(Received for publication, December 5, 1995; and in revised form, January 19, 1996)
From the
We have reconstituted nucleosomes containing the Xenopus
borealis 5 S rRNA gene, a single histone octamer, and 1 or 2
molecules of histone H1. We determine that the 1st molecule of histone
H1 to associate with the 5 S nucleosome binds with high affinity (K
2 nM), and the 2nd
molecule of H1 binds with a reduced affinity (K
10 nM). This latter binding is comparable with the
association of histone H1 with naked DNA. Neither molecule of histone
H1 alters the helical periodicity of DNA in the nucleosome as revealed
by hydroxyl radical cleavage. We conclude that although multiple
molecules of histone H1 can associate with nucleosomal DNA, there is
only a single high affinity binding site for histone H1 within the 5 S
nucleosome.
In vivo during the maturation of the chromatin of chicken erythrocytes, multiple molecules of linker histones have been shown to become associated with a single nucleosomal repeat length of DNA containing a single histone octamer(1, 2, 3, 4) . In contrast, the chromatin of mammalian tissue culture cells contains on average a single molecule of histone H1 per histone octamer(5, 6) . In vitro deconstruction and reconstruction experiments indicate that two H1-binding sites exist within each nucleosome and that the presence of 2 molecules of H1 per nucleosome generates a more compact structure than native chromatin(7) . The biological and structural significance of chromatin potentially consisting of a mixture of nucleosomes containing 0, 1, or 2 molecules of histone H1 has not been resolved.
More recently several investigators have made use of Xenopus or Drosophila chromatin assembly extracts to investigate the influence of chromatin structure on transcriptional regulation. These extracts are deficient in the normal somatic form of histone H1(8, 9, 10, 11, 12) and at least for Xenopus contain maternal H1 variants(13, 14) . Deficiency in the normal somatic form of histone H1 should have allowed these extracts to prove useful in determining the influence of this variant of histone H1 on transcription. However, the results of such experiments are controversial. Histone H1 has been variously reported to act as a general inhibitor of transcription within chromatin (10, 12, 15) or not to influence the transcription process (11, 16) or to selectively inhibit the transcription of particular genes(9, 17) . In vivo the selective inhibition of particular genes by the normal somatic variant of histone H1 is observed(18) .
One
possible explanation for the discrepancy in results is that chromatin
templates with different structural properties are being assembled as a
consequence of including histone H1 in varying stoichiometries. The
standard assay for the assembly of histone H1 into chromatin in the Xenopus and Drosophila assembly systems requires
measurement of the change in nucleosome spacing(19) . The
nucleosomal spacing increases from 180 bp ()without addition
of exogenous H1, to a repeat of 205 bp when 2 molecules of histone H1
are present per nucleosome, to a repeat of 220 bp when an excess of 5
molecules of histone H1 per nucleosome is present(19) . Without
histone H1, transcription in the Xenopus extract is repressed
by histone octamers alone only at very high densities (one per 160 bp),
but with histone H1 transcription was repressed at a ratio of 1.5
molecules of H1 per nucleosome, at a nucleosomal repeat length of 205
bp(10, 19) . Comparable results in which exogenous
histone H1 increases the spacing of nucleosomes were reported in Drosophila extracts; 1 molecule of histone H1 per nucleosome
increased nucleosome spacing from 190 to 210 bp, and 3 molecules per
nucleosome increased spacing to 220 bp(15, 16) .
However, transcription results were very different; Kamakaka and
colleagues (15) find that 3 molecules of H1 per nucleosome are
necessary to repress transcription, whereas Sandaltzopoulos and
colleagues (16) find that histone H1 makes a negligible
contribution to transcriptional repression.
A concern with these studies is whether an unusual or atypical chromatin structure is being assembled as a consequence of including much higher stoichiometries of histone H1 than are normally present in chromatin. The spacing of nucleosomes in a normal somatic cell is typically 180-200 bp(20) . Thus multiple molecules of histone H1 must be physically associated per nucleosome to generate the increase in nucleosome spacing to 220 bp. How might these additional molecules bind to nucleosomal DNA? We have investigated this issue using a positioned nucleosome containing a fragment of the Xenopus 5 S rRNA gene(21, 22, 23, 24, 25, 26) . We establish conditions under which a 2nd molecule of histone H1 can be stably incorporated into a 5 S nucleosome protecting additional linker DNA from micrococcal nuclease digestion. We find that the association of this 2nd molecule of histone H1 with nucleosomal DNA occurs with comparable affinity to the association of H1 with naked DNA. Thus, only a single high affinity histone H1 binding site exists on the 5 S nucleosome.
Figure 1: Histone H1 association with nucleosomal DNA. Gel retardation assays for H1 binding to a mixture of naked and octamer-associated DNA are shown. The 237-bp HpaII-DdeI fragment of pJHX1 was end-labeled using Klenow fragment of DNA polymerase at the HpaII site and reconstituted with purified core histones (see ``Materials and Methods'') such that approximately 50% of the DNA mass was associated with histone octamers. This mixture of reconstituted and naked DNA (50 ng total mass) was mixed with 0, 10, 20, 40, and 80 ng of histone H1 (lanes 2-6). Nucleoprotein complexes were resolved on a 0.7% agarose gel (see ``Materials and Methods''). An autoradiograph is shown. Lane 1 contains naked DNA alone (F). The positions of free DNA, octamer-bound (Oct) DNA, and nucleosomes containing 1 molecule of H1 (H1-Oct) or multiple molecules of H1 (2H1-Oct) and aggregates of H1 with DNA (Aggregates) are indicated.
We wished to further substantiate the stoichiometry of histone H1 association with nucleosomal DNA(21) . This was accomplished by recovery of nucleoprotein complexes from the non-denaturing gels followed by radioiodination of the associated proteins. Control iodinations of known masses of histone H1 and core histones allowed the stoichiometry of histone H1 to the histone octamer to be determined as 1 under the reconstitution conditions shown in Fig. 1, lane 3 (data not shown). The formation of aggregates of histone H1 with naked DNA that migrate as a diffuse smear (see Fig. 2C) prevents the exact stoichiometry of linker histones within the second octamer-containing complex to be determined; however, our iodination experiments indicate a stoichiometry of 2 or 3 molecules of histone H1 per octamer (not shown). These methodologies also allowed the demonstration that no histone H1 was associated with the DNA migrating at the position indicated as Free DNA in Fig. 1. We conclude that it is possible to form three types of nucleoprotein complexes in our H1 reconstitution experiments: complexes of the histone octamer and a single molecule of histone H1, complexes of the histone octamer and at least 2 molecules of histone H1, and complexes of histone H1 with naked 5 S DNA. We next examine both the nature and strength of association of histone H1 with DNA in these different nucleoprotein complexes.
Figure 2: Gel retardation assays for the measurement of the binding affinity of histone H1 to DNA when associated with a histone octamer (A) or when associated with a histone octamer and a single molecule of histone H1 (B) or when naked (C) (see ``Materials and Methods''). A, gel retardation assays for H1 binding to a mixture of naked and octamer-associated DNA (see ``Materials and Methods'' and note that carrier chromatin is present). Final histone H1 concentrations were 0, 0.19, 0.39, 1.0, 2.0, 3.0, 6.0, 13.0, 26.0, and 52.0 nM in lanes 1-10, respectively. B, gel retardation assays for H1 binding to DNA already associated with a histone octamer and a single molecule of H1 (see ``Materials and Methods'' and note that carrier chromatin is present). Lane 1 shows naked DNA; lane 2 shows DNA associated with a histone octamer; lanes 3-9 contain DNA associated with a histone octamer and final histone H1 concentrations of 10, 11, 13, 14, 17, 20, and 25 nM, respectively. C, gel retardation assays for H1 binding to naked DNA (see ``Materials and Methods'' and note that carrier DNA is present). Lane 1 shows naked DNA; lanes 2-9 contain histone H1 at concentrations of 11, 13, 14, 17, 20, 25, 33, and 50 nM, respectively.
Figure 3:
Binding titration for histone H1 binding
to naked DNA (), octamer-bound DNA (
), and
H1-octamer-bound DNA (
). Autoradiographs of gel mobility shifts
were scanned with a laser densitometer, and the fraction of histone
H1-bound DNA or nucleoprotein complex was plotted against the H1
concentration used.
Figure 4: Dissociation of histone H1 from nucleoprotein complexes. Left panel, dissociation of histone H1 from nucleosomal DNA. A mixture of naked and octamer-bound 5 S DNA (10 ng) (shown in lane 1) was incubated with 100 ng of histone H1 for 15 min before the addition of increasing amounts of linearized competitor DNA. Lanes 2-8 contain 0, 8, 16, 32, 63, 125, and 250 ng of competitor DNA, respectively. Right panel, as in left panel except 10 ng of naked 5 S DNA was used.
A feature of histone H1 association with 5 S nucleosomal DNA is that the additional linker DNA protected from micrococcal nuclease digestion is asymmetrically distributed with respect to the nucleosome core(21) . We next mapped the micrococcal nuclease digestion boundaries of the 175- and 200-bp DNA fragment using denaturing gel electrophoresis. We found the boundaries of histone-DNA interactions in the 175-bp DNA fragment to be tightly distributed as previously determined ((21) , not shown). However, the boundaries of the 200-bp fragment were much more diffuse and weaker than those obtained with the 175-bp fragment (not shown). The variation in the position of the boundaries of the 200-bp fragment indicates that either significant heterogeneity in the position of histone H1-DNA contacts exists or that these additional histone-DNA contacts are easily displaced during digestion.
Finally we examined the influence of incorporating 2 molecules of histone H1 into the nucleosome for the wrapping of DNA around the core histones. Previous work with 5 S monosomes and dinucleosomes had not detected any change in DNA structure on the surface of the histone octamer following inclusion of histone H1(21, 37, 38) . Hydroxyl cleavage of the 5 S nucleosome does not reveal any change in the helical periodicity of DNA on the surface of the histone octamer or in the extent of histone DNA interactions following inclusion of a single molecule of histone H1 (Fig. 5, compare lanes 3 and 4, and 8 and 9). Neither does addition of a 2nd molecule of histone H1 alter the wrapping of DNA within the nucleosome (Fig. 5, compare lanes 3 and 5, and lanes 8 and 10). We conclude that the incorporation of a 2nd molecule of histone H1 into the nucleosome does not significantly alter pre-existing contacts made by the core histones and histone H1 with DNA.
Figure 5: Structure of DNA in the 5 S nucleosome is not changed significantly by conclusion of multiple histone H1 molecules. Hydroxyl radical footprinting is shown. Reconstitution reactions as shown in Fig. 1, lanes 2, 3, and 5, were cleaved with hydroxyl radical (see ``Materials and Methods'') before resolution on a non-denaturing gel, excision of the appropriate complex, deproteinization, denaturation of DNA, and resolution on a denaturing gel (see ``Materials and Methods''). Lanes 1-5 and lanes 6-10 show the same samples subjected to electrophoresis for different times. Lanes 1 and 6 show a Maxam-Gilbert G reaction for markers (G); lanes 2 and 7 show hydroxyl radical cleavage of naked DNA (N); lanes 3 and 8 show cleavage of octamer-bound DNA (C); lanes 4 and 9 of H1-octamer show cleavage of bound DNA (A); and lanes 5 and 10 show cleavage of 2H1-octamer-bound DNA (B). The ellipsoids show the position of core (shaded) and chromatosome (open) boundaries for lanes 6-10. The asterisks mark the dyad position for the nucleosome core.
These experiments were designed to examine the nature of a defined sequence nucleosome containing 1 or more molecules of histone H1. The major conclusion is that although multiple molecules of histone H1 can bind to DNA wrapped around a single histone octamer, only a single preferential high affinity binding site exists for histone H1 (Fig. 1Fig. 2Fig. 3Fig. 4). Structural changes to this defined sequence nucleosome following inclusion of more than one histone H1 molecule are minor (Fig. 5). Although these results are obtained with a particular nucleosome they have general implications for studies interrelating the influence of histone H1 on chromatin structure and transcription.
The binding of a 2nd molecule of histone H1 to nucleosomes may occur in vivo(4) and has been documented in vitro (Refs. 7, 15, 16, and 19, and this work). The association of additional molecules of basic protein with nucleosomal DNA is not surprising, since additional histone octamers can also bind to the nucleosome(46) . The association of additional molecules of histone H1 during chromatin assembly does, however, lead to a decreased density of nucleosomes on the DNA molecule and an increased length of DNA bound by H1 away from core histone-DNA contacts(15, 16, 19) . The additional molecules of histone H1 bound to nucleosomal DNA under these conditions are likely to be bound more weakly than the 1st molecule to bind to the nucleosome (Fig. 1Fig. 2Fig. 3Fig. 4). Thus it is possible that the repressive character of chromatin will decrease as more histone H1 is reconstituted into chromatin. This result is in fact observed by Sandaltzopoulos et al.(16) .