Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome*

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones. Despite the renewed interest in histone H2A/H2B ubiquitination (1–3), the functional role of uH2A 1 still remains controversial. This is in contrast to ubiquitinated H2B where a strong correlation with transcriptional activation has long been established (2), and even a molecular mechanism involving a trans-histone regulatory pathway

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.
Despite the renewed interest in histone H2A/H2B ubiquitination (1)(2)(3), the functional role of uH2A 1 still remains controversial. This is in contrast to ubiquitinated H2B where a strong correlation with transcriptional activation has long been established (2), and even a molecular mechanism involving a transhistone regulatory pathway has been shown to be involved in this process (4,5). With uH2A, there is almost as much experimental evidence for its association to actively transcribing chromatin as there is to the opposite. For instance, it was shown that nucleosomes from the transcriptionally poised hsp70 and copia genes from Drosophila contain 50% uH2A (6). Also nucleosomes containing mono-and diubiquitinated H2A were found to preferentially occur near the 5Ј-end of the transcribed mouse dihydrofolate reductase gene (7). On the other hand, early studies with rat liver nuclei linked the disappearance of uH2A to increased transcription (8), and it was shown that Drosophila nucleosomes consisting of non-transcribed 1.705 satellite DNA showed an enrichment in uH2A (9). In support of these early data, a role for histone uH2A in Polycomb silencing has been demonstrated recently (10). Further-more uH2A has been shown to be concentrated in the heterochromatic inactive sex body in pachytene spermatocytes (11). Nevertheless the molecular mechanism(s) involved remains completely unknown.
Despite the controversy, ubiquitinated histones have been associated with chromatin partially depleted of linker histones (12) and transcriptionally active or poised sequences (1,2,6,7,13) that are thought to be have a reduced linker histone content or have altered association with linker histones (for a review, see Ref. 14). However, there is evidence that suggests that histone H2A ubiquitination does not interfere with linker histone binding. Two independent studies have shown that H1 can be cross-linked to uH2A in vitro (15) and in vivo (16). Moreover, in the cross-linking studies performed in mouse cells, the molar ratio of H1-uH2A to H1-H2A was the same as the molar ratio of uH2A to H2A (16).
Linker histones are required for the stabilization of well defined chromatin fibers (17)(18)(19), and interference with the binding of linker histones could affect chromatin condensation or the stability of the folded structures. Under physiological conditions, the structure of most linker histones is comprised of three domains: a strongly basic unstructured NH 2 -terminal tail, a nonpolar globular domain, and another strongly basic unstructured tail at the COOH terminus (20). The three-dimensional structure of the globular domain of histones H1 and H5 has been determined using two-dimensional NMR (21,22), and the structure of the globular domain of histone H5 was further resolved to 2.5 Å following the determination of its crystal structure (23). Interestingly the three-helix bundle structure of the globular domain of histone H5 has been reported to resemble that of the bacterial catabolite gene activator protein (22,23) as well as that of the DNA recognition motif of hepatocyte nuclear factor-3 (HNF-3), a Drosophila transcription factor (22). This structural similarity led to a model in which the primary binding site of linker histones to DNA is comprised of helix III of the globular domain binding to the major groove and binding of the ␤-hairpin to the adjacent minor groove (21,23).
Despite this, the position of the globular domain of a linker histone on the nucleosome has been quite controversial and may indeed be variable. The issue of the symmetrical or asymmetrical protection of the flanking DNA ends by histone H1 in the chromatosome remains unsettled (24,25). However, the precise location of the globular domain on the vicinity of the pseudodyad axis is now well established (for reviews, see Refs. 26 and 27).
The COOH terminus of H2A has been found to make contacts with the linker DNA in nucleosomes containing at least 167 bp of associated DNA (28,29). In the case of core particles lacking linker DNA, the H2A COOH tail repositions to contact the DNA of the dyad axis (29). The H2A COOH tail is therefore likely to have some influence on linker histone binding at the nucleosomal level since some DNA contact sites are shared. The crystal structure of the nucleosome (30) indicates that the H2A COOH-terminal tail emerges from the nucleosome near the proposed site of the linker histone globular domain. It has been shown that the contacts between the H2A COOH-terminal tail and the linker DNA are altered slightly in the presence of linker histone (31,32) indicating that the H2A tail domain is repositioned upon linker histone binding. Ubiquitination of H2A is a bulky modification that occurs at lysine 119 at the beginning of the COOH-terminal tail (1), and it would therefore be expected to interfere with linker histone binding.
In this work, we used a defined in vitro system using individually purified components to investigate the effects of histone ubiquitination on linker histone binding. The results obtained with this system indicate that, contrary to the expectations, ubiquitination of histone H2A did not preclude but rather favored the binding of linker histones to nucleosomes. This observation is in very good agreement with the latest transcriptional silencing role ascribed to this histone H2A modification (10).

Isolation of Chicken Erythrocyte Nuclei, Nucleosomes, and Core
Histones-Chicken nuclei and nucleosome core particles were prepared as described previously (33,34). Alternatively bulk nucleosomes were isolated from chicken erythrocyte nuclei following a protocol based on the method described by Olins et al. (35). Briefly in this method the nuclear chromatin (at approximately 6 mg/ml of DNA) in 0.1 M KCl, 50 mM Tris-HCl (pH 7.5), 1 mM CaCl 2 ) buffer is subjected to an extensive micrococcal nuclease digestion (150 units of micrococcal nuclease for 24 min at 37°C), and the whole nuclear digest is then hypotonically lysed by extensive dialysis against 0.25 mM EDTA. The dialysate is next centrifuged at 8,000 ϫ g for 20 min at 4°C, and the supernatant is dialyzed again against 0.1 M KCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5) to precipitate the linker histone-containing nucleosomes. Centrifugation under the same conditions as above leads to a supernatant consisting of linker histone-depleted nucleosome fraction and a linker histone fraction in the pellet. Histones from both fractions were extracted with 0.5 N HCl and precipitated overnight at 4°C with 6 volumes of acetone.
Purification of Linker Histones from Chicken Erythrocyte Nuclei-Extraction of linker histones from chicken erythrocyte nuclei was carried out using CM Sephadex C-25 as described by Garcia Ramirez and colleagues (36). Purified linker histones were stored in 50% (v/v) glycerol at Ϫ20°C and were found to remain stable for up to 1 year. Storage in glycerol counteracts the problem of histone precipitation that occurs upon repeated freezing and thawing of histone samples as reported by Kaplan et al. (37).
Fractionation and Purification of Calf uH2A-uH2A was purified from calf thymus histones with a slight modification of the protocols described elsewhere (38,39). Briefly ϳ250 mg of 0.4 N HCl-extracted histones were chromatographed at room temperature on a 4.5 ϫ 100-cm BioGel P60 (100 -200 mesh) column in 50 mM NaCl and 20 mM HCl buffer (40) at a flow rate of 40 ml/h. H2A fractions from three BioGel P60 column runs were pooled, adjusted to 6 M urea and 50 mM sodium acetate (pH 5.4), and loaded onto a 1.5 ϫ 20-cm Whatman CM 52 column equilibrated in 6 M urea, 50 mM NaCl, and 50 mM sodium acetate (pH 5.4). Bound proteins were eluted at room temperature with a linear salt gradient (125-175 mM NaCl) in the same buffer. Fractions were analyzed by SDS-PAGE, and those containing uH2A were pooled, dialyzed against 40 liters of deionized water, and lyophilized. Finally pooled fractions from three Whatman CM 52 columns were lyophilized; resuspended in 8 M urea, 2 mM Tris-HCl, and 10% (v/v) ␤-mercaptoethanol; and rolled for 4 h at 4°C. The pH was adjusted to 2.2 (39) with HCl, and the solution was applied to a 2.5 ϫ 100-cm BioGel P60 (100 -200 mesh) column equilibrated in 7 M urea and 2 mM Tris-HCl (pH 2.2). The flow rate used was 4 ml/h, and ϳ0.5-ml fractions were collected. Fractions containing pure uH2A were identified by SDS-PAGE analysis, pooled, and dialyzed against deionized water. Native 4.5% PAGE-Native 4.5% PAGE in 20 mM sodium acetate, 1 mM EDTA, 40 mM Tris-HCl (pH 7.2) buffer for the analysis of nucleosome core particles was carried out according to Yager and van Holde (41).
SDS-PAGE-Electrophoretic analysis of the histones was carried out by SDS-PAGE (20% (w/v) acrylamide, 0.1% (w/v) N,NЈ-methylenebis(acrylamide)) according to Ref. 42. On occasion (Western blots) the precast NuPAGE Novex bis-Tris gels from Invitrogen were used. Western Blot Analysis-Western blot analysis was carried out using a rabbit polyclonal antibody prepared in our laboratory. The primary antibody was used at a dilution of 1:1,000, and the dilution of the secondary (goat anti-rabbit) was 1:10,000.
Preparation of 167-bp Random Sequence DNA-Preparation of 167-bp nucleosome cores was carried out as described in Ref. 43. DNA was purified from these nucleosomes by phenol extraction.
Purification of Defined Sequence DNA Templates-p5S 208-12 plasmid containing 12 tandem repeats of a 208-bp sequence derived from the Lytechinus 5 S rRNA gene (44) was prepared as described elsewhere (45). The 208-12 DNA and 208 DNA were excised from the plasmid by HhaI and AvaI digestion, respectively. DNA fragments of interest were separated from the remainder of the plasmid by centrifugation through a linear 5-12% (w/v) sucrose gradient in TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) buffer for 16 h at 4°C at 111,000 ϫ g.
Reconstitution of Nucleosomes-Nucleosome cores were reconstituted onto purified 167-bp random sequence or sequence-defined 208-bp 5 S rDNA fragments using the reconstituted hybrid chicken erythrocyte octamers as a histone source containing either calf H2A (control) or calf uH2A and following the procedures already described (45).
Reconstitution of Linker Histones onto Nucleosome Cores-Nucleosomes were reconstituted with linker histones (chicken erythrocyte H1) by either direct addition (25) or by salt dialysis. In this latter instance, reconstituted nucleosome cores and linker histones in 550 mM NaC1, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4) were mixed in the ratios stated in the text and dialyzed for at least 4 h against 10 mM NaC1, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4).
The reconstituted products were purified by 5-20% (w/v) sucrose DNA Melting Profiles-Reconstituted nucleosome samples at a concentration of 50 g/ml in the buffer specified in the text were heated at a rate of 1°C/min from 30 to 100°C. Absorbance at 260 nm was recorded every 0.3°C using a Pye Unicam SP 1800 spectrophotometer equipped with a custom made temperature controller interfaced to an analog to digital device (Oasis/4, 3D Digital Design and Development Ltd., London, UK) and an IBM 386 computer. Where indicated, the first derivatives of A 260 versus temperature profiles were numerically deconvoluted by fitting a sum of Gaussian curves using a Marquardt-Levenberg algorithm. 2 Determination of Nucleosome and Chromatosome Positioning-The positioning of the histone octamer in the nucleosomes and chromatosomes reconstituted onto 208-bp 5 S rDNA was carried out as described elsewhere (46,47).

RESULTS
Histone uH2A Is Equally Distributed in Histone H1-containing and Histone H1-depleted Nucleosome Fractions-As mentioned in the Introduction there is some evidence that histone H1 does not preclude histone H2A ubiquitination (15,16). To further analyze this, chicken erythrocyte chromatin was extensively digested with micrococcal nuclease followed by dialysis in 0.1 M KCl. This is based on an early salt fractionation method described by Olins et al. (35) that allows fractionation of the linker histone-containing nucleosome fractions from the linker histone-depleted nucleosome fractions. The KCl-insoluble nucleosome fraction consisted of a DNA band centered around 165 bp (chromatosome) and a band centered at 120 bp resulting from nucleosome core particle overdigestion (see Fig.  1A, 2). Overdigestion of nucleosome DNA beyond 146 bp led to precipitation of the resulting subnucleosome particles. The DNA from the soluble fraction almost exclusively consisted of 146-bp DNA from nucleosome core particles (see Fig. 1A, 1). As it can also be seen in this figure, when a Western blot analysis was performed on an equivalent amount of histones obtained from both the 0.1 M KCl-soluble and -insoluble fractions using a ubiquitin-specific antibody, the intensity of the signal corresponding to uH2A was the same in both fractions (Fig. 1C).
Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding-Nucleosomes consisting of either H2A or uH2A were reconstituted onto random sequence 167-bp DNA, and the binding of histone H1 was assessed. Several unsuccessful attempts were made to reconstitute histone H1 by direct addition (results not shown). The failure of linker histone reconstitution by direct addition was attributed to the histone octamers adopting an equilibrium position on the 167-bp DNA that was unfavorable to successfully form chromatosomes. It has been shown previously that octamer position can affect linker histone binding (48). The short length (Յ22 bp) of the linker region available for H1 binding could also contribute to this problem.
Therefore, we decided to use a linker histone reconstitution technique that involves addition of the linker histones to nucleosomes or chromatin at elevated salt concentrations 2 P. Hü sler, unpublished.  1-6). The standards were total acid extracts of nuclei from chicken erythrocytes (CE) or calf thymus (CA). Ovalbumin was used as a protein carrier and was added to the samples just before precipitation. C, gels were scanned, and the quantity of H1 associated with each sample was determined by comparison with a total acid extract of calf thymus of similar intensity on the same gel. The data were fitted to lines representing the least squares. The solid line represents control 167-bp cores, and the dashed line represents uH2A hybrid 167-bp nucleosome cores. The data shown represent the average of four experiments. R 2 values were determined to be 0.981 and 0.989 for uH2A hybrid and control nucleosome cores, respectively. The ratio of H2A to H2B and H3 was used as an internal standard, and in all cases this ratio never deviated more than 5% compared with that of the standard calf thymus histones. H1s, H1 histones.
(0.5-0.6 M NaCl) and subsequent dialysis into low salt buffer (see "Materials and Methods"). At the high salt concentrations, the cationic charges of linker histones are shielded, and ionic interactions with the chromatin DNA are disrupted. If unfavorable octamer positions were inhibiting correct linker histone binding, this reconstitution protocol should allow repositioning of the octamer since it is known that octamers are mobile on DNA (49) especially at higher salt concentrations (50). The histone composition of the chromatosome peaks from the sucrose gradient fractionation following the reconstitution procedure are shown in Fig. 2, A and B. These results showed that uH2A did not prevent linker histone binding to 167-bp nucleosome cores in agreement with previous results (15,16) and that linker histone binding was slightly favored in the presence of uH2A. The H1 content of uH2A hybrid and control 167-bp cores reached the levels found in a total acid extract of calf thymus at a molar input ratio of 1 and 1.3 histone H1/167-bp nucleosome core DNA, respectively. A t test for independent samples with 95% confidence was performed at each histone H1 input ratio for the results shown in Fig. 2 using Statistica (Statsoft) software. The results indicated that there was no statistical difference between control and uH2A hybrid nucleosome cores except at a molar input ratio of 1.2 histone H1/nucleosome core.
Gel retardation assays were also used to monitor the re-association of histone H1 to control and uH2A hybrid 167-bp nucleosome cores by salt dialysis (Fig. 3). In agreement with Fig. 2, uH2A hybrid chromatosomes were formed at slightly lower histone H1 concentrations than those required to form control chromatosomes. Histone H2A Ubiquitination Does Not Lead to a Major Disruption of the Octamer-DNA Interactions in the Chromatosome-Control and uH2A hybrid 167-bp cores reconstituted at an input ratio of 1.2 mol of chicken erythrocyte histone H1/mol of nucleosome core (as above) were analyzed by thermal denaturation (Fig. 4). Under the conditions used, all samples exhibited a triphasic hyperchromicity curve. The first two transitions are thought to be associated with the melting of the DNA associated with the ends of the nucleosome core, while protein denaturation and melting of the DNA more tightly associated with core histones represent the main transition (51,52). In 167-bp control and uH2A hybrid nucleosome cores, the first transition occurred at 69 and 68°C and represented 13% (21 bp) and 15% (25 bp) of the DNA, respectively. This could reflect a slightly decreased interaction of histone tails with the ends of the nucleosome core DNA in the presence of uH2A. This decrease in protein-DNA interaction was more subtle than that caused by core histone acetylation where a decrease of 3°C in the T m of the first transition was observed (53). In the presence of linker histones, the first transition decreased in magnitude  lanes 1-6) by salt dialysis and analyzed on 0.7% (w/v) agarose gels in 0.5ϫ TBE. DNA represents free 167-bp DNA. Gels were stained with ethidium bromide. C, gel negatives were scanned, and bands were integrated using ImageQuant software to determine the percentage of chromatosomes at each linker histone input ratio. The solid line represents control 167-bp cores, and the dashed line represents uH2A hybrid 167-bp cores. and shifted to ϳ70°C for both control and uH2A hybrid chromatosomes. This decrease in magnitude of the first transition was slightly more pronounced for the uH2A hybrid chromatosomes than for control chromatosomes. The association of linker histones with the ends of the core DNA would be expected to stabilize this DNA, correlating with the observed increase in the T m value of the first transition of 1 and 2°C for control and uH2A hybrid chromatosomes, respectively. The second and third transitions of both samples occurred at 79 and 83.8°C, respectively, indicating that the presence of uH2A did not lead to large scale disruption of octamer-DNA interactions in good agreement with previous observations (38,54).
Histone H1 Equally Affects the Positioning of the Histone Octamer in the Chromatosome Regardless of the Ubiquitinated Nature of Histone H2A-We decided next to determine the effect of ubiquitination of H2A on the position of chromatosomes on 5 S rDNA. To this end, chicken histone H1 was reconstituted with control and uH2A hybrid nucleosomes by direct addition at 50 mM NaCl (25). As described above, this methodology was not suitable for reconstituting linker histones onto 167-bp nucleosome cores. However, direct addition of linker histones to 208-bp nucleosomes resulted in the formation of distinct chromatosome bands (see Fig. 5) suggesting that the additional linker DNA plays a role during linker histone binding.
To determine the affinity of the interaction between histone H1 and control and uH2A hybrid nucleosomes, the nu-cleoprotein gels such as those shown in Fig. 5, A and B, were scanned and integrated, correcting for UV quenching. In all cases the starting material consisted of ϳ80% nucleosomes and ϳ20% free DNA. From the Hill plots generated from this data (see Fig. 5), histone H1 was found to bind to control nucleosomes with an apparent equilibrium dissociation constant (K D ) of 33 nM and to uH2A hybrid nucleosomes with an apparent K D of 21.8 nM. From these results, it can be deduced that under the experimental conditions used, uH2A nucleosomes had a 1.5-fold increased affinity for histone H1 compared with control nucleosomes.
To confirm linker histone binding and to determine the influence of uH2A on chromatosome positioning, control and uH2A 208-bp nucleosomes were reconstituted with a molar input ratio of 0.75 histone H1/nucleosome and digested with micrococcal nuclease. Very definite pauses at 167 bp, characteristic of linker histone protection of DNA (55,56), were observed in both cases from the trial digests (not shown). Although kinetic stops at 167 bp are observed in nucleosomes (43,57,58), comparison of trial digests of nucleosomes reconstituted in the absence of H1 and chromatosomes demonstrated that 167-bp bands would no longer have been present in nucleosomes lacking linker histones at the optimal digestion time used to produce 167-bp bands from chromatosomes. Furthermore 208-bp uH2A or H2A reconstituted nucleosomes produced, under similar digestion conditions, only a 151-bp nucleosomal band. Fig. 6, A and B, shows the results obtained when the 167-bp bands were excised and digested with several restriction enzymes as described under "Materials and Methods." The results were essentially identical for control and uH2A hybrid nucleosomes. In the presence of histone H1, a dominant position (Ϫ4 to 163) was observed that is occupied by ϳ80% of chromatosomes (Fig. 6B, see position AЈ). This can be envisaged to correspond to a 10-bp protection of the DNA on either side of position A or a 20-bp protection downstream of position B (see Fig. 6B). This position and the shift from several other minor translationally related positions (such as that shown in Fig. 6B, position CЈ) are in close agreement with a major position of chromatosomes on n-mer arrays of 208 template DNA (47,59). A 10-bp pattern of protection on either side of the nucleosome (Fig. 6B, main position AЈ) agrees with earlier data (55,56,60,61). Under the experimental conditions used by us, no evidence could be found for asymmetric protection of linker DNA as has been observed with Xenopus 5 S rDNA (25,62,63). Interestingly this chromatosome position in Xenopus 5 S rRNA has been found more recently to be susceptible to sequence-specific artifacts, and another major chromatosome position that involves protection of 20 bp on one side of the nucleosome has also been reported (24,64,65). If this type of protection were the case here, it implies that the incorporation of histone H1 led to a shift in the equilibrium position of nucleosomes from position A to position B. Alternatively, since nucleosomes are more mobile in the absence of linker histones (66), it is possible that digestion with micrococcal nuclease could induce short range sliding of nucleosomes.
Since positions A and B are Ϫ10 bp apart, the rotational setting of the DNA relative to the histone octamer would be unchanged. A summary of nucleosome and chromatosome positions is shown in Fig. 6B. DISCUSSION Although the functional role of histone H2A ubiquitination still remains quite obscure, a strong correlation has been established over the years between this posttranslational modification and transcriptionally active chromatin (1-3). Transcriptionally active chromatin has long been associated to linker histone depletion (14). Hence it was of interest to deter- The results shown in Fig. 1 as well as other previously published data (15,16) clearly show that the occurrence of H2A ubiquitination can take place indistinctively in linker histonecontaining nucleosomes and chromatin. The in vitro studies conducted here on the binding of histone H1 to nucleosomes consisting of 167-bp random sequence or 208-bp sequence-defined (5 S rRNA) DNA (Fig. 3) are fully consistent with these findings.
It appears from the data that the presence of two uH2A molecules in nucleosomes does not preclude linker histone binding. These results are in agreement with earlier crosslinking studies that determined that the proximity of H1 to H2A was unchanged by ubiquitination (16). The interaction between high mobility group 14 and 17 is also not inhibited by nucleosomal uH2A (54).
Importantly, however, both the results from Hill plot analysis of the chromatosome formation on 208-bp DNA (Fig. 5) and the melting profiles that show a decrease in the first melting transition and a higher T m value (see Fig. 4) indicate that the occurrence of ubiquitination at the COOH end of H2A actually contributes to enhance the interaction of histone H1 with the nucleosome.
The COOH-terminal tail of H2A is known to contact the DNA at the ends of 167-bp nucleosome cores (28, 29) (see Fig. 7), and it undergoes some displacement upon linker histone binding (31,32). Since the site of ubiquitination of H2A (Lys-119) is in the COOH-terminal tail, it is possible that the presence of the ubiquitin moieties in the vicinity of the binding site of the globular domain of linker histones to the nucleosome (27) (Fig.  7) may create a more hydrophobic environment resulting in an enhancement of histone H1 binding. Alternatively this modification may lead to subtle repositioning of the C-terminal domain of linker histones to facilitate linker histone contacts with the linker DNA and affect chromatin folding. Such a situation may be more complex in vivo where the ionic environment and higher order chromatin structures might influence the position of the nucleosomes. Nevertheless this later possibility appears quite unlikely as we have already shown that the folding of nucleosome arrays is not affected by the occurrence of uH2A (33). Furthermore ubiquitination of histone H2A had no effect at all in nucleosome positioning (see Fig. 6).
The results of this study are highly reminiscent of those obtained with acetylated histones, another posttranslational modification of histones that has been strongly correlated with chromatin transcriptional activity (67)(68)(69). Similarly to what has been described here, it was found that core histone acetylation did not block the binding of linker histones to nucleosomes (70) and that this modification did not have any major represent nucleosomes reconstituted at molar input ratios of 0, 0.12, 0.37, 0.5, 0.62, and 0.75 histone H1/nucleosome, respectively. *, this band co-migrated with H1-bound 208-bp DNA. Gels were stained with ethidium bromide. C, Hill plot of histone H1 binding to control (black diamonds) and uH2A hybrid (empty squares) 208-bp nucleosomes. The fraction of H1bound nucleosomes (Y) was determined by integration of nucleoprotein gel scans. The results are the average of three experiments. The concentration of H1 was determined using a molecular weight of 21,782 g/mol (72). R 2 values of the linear regression lines for uH2A and control nucleosomes were determined to be 0.988 and 0.976, respectively. influence on either the mobility of the histone octamers or the positioning of the nucleosomes (71).
Our observations about the enhanced histone H1 binding to uH2A-containing nucleosomes are helpful in addressing the still controversial functional role of this histone H2A modification. They clearly support the early implications of a repressive role for uH2A (8,9,11) as well as its very recent involvement in Polycomb silencing (10). As we pointed out, the mechanisms by which uH2A contributes to PcG silencing are not known. In this regard, the higher histone H1 binding affinity for uH2A nucleosomes would clearly validate this in vivo observation while providing a potential contributing structural component of the molecular mechanism(s) involved. FIG. 7. Side view of a proposed molecular structure of a nucleosome consisting of two ubiquitinated H2A histones and the winged helix domain of histone H5. The DNA phosphodiester backbone is shown in blue; histones H2B, H3, and H4 are white; histone H2A is in yellow; ubiquitin is red; and H5 is in green. The structure of the nucleosome was obtained by using the crystal coordinates of the nucleosome core particle from Ref. 73. The winged helix structure of histone H5 was from Ref. 23, and the ubiquitin structure was from Ref. 74. The location and orientation of the histone H5 globular domain was as in Ref. 75.