Localization of the Sites Along Nucleosome DNA Which Interact with NH,-terminal Histone Regions*

Trypsin digestion of HeLa nucleosomes produces the same series of discrete histone breakdown products observed previously by others during digestion of chromatin; thus, trypsin excises the NH2-terminal ends of the histones from the chromatin core particle. The resulting nucleoprotein complex sediments at 9 S, has an increased molecular ellipticity at 280 nm, and has DNase I-susceptible sites at 10 nucleotide intervals. Nucleosomes containing a 32P label at the 5'-DNA termini were digested sequentially with trypsin and DNase I. Following trypsin digestion, the segments of nucleosome DNA 20 to 35 and 60 to 80 nucleotides from the 5' end became more susceptible to DNase I, suggesting that these segments interact with the trypsin-sensitive regions of the histones.

Trypsin digestion of HeLa nucleosomes produces the same series of discrete histone breakdown products observed previously by others during digestion of chromatin; thus, trypsin excises the NH,-terminal ends of the histones from the chromatin core particle. The resulting nucleoprotein complex sediments at 9 S, has an increased molecular ellipticity at 280 nm, and has DNase I-susceptible sites at 10 nucleotide intervals. Nucleosomes containing a :laP label at the 5'-DNA termini were digested sequentially with trypsin and DNase I. Following trypsin digestion, the segments of nucleosome DNA 20 to 35 and 60 to 80 nucleotides from the 5' end became more susceptible to DNase I, suggesting that these segments interact with the trypsin-sensitive regions of the histones.
The concept that eukaryotic chromatin is composed of nucleoprotein subunits, called % bodies" (1) or "nucleosomes" (2) seems well established, and a variety of models has been proposed for the structure of the chromatin subunit (1, 3-10). However, the details of nucleosome structure are not well understood; more complete knowledge of the protein-DNA interactions within the chromatin subunit is necessary in order to fully understand the mechanisms leading both to the packing of DNA within the nucleus and to the regulation of the expression of genetic information. For example, there may be only subtle differences between nucleosomes containing transcribed DNA sequences and those containing sequences which are repressed (11)(12)(13).
Therefore, we have developed methods for preparing homogeneous populations of intact nucleosomes (14)  The kinetics of digestion (not shown) indicates that the histones within the nucleosome are not all equally sensitive to the protease; H3 is degraded most rapidly, followed by H2A, H4, and finally H2B. Similar observations have been made previously (24-26). Fig. 1 shows that trypsin digestion of HeLa nucleosomes generates a characteristic population of cleavage fragments in a pattern which is virutally identical with those previously described (24,26,27). Analysis of such fragments by others has suggested that trypsin digestion results primar- Nucleosomes were digested for 0 min (left slot) or 128 min (right slot) and the reaction stopped with trypsin inhibitor.
The digest was made 1% in sodium dodecyl sulfate and 1% in /3-mercaptoethanol and was electrophoresed in a discontinuous 18% polyacrylamide gel. Migration was from top to bottom.
The positions of migration of the four smaller histones are indicated.
ily in the removal of 20 to 30 amino acid residues from the NH* end of each histone (24). We have isolated the trypsin-resistant nucleoprotein complex by gel filtration, using nucleosomes prepared from cells labeled in. uiuo with [3H]lysine and [3H]arginine. Fig. 2 shows that during gel filtration approximately half of the radioactivity in the digest passes through the column unretarded. This fraction contains all the input material absorbing at 260 nm and the proteins in this first peak have an electrophoretic pattern identical with that shown in Fig. 1. The protein/DNA ratio of these nucleoprotein complexes is 0.7 to 0.8 g/g, and they have an amino acid composition which is consistent with the idea that trypsin excises the NH,-terminal histone regions (Table I). Thus, these results, taken together, indicate that the organization of HeLa nucleoprotein, as measured by its digestion with trypsin, is indistinguishable from that of other tissues. Further, it seems that trypsin digestion of HeLa nucleosomes removes the lysine-and arginine-rich NH, ends of the histones, as first shown for chromatin by Weintraub and Van Lente (24).
Trypsin contains no detectable endonuclease or exonuclease contamination, as measured both by polyacrylamide gel electrophoresis of DNA purified from trypsin-digested nucleosomes and by measurement of acid-soluble radioactivity generated during trypsin digestion of nucleosomes labeled in viva for 128 min and the reaction stopped with trypsin inhibitor.
The mixture was made 10 rnM in MgCl, and layered over a column (1 x 5 cm) of Bio-Gel P-150 equilibrated with 10 mM MgCl,, 5 mM Tris/Cl, pH 8. Fractions were eluted with the above buffer and analyzed for radioactivity by liquid scintillation counting in Aquasol (New England Nuclear Corp.). solution after trypsin digestion. The results also reveal that trypsin digestion produces no increase in the heterogeneity of the nucleosome population (as measured by the breadth of bands on the gel), confirming the analysis of boundaries in the ultracentrifuge. Fig. 4 shows that the molecular ellipticity at 280 nm of trypsin-digested nucleosomes is about twice that of native particles, but is still less than half that of protein-free DNA. These results suggest that at least some of the DNA in trypsindigested nucleosomes has assumed a conformation more like that of protein-free DNA. We attempted to determine whether this increase in molecular ellipticity reflects an overall change in DNA conformation or whether certain regions of nucleosome DNA undergo a greater change than other regions; for example, following trypsin digestion, those DNA segments which interact in the native particle with the trypsin-sensitive regions of the nucleosomal histones might undergo a greater Thus, the trypsin-resistant histone regions must also play an important role in determining the general conformation of nucleosome DNA. Fig. 7 indicates that, for a given extent of DNase I digestion (as measured by acid solubility), the average DNA fragment size is smaller for trypsin-digested nucleosomes than for native particles.
This could be due either to (a) an overall increase in nuclease susceptibility for the entire nucleosome, or (6) an increase in the nuclease susceptibility of particular sites along the nucleosome. Examination of the autoradiogram (Fig. 8)  to several fundamental problems.
One involves the nature of the protein-DNA interactions required both for the Structure initial packing of the DNA into a nucleoprotein complex and for the subsequent maintenance of this complex as an ordered structure. A second involves the subtle alterations in this basic structure which may lead to the specific expression of genetic information.
The results of these experiments indicate that the complete removal of the trypsin-sensitive NH, ends of the histones leads to a change in the conformation of at least some of the DNA within the nucleosome (its increased molecular ellipticity and increased nuclease susceptibility) and yet does not destroy its structural feature of having nuclease-susceptible sites at lonucleotide intervals. These observations imply (a) that the NH2 ends of the histones are not required for the maintainance of the DNA in nucleosome-like conformation (although these NH, ends may be important in the initial folding of the DNA into a nucleoprotein complex) and (b) that the NH, ends do not, by themselves, determine the distribution of potential nuclease-susceptible sites along nucleosome DNA (although they do affect the relative susceptibilities of particular sites). These studies indicate that the nucleosome is a quite stable structure, since the rather drastic alterations in composition produced by trypsin digestion do not dramatically alter its structural features. Thus, the trypsin-resistant regions of the different histones must not only interact with each other to form the protein core of the nucleosome but must also have important interactions with nucleosome DNA.
Although removal of the NH, regions of the histones produces relatively minor changes in overall nucleosome structure, the nuclease susceptibility of certain sites along nucleosome DNA is substantially increased following excision of these regions. Following trypsin digestion, the sites 20 to 35, 60 to 80, and, likely, 105 to 120 nucleotides from the 5' end of the DNA, protected against DNase I digestion in the native particle, become more accessible to the nuclease. Our previous experiments suggested that the relative resistance of these sites to nuclease digestion arises from histone binding to DNA, preventing the formation of a productive metal. DNA. enzyme complex (18). Coupled with these earlier data, the findings presented here allow us to operationally define these particular sites along nucleosome DNA as loci which interact with the NH,-terminal regions of the four smaller histones. Trypsin digestion leads to a reduction in the sedimentation coefficient of the nucleosome from 10.7 to 9.0 S. In contrast, in the presence of 6 M urea, undigested core particles sediment at about 6 S (14). This suggests that the decrease in the sedimentation coefficient of trypsin-digested nucleosomes is primarily related to a loss of mass, rather than to any substantial change in overall conformation. This implies that the NH, ends of the histones are not required to maintain the nucleosome in its native compact shape. Thus, if the nucleosome is composed of two "half-nucleosomes" (lo), the NH, ends of the histones do not serve to bind the two halves together.
These experiments also show that the decrease in the interaction between nucleosome DNA and the NH, ends of the histones is accompanied by an increase in the interaction between the DNA and a different protein (in this case, a nuclease). This suggests the possibility that more subtle alterations in the interactions between nucleosome DNA and the NH, ends of the histones, such as those which might result from histone modification (28-331, might also increase the relative accessability of certain sites along DNA to other proteins, such as RNA polymerase, hormone.receptor com-plexes, or other regulatory macromolecules. Such decreases in the strength of interactions between DNA and the NH, ends of the histones might also play a role in the increased DNase I susceptibility of the transcribed regions of the genome (12, 13).