histone H1 in Chromatin and in H1 . DNA Complexes

Abstract

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 . DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the HI is complexed with the DNA rather than with the other histones. Microcalorimetric measurements reveal that the binding of H I to DNA is athermic or involves a heat of reaction which is very small indeed. This is thc second in a series of papers in which the role of the very-lysine-rich histone H1 in Chromatin structure is investigated by N M R and other methods. The first of the series [ l ] was devoted to the structure of the isolated histone.Chromatin can easily be extracted from calf thymus by isolation of clean nuclei, followed by gentle breakage of nuclear membrane to release the chromatin. The standard product of this process is a uniform gel having a DNA concentration in the region of 6 mgjg chromatin, together with the five histone fractions and a few percent of non-histone proteins. As has been reported previously [2] the gel may be caused to contract to about 15 ?< of its original volume by dialysing against sodium chloride solutions in the region of 0.05 -0.45 molar, while higher concentrations of sodium chloride remove the H1 and cause the contracted gel to relax to its original volume. The contraction of chromatin is dependent on the presence of H I , and the nuclear magnetic resonance (NMR) spectra of the gel may be attributed entirely to the HI component of the complex, the other histones apparently being bound much more tightly, so that their spectra are broadened and are unobservable under the conditions used. A more detailed study of the effects of various ions and of urea on chromatin gel Ahhreviurions. NMK, nuclear magnetic resonance; HI, hislone H1 (FI).has now been undertaken, and the results are presented in this communication. In addition studies have been made of the interaction between H1 and DNA alone, with the interesting result that both the NMR spectra and the macroscopic behaviour of the complex exactly parallel those in chromatin. This may be taken as an indication that the operation of HI in chromatin is largely or completely independent of the other histones. The formation of the H1 .

Conformational Studies of Two Non‐histone Chromosomal Proteins and Their Interactions with DNA

“…It is noteworthy that throughout the salt range over which the protein is bound to DNA (z 0-0.4 M NaCl) a welldefined protein spectrum is observed, suggesting that there is a portion of the molecule that is entirely free even under these conditions. This is in contrast to what is observed with histones, in particular H1 [15]. The presence of a section of protein 1 that always remains free from DNA could be related to the observation that DNA binds up to 10 times its own weight of high-mobility-group protein [3,4] and yet remains soluble.…”

“…The NMR spectrum of protein 1 was therefore observed in the presence of DNA at various ionic strengths at neutral pH. Previous experiments of this nature with histones have established that not only is the spectrum of the DNA so broad as to be unobservable under the normal spectrometer conditions, but so also is the spectrum of protein that is bound to the DNA [15]. Fig.8 shows the results for protein 1 together with the spectrum of free protein at the same ionic strengths and pH for comparison.…”

Closely Spaced Nucleosome Cores in Reconstituted Histone . DNA Complexes and Histone-H1-Depleted Chromatin

“…It is well known that these concentrations of mono-or divalent cations lead to a condensation of chromatin which is mediated by histone H1 [54]. Therefore we investigated the effect of removing histone H1 from chromatin on the degradation by DNase I1 in the presence and absence of divalent cations.…”

Studies on the Role and Mode of Operation of the Very-Lysine-Rich Histone H1 in Eukaryote Chromatin. The Isolation of the Globular and Non-Globular Regions of the Histone H1 Molecule

“…Interest in this histone fraction has been stimulated by the observation that they are apparently not, as are the other histones, involved in the basic subunit structure of the chromosome [2], but may be involved in its higher order structure. It is now quite clear that the H1 histones are the active agents in salt-induced chromatin condensation [3], and there is good evidence that they are also involved in mitotic chromosome condensation in vivo [4].…”

“…Interest in this histone fraction has been stimulated by the observation that they are apparently not, as are the other histones, involved in the basic subunit structure of the chromosome [2], but may be involved in its higher order structure. It is now quite clear that the H1 histones are the active agents in salt-induced chromatin condensation [3], and there is good evidence that they are also involved in mitotic chromosomePhysical studies on the H1 molecule in solution have shown that under physiological conditions of pH and ionic strength the molecule folds into a globular structure [S], and there is evidence that this structure does not involve the whole molecule. It would seem reasonable that this globular structure reflects some state of the H1 histone in chromatin, and we have invested some effort in attempts to isolate the globular portion of the molecule by chemical and enzymic cleavage.…”