Distinct Properties of the Two Putative “Globular Domains” of the Yeast Linker Histone, Hho1p

Distinct Properties of the Two Putative “GlobularDomains” of the Yeast Linker Histone, Hho1p

Tariq Ali and Jean O. Thomas*

Cambridge Centre forMolecular Recognition andDepartment of BiochemistryUniversity of Cambridge80 Tennis Court RoadCambridge CB2 1GA, UK

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has tworegions of sequence (GI and GII) that are homologous to the single globu-lar domains of linker histones H1 and H5 in higher eukaryotes. However,the two Hho1p “domains” differ with respect to the conservation of basicresidues corresponding to the two putative DNA-binding sites (sites Iand II) on opposite faces of the H5 globular domain. We find that GI canprotect chromatosome-length DNA, like the globular domains of H1 andH5 (GH1 and GH5), but GII does not protect. However, GII, like GH1and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor,and binds more tightly than GI to linker-histone-depleted chromatin.Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largelyunfolded, whereas GI, like GH1 and GH5, is structured, with a higha-helical content. However, in the presence of high concentrations oflarge tetrahedral anions (phosphate, sulphate, perchlorate) GII is alsofolded; the anions presumably mimic DNA in screening the positivecharge. This raises the possibility that chromatin-bound Hho1p may bebifunctional, with two folded nucleosome-binding domains.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: chromatin; chromatosome; four-way junction; intrinsicallyunstructured domain; linker histone*Corresponding author

Introduction

Chromatin contains, on average, about one copyof the linker histone H1 (or cell-type specific vari-ants such as H5) per nucleosome.1 H1 protects20 bp beyond the 146 bp of the nucleosome coreparticle from digestion with micrococcal nuclease2

to give the 166 bp chromatosome particle.Studies of salt-induced folding in vitro show thatlinker histones also promote the formation of anordered, stable higher-order structure in bulkchromatin3,4 and stabilise the folding of syntheticnucleosome core arrays.5 H1 has a tripartite struc-ture, with a central globular domain of about 80amino acid residues flanked by N-terminal, andlonger C-terminal, basic tails. The globular domainbinds to the body of the nucleosome and can

alone confer chromatosome protection,6 whereasthe C-terminal (and possibly the N-terminal) tailprovides counterions for the linker DNA, thusfacilitating salt-induced condensation.7,8 The twoDNA-binding sites inferred to exist on the globulardomain9,10 were proposed to be two clusters ofbasic residues on opposite faces of the domain11

and this was subsequently confirmed by muta-genesis.12 “Site I” appears to interact with DNAabout 5 bp from the end of chromatosomal DNA13

through a helix corresponding to the DNA-recognition helix in the sequence-specific,“winged-helix” proteins CAP and HNF3g, withwhich GH5 is structurally homologous;11,14 “siteII” interacts about 5 bp from the nucleosomedyad.13 Both site I and site II clusters of basicresidues are required for chromatosomeprotection, and for binding to four-way junctions,12

in which the juxtaposed arms presumably mimicthe closely apposed duplexes contacted in thenucleosome.12,15

The sequence of chromosome XVI of the yeastSaccharomyces cerevisiae16 revealed a candidatelinker histone, Hho1p,17,18 for which there hadbeen only circumstantial immunochemical

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: CD, circular dichroism; PCR,polymerase chain reaction; DTT, dithiothreitol; PMSF,phenylmethylsulphonyl fluoride; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; BSA, bovine serum albumin;dsDNA, double-stranded DNA; 1D, one-dimensional.

doi:10.1016/j.jmb.2004.02.029 J. Mol. Biol. (2004) 337, 1123–1135

evidence.19 The HHO1 gene was subsequentlyshown to be co-expressed with the core histonegenes in S-phase.20 Hho1p has an unusual domainstructure (Figure 1(a)), with two regions (GI andGII) homologous to the single globular domain ofH1 from higher eukaryotes (Figure 1(b)); GI andGII show 43% identity over ,90 amino acidresidues.17 They are linked by a region of about 40amino acid residues, similar in amino acid compo-sition and sequence to the much longer C-terminal“tail” of the canonical H1, which is missing inHho1p. The short basic N-terminal tail of Hho1presembles that of H1. Recombinant His-taggedHho1p binds to H1-depleted HeLa chromatinand protects ,166 bp DNA from micrococcalnuclease digestion.21 These observations takentogether support the suggestion based on sequencehomology that, despite its unusual domain organi-

sation, Hho1p is the yeast linker histone, associat-ing with the generally active and relativelyhighly acetylated22 yeast chromatin, in which thenucleosome repeat length is short (,165 bp23 or,160 bp24) and the linker length therefore essen-tially “zero”.

The presence of two regions in Hho1p withsequence homology to the globular domain ofthe canonical H1 from higher eukaryotes raisesobvious questions. A critical issue is whether bothregions represent functional globular domainswith the properties established for the globulardomains of H1 and H5 (e.g. chromatosome protec-tion, four-way junction binding). These propertiesrequire the two functional DNA-binding sites,sites I and II, characterised in GH5 as clusters ofthree and four basic residues, respectively,11,12

although replacement of single basic residues with

Figure 1. Hho1p and its domains. (a) Schematic of the domain structure of Hho1p (not to scale) and SDS/18% PAGEof full length Hho1p (lane 2) and the purified recombinant fragments NGL, GI and GII (lanes 3–5, respectively). Lanes6 and 7, recombinant GH5 (residues 24–97) and GH1 (residues 40–113). Lanes 1 and 8, protein molecular massmarkers (M). (b) A multiple-sequence alignment of the GI and GII domains of Hho1p and the globular domains oflinker histones from various species. (Alignment shown only for corresponding putative structured regions of the vari-ous globular domains, based on the known structures of GH5 and GH1; GH1 sequence is from the 1.1L H1 variant.)Numbers below the H5 sequence refer to the seven basic amino acid residues in the two putative DNA-binding sites,sites I and II; corresponding basic residues in the globular domains of the other proteins are shown in bold (six in theGI domain of Hho1p, four in GII). The secondary structure of GH511 is shown schematically below the GH5 sequence;arrows indicate b-strand.

1124 Yeast Linker Histone Domains

alanine had no obvious effect.25 The GI domainof Hho1p has basic residues at six of these sevenpositions and lacks only one residue at site I (corre-sponding to Lys69 of H5), which, however, is notconserved across all linker histones (Figure 1(b)).The GII domain, as well as lacking the same non-conserved residue at site I, also lacks two of thefour basic residues at site II, both situated in anexposed loop in the GH511 and GH126 structures.

In order to begin to address the structural roleof a second potential globular domain in Hho1p,we have asked whether the isolated GI and GIIdomains show the characteristic DNA and chroma-tin-binding properties of the globular domains ofH1 and H5. We find significant differences betweenGI and GII, as well as similarities. The isolateddomains, free of DNA, also show differences instructural stability.

Results

We have studied the properties of recombinantGI and GII from Hho1p, alongside NGL (GI withthe basic flanking regions; Figure 1(a)) and intactHho1p. The limits of the GI domain were deter-mined by characterisation of the stable productof tryptic digestion of Hho1p, which was shownby N-terminal sequence analysis and mass spec-trometry to be the 93-residue fragment comprisingresidues 38–130. No stable product correspondingto GII was detected, so residue 171 was chosen asthe N-terminal residue of the domain to be cloned,based on sequence homology with GI; the C termi-nus of the 88-residue fragment coincided with theC terminus of the protein (residue 258). The GIand GII domains and NGL (residues 1–170) thusdefined were cloned and the proteins expressedand purified (Figure 1(a)). Multiple-sequencealignment (Figure 1(b)) shows the high degree ofhomology between the GI and GII domains andthe globular domains of histone H1 from a rangeof species including fungi (Aspergillus, Ascolobus),as well as the globular domains of chicken H1 andH5 whose structures have been determined byNMR and X-ray crystallography, respectively.11,26

Basic residues corresponding to the basic clusters in

the two proposed DNA-binding sites (sites I and II)on GH5 are shown in bold.

Binding to DNA-cellulose

To compare the affinities of the proteins anddomains for general, mixed-sequence double-stranded DNA (dsDNA) they were bound toDNA-cellulose and eluted with increasing concen-trations of NaCl (Figure 2). Hho1p binds relativelytightly, with similar affinity to chicken erythrocyteH1 (Figure 2(a); 50% elution at ,400 mM NaCl forHho1p and 450 mM NaCl for H1). The GI and GIIdomains of Hho1p bind less tightly, as expected,50% elution occurring at ,125 mM NaCl for GIand ,225 mM for GII (Figure 2(b)); chicken GH1has a slightly higher affinity. NGL, which encom-passes GI (see Figure 1(a)), binds with significantlyhigher affinity than GI itself (50% elution at300 mM; Figure 2(b)), due to the addition of basicregions flanking GI. The lower affinity of GI thanof GII is consistent with the lower net positivecharge on GI (þ8 compared with þ12 for GII).

Binding to four-way junction DNA

Both H127 and its globular domain15 bind prefer-entially and “structure-specifically” to four-wayDNA junctions relative to linear DNA, for whichit has no significant sequence-specificity. To assessthe binding of Hho1p and its domains to four-wayjunctions, radiolabelled four-way junction DNAwas titrated with increasing amounts of Hho1p,GI, GII and NGL in the presence of a large excessof unlabelled sperm salmon DNA as a competitorin gel-retardation assays (Figure 3). Hho1p, NGLand GII all bind to the DNA to produce a singlecomplex, with half-maximal shift of the DNA atabout 50 nM, 750 nM and .5 mM, respectively(Figure 3(a)). GI does not bind to four-way junc-tions at all under the same conditions. However,when the concentration of competitor DNA is low-ered from 50 mg/ml to 50 ng/ml, GI does bind(Figure 3(b)) and behaves similarly to GII in thepresence of the higher concentration of competitor(Figure 3(a)). In the absence of any competitor

Figure 2. DNA-cellulose bindingassay. Comparison of the salt-dependent elution from DNA-cellu-lose of (a) Hho1p and H1; (b) GIand GII domains, and NGL, ofHho1p and the globular domainof H1 (GH1). Eluted protein wasquantified by densitometry ðn ¼ 2Þof stained SDS/18% polyacryl-amide gels.

Yeast Linker Histone Domains 1125

(Figure 3(b)) GI binds to give two complexes, prob-ably corresponding to two protein moleculesbound (non-cooperatively) to the junction; thehalf-maximal shift occurred at ,1.5 mM. Bindingof the GI domain with high affinity and specificityin the presence of excess competitor when it is pre-sent as NGL (Figure 3(a)), is presumably due toelectrostatic stabilisation of binding by the basicflanking regions. In summary, although they bind

with different affinities, the GI and GII domainsboth recognise four-way junction DNA and showa preference for this over linear DNA.

Binding to chromatin andchromatosome protection

To assess the ability of Hho1p and its domainsto bind to linker-histone-depleted chromatin,

Figure 3. Gel-retardation assay of the binding of Hho1p and its “domains” to four-way junction DNA. 32P-labelledfour-way junction DNA (2.5 nM) incubated with increasing concentrations of the various proteins (a) in the presenceof 50 mg/ml of competitor salmon sperm DNA, (b) in the presence of 50 ng/ml, or no, competitor, was analysed innative 6.5% polyacrylamide gels. Autoradiograms are shown.


H1/H5-depleted chicken erythrocyte chromatinthat had been incubated with Hho1p, NGL, GI orGII at one molecule per nucleosome was centri-fuged through 5% to 30% (w/v) sucrose gradientscontaining either 5 mM or 80 mM NaCl, to removeunbound proteins. The globular domain of H5,GH5, was used as a “control”.12 The chromatin-containing fractions identified by monitoring the

absorbance at 280 nm (A280) were analysed bySDS/18% PAGE to visualise bound proteins.Figure 4(a) (lanes 8, 9 and 11, 12) shows thatHho1p, NGL, GI and GII all bind to chromatin atlow ionic strength (10 mM Tris–HCl (pH 7.5),5 mM NaCl). Hho1p, NGL and GII also bind inthe presence of 80 mM NaCl (Figure 4(b), lanes 8,9 and 11, respectively), but GI (lane 12) showed

Figure 4. Sedimentation assay for binding of Hho1p and its “domains” to chromatin. H1/H5- depleted chickenerythrocyte chromatin incubated with Hho1p, NGL, GI or GII, or with the globular domain of H5 (GH5), was centri-fuged through 5% to 30% (w/v) sucrose gradients containing either (a) 5 mM NaCl or (b) 80 mM NaCl. The chroma-tin-containing peak fractions from the gradients (lanes 8–12) and the corresponding input (non-centrifuged) samples(lanes 1–5) were analysed by SDS/18% PAGE. Lanes 7 and 14 (dep.), linker-histone-depleted chromatin alone, withoutand with centrifugation, respectively; lanes 6 and 13 (native), unstripped chromatin, without and with centrifugation.(c) Gradient peak fractions for chromatin incubated with GH5 (lanes 1 and 4), GII (lanes 2 and 5) and GI (lanes 3 and6) at 5 mM NaCl (lanes 1–3) or 80 mM NaCl (lanes 4–6), resolved on a longer gel.


little or no binding at this salt concentration. This isshown more clearly in Figure 4(c), where the gelwas run for longer to give better resolution of GIfrom H4. Thus, the GI domain binds (presumablylargely electrostatically) to chromatin more weaklythan GII, as it did to DNA-cellulose (Figure 2(b)).

The ability of Hho1p and its domains to conferprotection from micrococcal nuclease on anadditional 20 bp beyond the core particle DNAlength and so generate a “chromatosome stop”was next tested. Hho1p and its domains were incu-bated with H1/H5-depleted chicken erythrocyteoligonucleosomes under conditions in which bind-ing had been demonstrated (Figure 4(a)) and thechromatin was then digested with micrococcalnuclease. Representative results are shown inFigure 5, which also shows the positive (GH5) andnegative (no added protein) controls (Figure 5(a)).GH5 protects the chromatosome-length DNA of,166 bp (indicated by p p ), whereas there is noprotection in the absence of any added proteinand the limit product of digestion is the ,146 bpnucleosome core-particle (indicated by p ). Hho1pand NGL, like GH5, show strong protection ofchromatosome-length DNA (,166 bp), as reportedfor Hho1p21 (Figure 5(b)). Figure 5(c) shows thatthe GI domain of Hho1p also affords protection of,166 bp, whereas GII consistently does not protectDNA longer than ,146 bp despite binding tochromatin more tightly than GI (Figure 4(c)), andbehaves similarly to the negative (“no proteinadded” control).

Folding of Hho1p, GII and NGL and the effectof various anions

In view of their different DNA-binding andchromatin-binding properties, GI and GII ofHho1p were tested for gross structural differencesby circular dichroism (CD) spectroscopy and one-dimensional (1D) 1H NMR spectroscopy. Far UVCD spectra measured in 10 mM sodium phosphate(pH 7.0), conditions under which the globulardomain of histone H1 is stabilised,28 showed sur-prising differences (Figure 6(a)). GI, like GH5,11,12,25

is largely a-helical as shown by the negativemolar ellipticity at 222 nm and 208 nm. In contrast,

GII has significantly less a-helix, and some randomcoil as indicated by the negative molar ellipticity at204 nm. Hho1p and NGL both contain a muchlower proportion of a-helix than GI, probably dueto an unstructured N-terminal region and inter-domain linker and, in the case of the full-lengthprotein, a largely unfolded GII domain. (The pro-portion of helix in Hho1p is consistently slightlylower than it is in NGL (Figure 6(a)), whereas itwould be significantly higher than in NGL if GIIwere folded.)

Previous studies have shown that large tetra-hedral anions induce secondary structure in intactH128 and in the C-terminal tails of H1 and H5,29,30

presumably mimicking the charge-screeningeffects that occur on DNA binding. We thereforetested the effects of various anions on GIIstructure. Figure 6(b)–(e) show CD spectra for GI,GII, NGL and Hho1p, respectively, in 10 mMsodium phosphate (pH 7.0), containing high con-centrations of sodium chloride (0.5 M), sodiumsulphate (0.5 M) or sodium perchlorate (1 M). Thespectra in buffer alone (“no salt”) are shown forcomparison. The spectrum of GI is essentiallyunchanged in the various salt conditions (Figure6(b)). In contrast there are significant structuralchanges in GII, NGL and Hho1p (Figure 6(c), (d)and (e), respectively) in the presence of sodiumsulphate and perchlorate but not 0.5 M sodiumchloride, which induces very little change. Figure6(f) compares the spectra of all the proteins in thepresence of 1 M sodium perchlorate, in which allshow significant a-helical structure. Under theseconditions, in contrast to 10 mM sodium phos-phate alone (Figure 6(a)), GII achieves an a-helixcontent similar to that of GI. The spectra alsosuggest that in NGL, and therefore probably inHho1p, the basic regions flanking the GI domain(i.e. basic N-terminal region and the inter-domainlinker in Hho1p) also adopt helical structure in thepresence of the large anions.

Similar conclusions about the structures of, andstructural changes in, GI and GII were drawnfrom 1D 1H NMR spectra. In 10 mM sodium phos-phate (pH 7.0) (Figure 7(a)), the GI spectrum(like that of GH5; data not shown) shows widedispersion of the amide backbone resonances at

Figure 5. “Chromatosome stop”assay. H1/H5-depleted chickenerythrocyte chromatin (weightaverage ,6–8 nucleosomes) wasincubated (a) with GH5 (as “posi-tive control”) or with no addedprotein (as “negative control”); (b)with Hho1p or NGL; (c) with GIor GII, and then digested withmicrococcal nuclease for varioustimes. DNA extracted from

samples taken at zero time (or one minute in the case of Hho1p and GII) (lane 1) and at two, five, ten and 15 minutes(lanes 2–5) was analysed in a 6.5% polyacrylamide gel; the mononucleosomal DNA region is shown. M, pBR322/Pst I DNA size marker (160 bp and 147 bp bands marked). ( p p ) and ( p ) indicate the approximate positions ofchromatosome (,166 bp) and core particle (,146 bp) DNA.


Figure 6. Circular dichroism (CD) spectra of Hho1p and of GI, GII and NGL in different ionic conditions. (a) and (f), GI, GII, NGL and Hho1p in 10 mM sodium phosphate(pH 7.0) (“no salt”) and 10 mM sodium phosphate plus 1 M NaClO4, respectively. (b)–(e), GI, GII, NGL and Hho1p in 10 mM sodium phosphate (pH 7.0) (“no salt”), and plus0.5 M Na2SO4 (black), 0.5 M NaCl (blue) or 1 M NaClO4 (green).

,7–10 ppm and sharp upfield-shifted methylresonances at 0–0.6 ppm, both characteristicfeatures of a folded protein; there were no signifi-cant salt-induced changes in the spectrum (notshown). For the GII domain in 10 mM phosphateboth these features are much less evident,suggesting that GII is largely unfolded. In contrast,in 0.1 M sodium phosphate (Figure 7(b)) the GI

and GII spectra are very similar, with well-dis-persed amide resonances and distinct upfieldmethyl resonances, indicating significant stabilis-ation of a folded GII domain under these con-ditions. There is no such folding of GII in 0.5 MNaCl (data not shown) but it is particularlymarked in 1 M sodium perchlorate (Figure 7(c));similar results were obtained in 0.5 M and 0.125 M

Figure 7. 1D 1H NMR spectra ofthe GI and GII domains of Hho1pin different ionic conditions. (a) GIand GII in 10 mM sodium phos-phate (pH 7.0); (b) GI and GII in100 mM sodium phosphate (pH7.0); (c) GII in 1 M sodium per-chlorate compared with GII in10 mM sodium phosphate.


sodium perchlorate as well as in 0.5 M sodiumsulphate (data not shown).

Discussion

Hho1p in S. cerevisiae contains two regions ofsequence homology to the central globular domainof the canonical histone H1 and is a candidatelinker histone, albeit with unusual domain organi-sation. An H1 homologue might be needed tostabilise the winding of two full superhelical turnsof DNA (,166 bp) around the histone octamerwhich, in the case of H1, is normally a property ofthe globular domain.6 The essentially zero inter-nucleosomal linker length in yeast23,24 obviates theneed for any significant charge neutralisation oflinker DNA; this is consistent with the absence ofa long basic C-terminal tail in Hho1p, althoughthe region between GI and GII in Hho1p is similarin amino acid composition and sequence to that ofthe canonical H1 tail and might perform a similarrole.

The existence of two putative globular domains,rather than one, in Hho1p raises the question ofwhether both are functional in terms of nucleo-some binding and chromatosome protection, andin essence whether Hho1p might be a bifunctionallinker histone. Both GI and GII have two of thethree basic residues originally proposed for theDNA-binding site I in GH5;11 the missing basicresidue (corresponding to Lys69 in GH5) is notconserved in H1 subtypes and variants acrossspecies (asparagine is often found at this position;Figure 1(b)) and is thus not an essential feature. GIand GII differ at the putative DNA-binding site II.In GI this contains four basic residues, as in GH5and GH1, whereas GII has only two (Figure 1(b)).We have assessed the extent to which theindividual GI and GII domains of Hho1p showthe DNA and chromatin-binding properties ofthe globular domain of the canonical histone H1,and find significant differences, as well assimilarities.

Like full-length Hho1p (and NGL) (Figure 5(b)),the isolated GI domain confers chromatosomeprotection during micrococcal nuclease digestion(Figure 5(c)) and, in addition, can form discretecomplexes with four-way DNA junctions (Figure3), although it binds with relatively low affinity toboth junctions and chromatin (Figure 4(c)) and isreadily sequestered from junctions on to an excessof competing linear DNA (Figure 3(a)). When GIis flanked by basic regions in NGL, it binds withhigh selectivity and affinity to four-way junctionsin the presence of excess competitor DNA (Figure3(a)). Although GII does not afford chromatosomeprotection (Figure 5(c)), in contrast to GI it bindswith high affinity to four-way junctions (Figure3(a)), probably at least in part because it is morebasic than GI. The junction/GII complex persistseven in the presence of a large excess of linear com-petitor DNA, unlike the complexes formed with

GI, which are abolished (Figure 3(a)). Taken atface value, these studies might suggest that two ofthe four basic residues at site II are sufficient forstructure-specific four-way junction binding byGII, and for chromatin binding, but not for chro-matosome protection.

The properties of both chromatosome protectionand preferential four-way junction binding in thecase of GI (the latter more obvious in NGL), andof junction binding in the case of GII, suggest thatGII as well as GI is likely to be folded in thepresence of chromatin and four-way junctions,although GII does not confer chromatosome pro-tection. This contrasts with the behaviour of GIand GII free in solution at low ionic strength, inthe absence of chromatin or DNA, where distinctdifferences in folding are apparent. CD (Figure 6)and 1D 1H NMR spectroscopy (Figure 7) showthat GII is largely unstructured in 10 mM sodiumphosphate, whereas GI is folded, as is the globulardomain of H1.28 The CD spectrum of Hho1p, aswell as the lack of resistance of GII to tryptic diges-tion of Hho1p (see Results), strongly suggests thatGII is also unstructured within the intact proteinunder these conditions, ruling out the possibilitythat the isolated recombinant GII domain mightbe unfolded because it is unduly truncated at theN terminus (the C terminus is that of Hho1p.). GIIthus appears to be one of a growing number of“intrinsically unstructured” proteins/domains.31

However, GII is structured in the presence of highconcentrations of large tetrahedral anions, whichprobably mimic the counterion properties of the ofDNA phosphate groups; this was also suggestedearlier for the C-terminal tails of H1 and H5,which assume a high a-helical content in thepresence of sodium perchlorate, but not sodiumchloride.29,30 We have recently determined thesolution structures of the isolated GI and GIIdomains in a relatively high concentration ofsodium phosphate32 and shown that the structuresare indeed very similar, containing a winged-helixfold as in GH1 and GH5; similar folding of GIImight occur when it binds to four-way junctionDNA and chromatin. A very similar structure forGI was also reported by another group33 when themanuscript was under review. GII was found tobe “virtually unstructured” in 10 mM sodiumphosphate, 100 mM sodium chloride (pH 7.2), inagreement with the results reported here; struc-turing conditions were not explored.

If the GI and GII domains in chromatin-boundHho1p are both folded they could, in principle,interact with nucleosomes in one of the two ways.First, the two domains might bind to adjacentnucleosomes in the short-repeat yeast chromatin,possibly stabilising nucleosome arrays. Second,both domains might bind within (and thus pos-sibly stabilise) the same nucleosome, each domainbinding asymmetrically, slightly off-dyad as forGH5,13 to a symmetry-related pair of sites. In thecase of H1 only one of these is normally occupied,1

although binding to a second, lower-affinity site


has been reported when an excess of H1 is addedto H1-depleted chromatin.34

If GII does indeed fold when it binds to linker-histone-depleted chromatin, it evidently does notbind in such a way as to protect 166 bp of DNAagainst micrococcal nuclease digestion, i.e. it doesnot generate a “chromatosome stop”. The mostobvious explanation would be that an impairedsite II does not locate correctly on the nucleosome.The lack of basic residues corresponding to K40and R42 of H5 (which lie in an exposed loop)might be particularly important, since residue 41of H5 is in close proximity to the DNA near thenucleosome dyad, as shown by site-specific pro-tein–DNA cross-linking.13 However, introductionof the missing basic residues at the putative site IIdid not result in a chromatosome stop under theusual conditions (S. Cooper & J.O.T., unpublishedresults), suggesting that the situation may be morecomplicated than simply a lack of two basicresidues.

Hho1p is clearly the yeast homologue of histoneH1 in higher eukaryotes. It is relatively abundantin yeast35 (T.A. & J.O.T., unpublished results)and widely distributed across chromosomalsequences35,36 (T.A. & J.O.T., unpublished results).It shows many of the properties of H1 in vitro,such as chromatosome protection and four-wayjunction binding. It is likely that it stabilises yeastnucleosomes but whether it plays a role in anyhigher-order structure that the short-repeat yeastnucleosome filament may adopt37 is not clear.Yeast strains in which the HHO1 gene is deletedare viable and were reported to have no detectablephenotype;18,21,38 however, loss of Hho1p hasrecently been reported to increase the rate atwhich DNA damage is repaired by homologousrecombination in S. cerevisiae,35 consistent with arole for Hho1p in maintaining chromatin structure,which might need to be loosened (e.g. by post-translational modification of Hho1p) to allowrecombination. Deletion of HHO1 in S. cerevisiaealso decreases the life span,35 as does silencingof the H1 gene in Ascolobus,39 possibly due to anincrease in homologous recombination, especiallyat ribosomal genes35 where, in yeast, an enrichmentof Hho1p has been reported.36 Microarray analysishas shown that transcription in a DHHO1 strain isgenerally modestly reduced (although only 27 ofthe 6216 genes examined had mRNA levelsreduced by more than twofold)40 so Hho1p is evi-dently not a general repressor and indeed appearsto be needed for the activation of some genes.Only a small number of genes showed an increase(less than 50%) in expression. Gene-specific effectsof linker histones on transcription41 have also beenreported in other organisms (e.g. in Tetrahymena42

which, however, has an atypical “linker histone”with no globular domain,43 and in Xenopus44).Deletion of Hho1p has no effect on the bulknucleosome repeat length or on the micrococcalnuclease cleavage pattern in two selected regions,21

and no effect on nucleosome positioning at a

particular locus.45 However, one possibility is thatHho1p does affect nucleosome positioning aroundcertain promoters and consequent exposure orocclusion of regulatory DNA sequences, throughan effect of limiting nucleosome mobility, aproperty that has been demonstrated for histoneH1 in vitro.46 In the case of Hho1p, this interpret-ation would be consistent with the microarrayanalysis of the effect of HHO1 deletion ontranscription.40 The role of the two potentialglobular domains in the function(s) of Hho1p andthe biological consequences, if any, of an intrinsi-cally unstructured GII domain remains to bedetermined.

Materials and Methods

Cloning of Hho1p and its domains (NGL, GI and GII)

PCR using Pwo polymerase (Boehringer Mannheim)was used to amplify the region corresponding to theHHO1 gene from yeast genomic DNA (yeast strainX4003-5B (MATa leu2, ade1, his4, met2, ura3, trp5, gal1,mal); Yeast Genetic Stock Centre). An Nde I restrictionsite encompassing the ATG start codon was introducedthrough the forward primer, and an in-frame stopcodon (TAA) and an Xho I restriction site through thereverse primer. The primers (restriction site nucleotidesshown in bold) were:

forward: 50-GGAATTCCATATGGCACCCAAGAAATCCACTACCAAGACC-30

reverse: 50-GCCGCTCGAGTTACGTGGAGAGTTTGACCTTCTTCTTGT0-30

The ,800 bp PCR product was digested sequentiallywith Nde I and Xho I, purified from an agarose gel andthen ligated with Nde I/Xho I-digested pET17b. The newconstruct was designated pET17b-HHO1.

The regions corresponding to NGL (residues 1–170),GI (residues 38–130) and GII (residues 171–258) weresimilarly cloned into pET17b, starting from pET17b-HHO1 as a template for PCR. The forward and reverseprimers were:

NGL forward: 50-GGAATTCCATATGGCACCCAAGAAATCCACTACCAAGACC-30

reverse: 50-GCCGCTCGAGTTACTTGGCGGTAACAGTAGGCG-30

GI forward: 50-GGCGGAATTCCATATGAAAAAGGAGGAAGCTTCCTCC-30

reverse: 50-GCCGCTCGAGTTAACTGACCTCTTTTTCTTTCTTTAC-30

GII forward: 50-GGAATTCCATATGAAGGCCTCTTCGCCTTCTTCATTGAC-30

reverse: 50-GCCGCTCGAGTTACGTGGAGAGTTTGACCTTCTTCTTGT0-30

All plasmid inserts were verified by automated DNAsequencing of both strands.


Expression, purification and characterisation ofHho1p and its domains

Escherichia coli BL21(DE3) cells47 containing the appro-priate pET17b plasmid encoding Hho1p or its domainswere grown (in terrific broth (TB) in the case of Hho1pand LB medium for the domains) to an absorbancemeasured at 600 nm (A600) of 0.5–0.6 at 37 8C with vigor-ous shaking, and expression was induced with 1 mMisopropyl-b-D-thiogalactoside; in the case of Hho1p thecultures were cooled to 23 8C immediately after induc-tion. The cells were harvested three hours after inductionand stored at 220 8C until needed. Cells were thawed inlysis buffer (10 mM sodium phosphate (pH 7.0), 1 mMEDTA, 1 mM dithiothreitol (DTT), 1 M NaCl, 0.5 mMphenylmethylsulphonyl fluoride (PMSF), 100 mg/mlbenzamidine, 100 mg/ml Na-p-tosyl-L-lysine chloro-methyl ketone (TLCK)) and lysed by passing twicethrough a French press. The lysate was centrifuged in aBeckman SW40 rotor at 35,000 rpm for one hour at 4 8C.The supernatant was diluted tenfold with buffer A(10 mM sodium phosphate (pH 7.0), 1 mM EDTA, 1 mMDTT, 0.5 mM PMSF) to a final NaCl concentration of0.1 M and loaded onto a 10 ml S-Sepharose Fast-FlowFPLC column (Pharmacia), pre-equilibrated with bufferA. Bound proteins were eluted with a linear gradient(eight column volumes) from buffer A to buffer B (bufferA plus 1 M NaCl) and fractions containing Hho1p, NGL,GI or GII, identified by SDS/18% PAGE,48 were pooled.Ammonium sulphate was added to a final concentrationof 2.5 M at 4 8C and the resulting suspension was centri-fuged at 7000 rpm in a Sorvall SS34 rotor for 30 minutesat 4 8C. The supernatant was then loaded onto a 20 mlPhenylSepharose FPLC column (Pharmacia), pre-equili-brated with buffer C (buffer A plus 2.5 M ammoniumsulphate). Proteins were eluted with a linear gradient(eight column volumes) from buffer C to buffer A andappropriate fractions identified by SDS-PAGE werepooled, dialysed against buffer A, and then loaded ontoa 1 ml Resource-S column (Pharmacia) pre-equilibratedwith buffer A. Proteins were eluted with a lineargradient (55 column volumes) from buffer A to bufferB. Fractions containing the pure proteins (Hho1p, NGL,GI or GII) were pooled, dialysed against 10 mM sodiumphosphate (pH 7.0), 1 mM EDTA, 1 mM DTT and storedin aliquots at 280 8C.

The proteins were characterised by matrix-assistedlaser desorption ionisation time-of-flight (MALDI-TOF)mass spectrometry and limited N-terminal sequencing,and their concentrations were determined from theamino acid analysis in conjunction with the knownamino acid sequence.49

DNA-cellulose-binding assay

Proteins (20 mg) were bound to DNA-cellulose (Sigma)pre-equilibrated with 10 mM Tris–HCl (pH 7.5), 0.1 mMEDTA, 0.5 mM DTT, 15 mg/ml BSA and eluted stepwisewith increasing concentrations of NaCl (0–700 mM forNGL, GI, GII and GH1; 0–1 M for Hho1p and H1) in thesame buffer.50,51 The proteins were precipitated with 25%(w/v) trichloroacetic acid and quantified by densitometryof stained SDS/18% polyacrylamide gels, with normalisa-tion for any variations in loading by reference to BSA.50,51

Gel-retardation assay with four-way junction DNA

Labelled four-way junction DNA (2.5 nM), formed by

annealing four 32P-end-labelled 30-mer oligonucleo-tides,52 was incubated (in a final volume of 10 ml) withvarious amounts of Hho1p or its domains (NGL, GI andGII) in binding buffer (10 mM Tris–HCl (pH 7.5),50 mM NaCl, 10% (v/v) glycerol, 100 mg/ml BSA, 1 mMDTT containing 50 mg/ml or 50 ng/ml (or no) sonicatedsalmon sperm DNA (800–1200 bp; Sigma) as indicated)at 23 8C for ten minutes, followed by 15 minutes at 4 8C.The samples were analysed by electrophoresis in 6.5%polyacrylamide gels containing TBE (89 mM Tris base,89 mM boric acid, 2 mM EDTA) which had been pre-run at 4 8C overnight at 50 V. Electrophoresis was carriedout at 4 8C for 1.5 hours at 150 V; vacuum-dried gelswere autoradiographed using pre-flashed Fuji-RX filmwith intensifying screens at 280 8C for about 16 hours.

Binding of proteins to H1/H5-depleted oligonucleosomes

Sedimentation assay

H1/H5-depleted chromatin (average length ,6–8nucleosomes), prepared by micrococcal nuclease diges-tion of chicken erythrocyte nuclei and sedimentationthrough 5% to 30% (w/v) sucrose gradients containing0.65 M NaCl,53 was dialysed against 10 mM Tris–HCl(pH 7.5), 0.2 mM EDTA, 5 mM NaCl, 0.5 mM PMSF.Hho1p or the various domains were added at one mol-ecule per nucleosome to the H1/H5-depleted oligonu-cleosomes (0.75 A260 unit) in 400 ml of 10 mM Tris–HCl(pH 7.5), 1 mM EDTA, 5 mM NaCl. The samples wereincubated for 30 minutes on ice and then analysed in5% to 30% (w/v) sucrose gradients containing 10 mMTris–HCl (pH 7.5), 1 mM EDTA, 0.5 mM PMSF andeither 5 mM or 80 mM NaCl, which were centrifuged ina Beckman SW28 rotor at 4 8C for 13.8 hours at28,000 rpm. The gradients were fractionated and moni-tored at 280 nm, and the peak fractions analysed forprotein content by SDS/18% PAGE.48

Chromatosome protection assay

Hho1p or its domains were added at one molecule pernucleosome to 2 A260 units of H1/H5-depleted oligo-nucleosomes in 400 ml buffer, and the samples were incu-bated on ice for 30 minutes and then for ten minutes at24 8C. CaCl2 was added to a final concentration of 2 mMfrom a 0.1 M stock, followed by 150 i.u. of micrococcalnuclease (Worthington). Samples were removed aftervarious times and digestion was stopped by the additionof 0.1 M EDTA (pH 8.0), to a final concentration of10 mM and chilling on ice. The deproteinised DNA wasanalysed by electrophoresis in 6.5% polyacrylamide gelscontaining TBE at 100 V for four hours. Gels werestained with ethidium bromide (0.5 mg/ml) and photo-graphed, with short wavelength UV transillumination,through a red filter using 35 mm (TMAX 400) film.

CD spectroscopy

CD spectra were recorded for proteins at 0.1 mg/ml in10 mM sodium phosphate (pH 7.0), and in the same buf-fer containing 0.5 M NaCl, 1 M NaClO4 or 0.5 M Na2SO4.The spectra were recorded in cuvettes with a 1 mm pathlength on a Jobin Yvon CD6 spectropolarimeter at 25 8Cover the range 190–250 nm, with measurements every0.5 nm. The average of four scans was subsequently pro-cessed for baseline subtraction using the manufacturer’ssoftware; the resulting spectra are represented as molar


ellipticity, [u], based on the appropriate mean residueweights. Spectra were smoothed using Kaleidagraph(Synergy Software).

1D 1H NMR spectroscopy

Samples (500 ml) contained 0.2–1 mM protein in10 mM sodium phosphate (pH 7.0), 10% (v/v)2H2O. NMR spectra were recorded at 298 K on a BrukerDRX 500 MHz spectrometer equipped with a tripleresonance probe with actively shielded z-gradients. Thespectral width was 10 kHz and 4K data points werecollected.

Acknowledgements

We thank Katherine Stott for recording the 1D 1HNMR spectra, Katherine Stott and Tim Stevens forgenerous help with some of the Figures and help-ful discussion, and Jim Murray for the yeast strain.We acknowledge the support of the Biotechnologyand Biological Sciences Research Council of theUK (BBSRC), and of the Engineering and PhysicalSciences Research Council (studentship for T.A).Amino acid analysis, protein sequencing, massspectrometry, oligonucleotide synthesis and DNAsequencing were carried out in the Protein andNucleic Acid Chemistry Facility and DNASequencing Facility of the Cambridge Centre forMolecular Recognition, which was supported bythe BBSRC and the Wellcome Trust.

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Edited by M. Yaniv

(Received 8 December 2003; received in revised form 7 February 2004; accepted 11 February 2004)


Documents

Distinct Properties of the Two Putative “Globular Domains” of the Yeast Linker Histone, Hho1p