DNA AND CELL BIOLOGYVolume 10, Number 4, 1991Mary Ann Liebert, Inc., PublishersPp. 239-248

REVIEWHistone Hl-DNA Interactions and Their Relation to

Chromatin Structure and Function

JORDANKA ZLATANOVA and JULIA YANEVA*

ABSTRACT

The belief that histone HI interacts primarily with DNA in chromatin and much less with the protein com-

ponent has led to numerous studies of artificial Hl-DNA complexes. This review summarizes and discussesthe data on different aspects of the interaction between the linker histone and naked DNA, including coop-erativity of binding, preference for supercoiled DNA, selectivity with respect to base composition and nu-cleotide sequence, and effect of HI binding on the conformation of the underlying DNA. The nature of theinteraction, the structure of the complexes, and the role histone HI exerts in chromatin are also discussed.

INTRODUCTION

The basic structure of interphase chromatin is well es-tablished. Histones H2A, H2B, H3, and H4 form the

core of the nucleosome while the fifth histone class —thelysine-rich histone HI—binds to the linker DNA sealingtwo turns of DNA around the nucleosomal core (Allan etai, 1980). Histone HI is also involved in forming higher-order, structures of the chromatin fiber (for reviews, see

Igo-Kemenes et ai, 1982; Thomas, 1984). Some authorshave implicated histone HI as a factor in the regulation ofgene activity as well (Weintraub, 1985; Zlatanova, 1990b).

The belief that histone HI interacts primarily with DNAin chromatin has led to intensive studies of artificial Hl-DNA complexes as model systems. Interest in such com-

plexes was based on correspondence between the salt-in-duced compaction of the nucleosomal fiber and the transi-tion from noncooperative to cooperative binding that isobserved under similar salt conditions. The usefulness ofHl-DNA complexes as a model for the interaction of thehistone with chromatin led to numerous studies of variousaspects of the protein-DNA interaction. Despite the largeliterature, however, general agreement has been reachedonly on a few points. This review will summarize the exist-ing data concerning artificial Hl-DNA complexes and dis-cuss those points which, in our view, are still controversialand require additional experimental work.

GENERAL CHARACTERISTICSOF HISTONE HI

The primary structure of histone HI is characterized byan uneven distribution of charged amino acid residuesalong the polypeptide chain. The amino and the carboxyltermini contain a great number of lysine, arginine, andproline residues. The central part of the molecule is consid-erably less basic and contains the bulk of the hydrophobicamino acid residues. This primary structure is relativelywell conserved during evolution but is more variable thanthat of the core histones (von Holt et ai, 1979; Wu et ai,1986).

The asymmetrical distribution of amino acids along thepolypeptide chain is directly reflected in the secondary andtertiary structure of the histone molecule. Protease diges-tion studies and physicochemical analysis indicate a three-domain structure (Cary et ai, 1981), consisting of a basicrandom-coiled amino terminus of 20-30 amino acids, acentral globular domain of about 80 mainly hydrophobicresidues, and a basically charged random-coiled C-tail ofabout 100 amino acid residues. The central globular do-main is conserved in evolution while the N-"nose" and theC-"tail" are responsible for the observed evolutionary andsubtype variability.

Histone HI is usually present in each cell as a family ofclosely related molecular species. The HI complement is

Institute of Genetics and »Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

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240 ZLATANOVA AND YANEVA

tissue specific and species specific and changes during thecourse of development and differentiation. In addition tothe "classical" HI subtypes present in most cells, there are

some HI subfractions only found in terminally differenti-ated cells. Examples include the male gamete-specific HI,histone HI0, characteristically present in nondividing tis-sues (Panyim and Chalkley, 1969), histone H5 in the nucle-ated erythrocytes of birds and some fish (Neelin et ai,1964; Miki and Neelin, 1975), and Hls in erythrocytes andliver of reptiles and amphibians (Brown et ai, 1981).These differentiation-specific subtypes contain more argi-nine and less lysine than the conventional set of HI. Thoserepresentatives of the differentiation-specific proteinswhose secondary structures have been studied (Hl0, H5)also have a typical nose-head-tail configuration. This con-

figuration seems to be important for histone function.

FORMATION AND APPEARANCE OFHl-DNA COMPLEXES

There are two approaches generally used for preparingHl-DNA complexes. The first consists in direct mixing ofthe two components in solutions of the final ionic strength.The second approach is based on step-dialysis of the mix-ture from an ionic strength high enough to prevent interac-tions to the selected final salt concentration. Both methodsgive comparable results.

The appearance of the complexes formed under a vari-ety of conditions has been studied by electron microscopyand was shown to differ as a function of the ionic condi-tions and the histone/DNA ratio. At low salt concentra-tions (usually below 20 mM Na*) and at relatively low HI/DNA ratios, the complexes appear as thin filaments ordouble fibers formed by parallel alignment of two DNAmolecules bridged by the histone or by intramolecularbackfolding of the DNA double helix (von Mickwitz et al.,1979, 1988; Bottger et ai, 1981, 1984; De Bernardin et ai,1986; Clark and Thomas, 1986, 1988). Increasing the his-tone/DNA ratio or the salt concentration leads to the ap-pearance of rods or cable-like structures thought to consistof thin filaments packed side by side (von Mickwitz et al.,1979; Bottger et ai, 1981; Clark and Thomas, 1986; DeBernardin et ai, 1986). If DNA is long enough, the rodsare circulized (Clark and Thomas, 1986, 1988); interest-ingly, histone HI, H5, and the sea urchin sperm-specificvariant spHl differ in the efficiency with which they formcircles (Clark and Thomas, 1988). Finally, "doughnut"-shaped complexes form at high ionic strength (0.17 MNaCl) under conditions of extensive neutralization of thenegative charges of the DNA molecule (Hl/DNA ratio of1.3) (Hsiang and Cole, 1977).

COOPERATIVITY OF INTERACTION ANDPREFERENCE FOR LONG DNA FRAGMENTS

The term "cooperativity of interaction" of HI with DNAis widely accepted to mean that, under specific conditions,HI binds extensively to some DNA molecules but only par-

tially or not at all to the rest (Renz and Day, 1976). The in-teraction of HI with DNA appears to be noncooperative atsalt concentrations below 20 mM Na* (Renz and Day,1976; Clark and Thomas, 1986; De Bernardin et ai, 1986);in the range of 20 to 50 mM there is a sharp transition to

cooperative binding. The complexes formed through coop-erative interactions can also be formed at low salt concen-

trations at sufficiently high protein/DNA ratios (Clark andThomas, 1986; De Bernardin et ai, 1986). Recently, we

showed that the interaction at 50 mM NaCl can still be ofthe noncooperative nature characteristic of low-ionic-strength buffers, even at high protein/DNA ratios (Panevaet ai, 1990). It is evident that salt concentration, his-tone/DNA ratios, and DNA size are not the only factorsinvolved in determining the cooperativity of interaction.

The term "cooperativity of interaction" has been used byother authors in a broader sense to denote the interactionleading to the simultaneous presence of two or more dis-tinct Hl-DNA complexes in a mixture. Two such subpop-ulations (slow and fast) of Hl-DNA complexes have beenobserved at 20 mMby Renz and Day (1976) and by Bottgeret ai (1981), who described complexes of 25S (double fi-bers) and 120S (bundles of double fibers). The latterauthors argued that the formation of these two types ofcomplexes resulted from cooperative interactive processes.

Several studies investigated the structure of Hl-DNAcomplexes formed under either cooperative or noncooper-ative conditions. De Bernardin et al. (1986) demonstratedthat histone HI could form two different kinds of com-

plexes with superhelical DNA. The first consists of solublecomplexes containing different amounts of HI (dependenton the input Hl/DNA ratio) bound to one DNA molecule.These were formed with low cooperativity; HI appearedto react preferentially in the vicinity of HI already boundto individual DNA molecules. The authors suggest that thebinding to DNA may be driven by the distribution anddensity of charge on the DNA, which would favor reactionwith DNA having two helically twisted duplex strands.Preexisting DNA supercoils are stabilized by HI: theDNA-relaxing enzyme did not affect preformed Hl-DNAcomplexes at relatively high Hl/DNA ratios (more than0.4); i.e., HI interfered with the action of the relaxingenzyme, thus supporting this view (Bina-Stein and Singer,1977).

The second kind of complex —insoluble aggregates—

was formed as soon as a critical ratio (dependent on theionic strength) of HI to DNA was exceeded (De Bernardinet ai, 1986). These had a cable-like appearance and were

formed cooperatively as they coexisted with free DNAmolecules. Similar types of complexes were observed byClark and Thomas (1986), who studied complex formationbetween HI and linear DNA fragments as a function ofionic strength and input protein/DNA ratio. At low ionicstrength, HI molecules bound distributively to all DNAmolecules generating soluble complexes containing twoDNA duplexes. As the ionic strength increased, anothertype of complex formed that existed together with protein-free DNA molecules; it contained several DNA molecules(apparently aligned in register) and closely apposed HImolecules, being capable of chemical cross-linking.

HISTONE Hl-DNA INTERACTIONS 241

Clark and Thomas (1986) suggested that the HI-HI in-teractions involved in the cooperative binding of HI toDNA may be either direct hydrophobic interactions be-tween globular domains or indirect interactions mediatedthrough local changes in DNA conformation, such as

bending. The view that direct hydrophobic interactions be-tween HI molecules underlie the cooperativity of bindingis somewhat contradictory to the conclusion of Singer andSinger (1978) that the globular domain (which is the hydro-phobic domain of the molecule) is not necessary for coop-erativity.

The cooperative binding of HI to supercoiled and re-laxed DNA has also been studied by Aviles et al. (1978),Watanabe (1986), and Triebel et ai (1988). Watanabe(1989) has demonstrated that the binding of HI to solublerat liver chromatin is also cooperative, in agreement withearlier data of Renz et ai (1977), but contrary to the viewsof Caron and Thomas (1981) and of Huang and Cole(1984). However, the studies of Renz et ai (1977) and ofWatanabe (1989) were performed in a severalfold excess ofHI over its normal amount in chromatin, so the resultsmight not represent the situation in vivo. Cooperativebinding of HI to chromatin is of great importance tounderstanding the action of HI in the structure and func-tion of chromatin; in view of the above controversies, itshould be studied further.

An observation that is directly linked to the cooperativ-ity of Hl/DNA interaction is the dependence of the bind-ing reaction on DNA size. Renz (1975) demonstrated thatthe histone, when given the chance to choose betweenDNA fragments of different sizes, favored the larger mole-cules. Renz and Day (1976) found that the range of DNAsizes over which HI preferred larger fragments dependedon the base composition of DNA. The preferential affinityof HI (or its carboxyterminal fragment) to high-molecular-mass fragments is related to the cooperativity of interac-tion; the cooperative distribution along the DNA back-bone appears to be energetically more favored for longDNA fragments than for shorter ones (Aviles et ai, 1978).

PREFERENTIAL BINDING TOSUPERCOILED DNA

One of the main features of the interaction of HI withDNA is the preference the histone possesses for super-coiled DNA. The first reports (Vogel and Singer, 1975,1976; Singer and Singer, 1976) indicated that HI has a

higher affinity for superhelical than for relaxed circular orlinear DNA. Vogel and Singer performed the original ex-periments at 100 mM NaCl. At this ionic strength, the co-operative binding of HI to DNA was lowered by the super-helicity of DNA, leading to retention of more supercoiledmolecules on the nitrocellulose filters. Knippers et ai(1978) regarded this ionic effect as the cause of the appar-ent preference for supercoiled DNA. Iovcheva and Dessev(1980) also argued that the efficiency of complex forma-tion between histone HI and different forms of DNAcould not serve as a reliable measure of affinity under theconditions used. Thus, to distinguish any preference that

HI might have for one form of DNA over the other, directcompetition experiments were needed. Such experimentswere performed by Iovcheva and Dessev (1980) and re-

cently by us (Yaneva et ai, 1990). The results show thathistone Hl (HlAB and HI0) possesses a strong bindingpreference for supercoiled DNA rather than linear DNA ofthe same molecular mass. In contrast, histone H5 seems

not to distinguish the different DNA forms (Iovcheva andDessev, 1980), contrary to earlier reports (Bina-Stein et ai,1976.

SELECTIVITY WITH RESPECT TO BASECOMPOSITION: POSSIBILITIES FORSEQUENCE-SPECIFIC INTERACTIONS

It was once thought that binding of HI to DNA was notselective. However, Mazen and Champagne (1968) andSponar and Sormova (1972) showed that HI bound prefer-entially to AT-rich regions. These data were confirmed byseveral groups (Bram et ai, 1974; Panusz et ai, 1974; Plu-cienniczak et ai, 1974; Renz and Day, 1976; Blumenfeld etai, 1978; Sawecka et al., 1978). Hwan et al. (1975) showedthat histone H5 also preferred AT-rich regions. Izaurraldeet ai (1989) and Kas et ai (1989) have now shown that his-tone HI binds highly preferentially to the AT-rich scaf-fold-attachment regions (SARs); the binding is not di-rected by a strict consensus in the regions but results fromthe overall structure and/or conformation of oligo-(dA) • oligo(dT) tracts. They suggested that SARs be con-sidered as cis-acting sequences that nucleate cooperativeHI assembly along the SAR into the flanking non-SARDNA, and control the conformation of chromatin do-mains.

Using isolated fragments of HI, Marekov et ai (1978)found that the preference for AT-rich DNAs (at ionicstrength exceeding 20 mM NaCl) was due to the structuredglobular part of the histone. However, the physical basisand/or significance of the selective binding of HI to AT-rich DNA is not clear, as poly(L-lysine) possesses the same

selectivity (Leng and Felsenfeld, 1966).The filter binding experiments of Renz (1975) provided

the first evidence that histone HI exhibits a preference foreukaryotic versus prokaryotic DNA. When a mixture ofdifferentially radiolabeled calf lymphocyte and Escherichiacoli DNA was incubated with histone HI, the histonebound preferentially to the eukaryotic DNA. The selectivebinding was observed under conditions favoring the coop-erative mode of interaction. Most of the 2 x 106 daltonfragments of mammalian DNA contained at least one pref-erential histone HI binding site whereas most of the 2 x105 dalton fragments had none. The proposed specificityof histone HI for mammalian DNA might be based largelyon AT content and size coupled with cooperativity (Renzand Day, 1976). Diez-Caballero et ai (1981) furthershowed that HI from calf thymus selectively precipitated asmall fraction of homologous (calf thymus) or heterolo-gous (herring, trout) eukaryotic DNA, but not phage X orE. coli DNA. The number of binding sites was limited.Such specific complexes could serve as nucleation pointsduring chromatin condensation.

242 ZLATANOVA AND YANEVA

The observations obtained with total DNA preparationswere recently confirmed and extended with well-defined in-dividual DNA sequences. Izaurralde et ai (1989) and Kaset ai (1989) demonstrated that under conditions of strongcooperativity, HI bound highly selectively to plasmids con-

taining scaffold-attached regions but not to pure bacterialplasmids. Similarly, we found that a plasmid containingthe mouse a-globin gene was retained substantially on ni-trocellulose filters by HI binding while the parental plas-mid was retained negligibly (Yaneva et ai, 1990). The HIpreferred the eukaryotic insert over the plasmid body, andthe preference could not be explained by a higher AT con-tent of the eukaryotic fragment. The preference of HI forsequences present in eukaryotic DNA was also evidentfrom gel retardation and competition analysis (Yaneva andZlatanova, unpublished data).

Data are also accumulating suggesting that HI preferssome eukaryotic sequences over others, and that this pref-erence is not connected to the AT content of the se-quences. Berent and Sevall (1984) found specific bindingof HI to a region at the 5' end of the rat albumin gene.The authors characterized the restriction fragments of thecloned gene retained on nitrocellulose filters following in-cubation with various protein extracts of chromatin. TheDNA fragment binding selectively to components of the0.35-1 M NaCl extract was subcloned and used for purifi-cation of the selectively bound protein, which was identi-fied as histone HI. This DNA fragment contained the firsttwo exons of the rat albumin gene plus 440 bp of the 5'-flanking region. Sevall (1988) localized the interaction sitewithin a 346-bp restriction fragment that included the bor-der between the first exon and the first intron. She showedthat the regions of HI binding within this fragment werenot particularly AT rich, in contrast to those between themwhich contained more than 80% AT. These data con-firmed the results of Diez-Caballero et ai (1981) and dem-onstrated that specific interactions between HI and certaingene regions might have a bearing on the role of HI in thefine regulation of gene expression.

Ristiniemi and Oikarinen (1989) reported that HI boundto the putative nuclear factor I (NF I) recognition sequencein the promoter of the mouse a2(I)-collagen gene. The evi-dence that the purified NF I is identical to histone HI is,however, not convincing. In view of this, we have recentlyperformed a series of experiments using a chemically syn-thesized oligonucleotide containing the natural NF I recog-nition sequence of the adenovirus origin of replication andpurified mouse liver histone HI. None of the three inde-pendent approaches that were used gave any indication ofspecific interaction of HI with the NF I recognition se-

quence. In the end, three groups have recently reported onthe cloning of NF I genes (Meisterernst et ai, 1988;Paonessa et ai, 1988; Santoro et ai, 1988). In ail cases theisolated genes code for proteins that are much bigger thanHI and in none of the sequences has any resemblance toHI been found. So it is evident that the issue of whetherHI is identical to some of the transcription factors orwhether it binds to sequences specifically recognized bysuch factors has to be elucidated further.

Pauli et al. (1989) offered more evidence for sequence-

specific Hl-DNA interactions by examining the binding ofend-labeled DNA fragments to Western blots of nuclearproteins. They found that histone HI bound specifically a

DNA fragment between nucleotides -749 and -651 of thedistal promoter of a cell cycle-dependent human H4 his-tone gene; this sequence had previously been identified as a"dehancer" (silencer) sequence. The binding site for HI isflanked by binding sites for a 45-kD nonhistone protein,and these DNA fragments possess some of the featurescharacteristic of known scaffold-attachment regions.

We have found that regions in the proximal 5'-flankingsequence and in the first half of the coding sequence of themouse a-globin gene are preferentially bound by HI as

compared to sequences in the more remote 5'-flanking por-tion and in the 3'-flanking region (J. Yaneva and J. Zlata-nova, unpublished results). Selectivity was strongly en-hanced under conditions of noncooperative binding (lowionic strength).

The paucity of data concerning the selectivity of interac-tion of HI with defined DNA sequences still does not per-mit a firm conclusion on this extremely important issue(for discussion, see Zlatanova, 1990a).

EFFECT OF HI BINDING ONDNA CONFORMATION

Although the B-form of DNA is preserved in Hl-DNAcomplexes (Olins, 1969; Taillandier et ai, 1979), the bind-ing of lysine-rich histones leads to some distortion of thecannonical structure of the double helix. For example, itinduces a deeply negative band in the circular dichroism(CD) curve of B-DNA interpreted as being due to the for-mation of the so-called ¥-DNA, which is believed to resultfrom condensation of DNA molecules into ordered aggre-gates (Tinoco et ai, 1980; Russo et ai, 1983; Mura andStollar, 1984; Moran et ai, 1989). To characterize whichdomain of the histone molecule was responsible for this ef-fect (and presumably for chromatin condensation), Russoet ai (1983) studied the binding of isolated globular do-main and of individual members of the HI family differingin length and amino acid composition of their carboxyl ter-mini. They found a straightforward correlation betweenthe value of ellipticity at 259 nm and the mole-percent ofarginine. The globular domain by itself caused no distor-tion of the CD spectrum of the B-form, suggesting that itwas the flanking amino- and carboxy-terminal domainsthat associate with DNA and induce the formation of ¥-DNA. The participation of the C-domain in *-DNA struc-ture formation was also demonstrated by Moran et al.(1989). Complexes of histone H5 with poly(dA-dT) displaya distinctive inverted CD spectrum that is not achievedwith HI (Mura and Stollar, 1984). This observation indi-cates the ability of the different lysine-rich histones tocause varying conformational changes in regions of chro-matin possessing highly biased base sequence.

Decreasing the relative humidity of DNA samples underrigorously controlled conditions of ionic strength leads toB—A transition. Taillandier et al. (1979) compared the ef-fect of the core histones and that of histone HI on the

HISTONE Hl-DNA INTERACTIONS 243

B—A transition as studied by infrared linear dichroism:While the core histones inhibited this transition, histoneHI left the DNA in the complex free to adopt an A con-

formation. These results suggest that HI has no influenceon the flexibility of DNA and that the differences in thepacking of chromatin are not necessarily due to the releaseof HI.

Both cellular DNA and synthetic polydeoxyribonucleo-tides can adopt the Z conformation (Nordheim et ai,1981; van de Sande and Jovin, 1982). Whether the Z con-formation can be induced or its stability altered by HIbinding has been studied using poly(dGdC), which can beexperimentally induced to adopt the Z conformation(Russo et ai, 1983; Mura and Stollar, 1984). Histone HIcannot by itself induce the Z conformation, but it does in-fluence the kinetics of the B to Z transition (Russo et ai,1983). Mura and Stollar (1984) observed that the lysine-rich histones HI and H5 brought about a reduction in thecharacteristic CD spectral features of Z-DNA, with H5having a larger effect. The quantitative difference in theeffect of HI and H5 may be related to the higher adapta-tion of H5 for formation of highly condensed inactivechromatin due to its higher arginine content (Mura andStollar, 1984). The conformation changes brought by HIbinding also depend on factors that determine how theDNA-histone association forms; simple mixing of poly-(dA-dT) with histone H5 led to a -spectrum, whereasmixing at high ionic strength and gradient dialysis led to amore distinctive structure and spectrum (Mura and Stollar,1984).

Histone HI binds preferentially to supercoiled DNA. Itis not known if the histone itself is capable of inducingsuperhelicity in relaxed closed circular DNA molecules orif it is capable of changing the superhelical density ofsupercoiled DNA forms. The answer to this questionseems to be negative. Bottger et ai (1976) reported that theequivalence point (the concentration of ethidium bromideat which the net superhelical density equals 0) did notchange at relatively low concentrations of HI, suggestingthat small amounts of HI did not change the superhelicityof DNA. Bina-Stein and Singer (1977) showed that HIcannot induce superhelicity in relaxed DNA rings. Rod-riguez-Campos et ai (1989), in a study on the assemblyand properties of chromatin containing HI, showed that0.8 molecules of HI per nucleosome in minichromosomesdid not alter the average linking number change per nu-cleosome (-AL/m), this value remaining equal to 1.0 as itis in minichromosomes lacking HI. Thus, the binding ofHI both to pure DNA and to minichromosomes is not ac-

companied by a large change in the superhelical state ofthe underlying DNA. Morse and Cantor (1986) reached asimilar conclusion for histone H5 by studying the effect ofits addition on the linking number of DNA in reconsti-tuted minichromsomes.

Finally, on the basis of a theoretical analysis Belmontand Nicolini (1981) suggested that the electrostatic interac-tion of HI with DNA may lead to spontaneous bending ofDNA, which, in its turn, may account for the observedfolding of polynucleosomes into the "solenoid" regularstructure.

NATURE OF THE INTERACTION

It is generally accepted that the interaction between HIand DNA is essentially electrostatic (Lawrence et ai, 1980;Watanabe, 1986). However, only a small number of the e-

amino groups interact with the phosphates of DNA at lowionic strength; at physiological salt concentrations, most ofthe e-amino groups participate in the interaction (Bradburyet ai, 1975a; Lawrence et ai, 1980). The turbidimetrystudies of Glotov et ai (1978) suggested that, in additionto general electrostatic interactions, specific histone-DNAinteractions involving hydrogen and/or hydrophobicbonds participate in the in vitro condensation process. DePetrocellis et ai (1986) have suggested that HI moleculespossess high-specific-activity binding sites for phosphategroups that might participate in the correct binding of HIto DNA in the organization of chromatin.

Most data indicate that the strongest interaction ofDNA is with the basic carboxy-terminal tail of histone HI.Thus, proton nuclear magnetic resonance spectra of DNAcomplexed with isolated amino- and carboxy-terminalhalves of the molecule showed that the amino-terminalfragment binds to DNA very weakly, but that the carboxy-terminal fragment binds to DNA as strongly as the wholemolecule (Bradbury et ai, 1975b). The spectra showed an

increase in binding with increased salt concentration,exactly the opposite of what would be expected on electro-static grounds. This suggested that there might be a con-nection between the conformational change in the car-

boxy-terminal fragment bound to DNA and the condensa-tion of Hl-DNA complexes or chromatin, as both theseevents occur under the same ionic conditions. Glotov et ai(1977) reported similar findings with fluorescence polariza-tion. Aviles et al. (1978) also found that the carboxy-termi-nal region is mainly responsible for the interaction of HIwith DNA. Lawrence et ai (1980) differentially spin-la-beled either the e-amino groups of lysine and arginine orthe unique tyrosine located in the globular part of HI. Thestate of immobilization of the two labels upon binding ofHI to DNA was followed by electron spin-resonance mea-

surements, showing different roles for the amino and car-

boxyl termini and the globular part, the latter being a poorcandidate for DNA binding.

The interaction of the globular domain with DNA maybe important in the recognition of superhelical DNA (Sin-ger and Singer, 1976). The COOH-fragment 72-217 (en-compassing most of the globular domain) resembles nativeHI in differentially binding to superhelical rather than re-laxed DNA. By contrast, fragment 106-212 has lost thisspecificity, binding superhelical and relaxed DNA equallywell. Similarly, intact HI containing an unfolded globularregion binds both forms of DNA to the same extent. Theinteraction of HI with superhelical DNA has two compo-nents: (i) recognition of superhelicity by the globular re-

gion of the molecule and (ii) interaction between the DNAand the lysine-rich portion in the carboxyl terminus. Theoverall reaction may involve a conformational change inHI. Bradbury et ai (1975b) also proposed that binding in-duced conformational change in HI. Similarly, the globu-lar part of HI has been implicated in the ionic strength-de-

244 ZLATANOVA AND YANEVA

pendent specific binding to AT-rich regions (Marekov etai, 1978).

It is not clear if histone HI possesses specific structuralfeatures that permit sequence-specific interactions. Ris-tiniemi and Oikarinen (1988) demonstrated that the globu-lar domain of HI is homologous to the nucleotide-bindingdomain of adenine nucleotide-binding proteins, suggestingthat histone HI possesses domains conferring sequence-specific binding. Turnell et ai (1988) identified a decapep-tide in the globular part of HI that forms an a-helicalmotif capable of specifically recognizing a narrow minorgroove of DNA of the kind appearing in oligo(dA) • oligo-(dT). Suzuki (1989a) identified the repeating tetrapeptideSer-Pro-Lys-Lys (SPKK) in the amino termini of seaurchin spermatogeneous histones HI and H2B as a newnucleic acid-binding unit. He concluded that the SPKK re-

peat bound to linker DNA and suggested that similar tetra-peptides (SPXX, where X is a hydrophilic but not a basicresidue) were frequently found in transcription factors(Suzuki, 1989b). As the binding constants of SPXX areless than those of SPKK in HI, the binding of HI shouldinhibit binding of transcription factors. Thus, the usage ofSPXX and SPKK may serve as a switch for gene regula-tion.

In the absence of X-ray crystallographic data, knowl-edge of the DNA structural elements involved in interac-tion with HI is incomplete. The phosphates in the DNAbackbone participate in ionic interactions with the basicamino acid residues of the histone. Hl-DNA binding mayalso occur through specific interaction with the carbonylgroup of thymine (Chinsky and Turpin, 1982). Izaurraldeet ai (1989) suggested that the oligo(dA) • oligo(dT) tractsin scaffold-attachment regions (see above) may be ele-ments responsible for specific HI binding. This specificbinding would be due to the narrow minor groove and tothe rigid conformation of such tracts. However, Sevall(1988) has suggested that HI does not bind to the regionsof very high AT content, which are characterized by a rigidbent conformation, but instead binds to more flexible re-gions of DNA.

Hl-DNA INTERACTIONS IN CHROMATIN:IMPLICATIONS FOR THE ROLE

OF HI IN VIVO

Several lines of evidence show that artificial Hl-DNAcomplexes can be used as a model system for studying therole of HI in chromatin. (i) HI interacts primarily with theDNA in chromatin and much less with the protein compo-nents. HI interacts with the linker DNA independently ofthe other histones, and is situated with DNA on the out-side of the nucleosomal protein core (Bradbury et ai,1975a; Varshavsky et ai, 1976; Noll and Kornberg, 1977).In this respect the interactions of HI in chromatin are simi-lar to those of HI in artificial complexes with pure DNA(Bram et ai, 1974; Baldwin et ai, 1975). (ii) The locationof the histone molecules in the fiber allows chemical cross-linking by bifunctional reagents with the formation of HIhomopolymers (Thomas and Khabaza, 1980; Ring and

Cole, 1983). Similar homopolymers were found in cooper-atively formed complexes of HI and linear double-stranded DNA (Clark and Thomas, 1986). (iii) The satura-tion ratio (bound Hl-DNA) is about the same for nakedDNA and for DNA in chromatin (Diez-Caballero et ai,1981). Also, the average packing densities of different mo-lecular species of HI in their complexes with DNA corre-late well with the average length of linker DNA of thechromatins with which the respective histone types are as-sociated (Clark and Thomas, 1988). (iv) The salt-inducedcompaction of the nucleosomal fiber correlates with thetransition from noncooperative to cooperative binding,taking place under similar salt conditions (Bradbury et ai,1975a; Renz and Day, 1976; Triebel et ai, 1988). (v) Thesalt dependence of the proton NMR spectra of gels ofchromatin and of Hl-DNA complexes is very similar(Bradbury et ai, 1975a,b). (vi) The interaction of the ly-sine residues and DNA is the same when HI is bound tolinear purified DNA or to HI-depleted chromatin (Girar-det and Lawrence, 1983).

The interaction of HI with chromatin has been studiedextensively. We will only summarize the major conclusionsabout the action of histone HI in chromatin based on ex-

periments with Hl-DNA complexes.Whether the interaction of HI with chromatin is coop-

erative or not is important both for the formation ofhigher-order structures of the chromatin fiber and for thepossible gene regulation via HI (see Zlatanova, 1990b).The cooperativity of HI binding may be important in theregional heterogeneity in the chromatin structure and func-tion. As discussed above, this issue is still controversial. IfHI binding to chromatin in vivo is cooperative, the inacti-vation mechanism must also be cooperative, which meansthat HI binding leading to the creation of superstructurescan only function as a long-range, general repressor mech-anism (Weintraub, 1985). However, the cooperativity mayinvolve only a few HI molecules, changing the structure ofa limited region of DNA. For example, six Hl-containingnucleosomes in a row are sufficient to form a higher-orderstructure (Bates et ai, 1981; McGhee et ai, 1983). Alter-natively, the cooperative binding may involve different HIsubtypes, only some of which can form higher-order struc-tures (the latter possibility is discussed by Lennox, 1984).

Whether the preferential binding to supercoiled DNAhas any functional significance is still not known. Eukary-otic DNA is probably negatively supercoiled in the loopsformed by the attachment of DNA to the nuclear matrix.It is not known whether superhelicity or torsional stress isrequired for transcription or is simply a consequence oftranscription itself. If template superhelicity is needed fortranscription, it might provide conditions for differentialbinding of regulatory molecules (i.e., histone HI versustranscription factors) to DNA of different superhelicaldensities. Thus, fine changes in the superhelical density ofDNA in defined chromatin loops might facilitate changesin protein binding, thus affecting gene expression. The dif-ferences in the binding of the various subfractions of HIto differently supercoiled DNA and the close physical link-age between the HI-binding region and the regions en-riched in topo II cleavage consensus sequences in the cell-

HISTONE Hl-DNA INTERACTIONS 245

cycle-regulated human histone H4 gene (Pauli et ai, 1989)may also be significant.

The significance of the selectivity of HI binding to DNAregions of specific base composition or sequences is notclear either. The preference of HI for AT-rich regionsmight be of importance as such regions are dispersed overthe genome, often located in regulatory gene elements.AT-rich scaffold-attachment regions might serve as cz's-act-ing sequences that nucleate cooperative assembly of HIinto neighboring DNA sequences (Izaurralde et ai, 1989).Such an interaction of HI with DNA of specific loops, ini-tiated at DNA attachment sites to the nuclear matrix maycontrol the conformation of the respective domains and setup the potential transcriptional repertoire of the cell. Thepreferential HI binding to AT-rich satellite DNA mightalso be involved in the mechanism of compaction andhence transcriptional inactivation of heterochromatin (Blu-menfeld et al., 1978).

The possibility of sequence-specific interactions showsthat if the interactions documented in vitro occur in vivothey can constitute a basic mechanism of gene regulation.They could directly influence gene expression by HI bind-ing to control regions which can be either positive or nega-tive (Zlatanova, 1990b) or they could control gene activityby folding distinct DNA regions of chromatin by H1 bind-ing (Diez-Caballero et ai, 1981). Such folding could becontrolled by specific nucleotide sequences in the relevantchromatin regions and by changes in the ionic environmentaccompanying different stages of the cell life cycle.

HI binding may confer changes in DNA conformation,altering chromatin structure and function. HI binding hasno influence on the flexibility of DNA, suggesting that dif-ferences in chromatin packaging may not require release ofHI. HI does not change the superhelicity of underlyingDNA, indicating that superhelicity may be determinativefor preferential HI binding but is not regulated by it.

Different HI subfractions differ in their ability to alterDNA or chromatin structure (for review, see Cole, 1987)and some of the HI subtypes present in a mammalian cellmay be capable of creating higher-order structures whileothers may not be, as suggested by Lennox (1984). If thedifferent subtypes of the histone molecule exert a differen-tial effect on chromatin structure and function, one wouldexpect a nonrandom distribution of the subtypes along thechromatin fiber (for review, see Zlatanova, 1990a) or anuneven distribution among different solubility classes ofchromatin fragments (for review, see Cole, 1987).

Phosphorylation of HI is a complex modification lead-ing to the appearance of a set of molecules modified at dif-ferent seryl- and threonyl-side chains under different phys-iological conditions. The role(s) of this modification is stillunclear. Studies performed on artificial phosphorylatedHl-DNA complexes have led to several conclusions: (i)Phosphorylation reduces the cooperativity of binding toDNA (Knippers et ai, 1989). (ii) The phosphorylatedforms retain the ability to discriminate between superheli-cal and relaxed DNA (Singer and Singer, 1978). (iii) Atmoderate ionic strength (0.1-0.3 M NaCl) phosphorylatedHI is more effective at cross-linking DNA than is the un-modified form, irrespectively of whether the HI was phos-

phorylated in vitro (Matthews and Bradbury, 1978) or invivo (Corbett et ai, 1980), suggesting that HI phosphor-ylation initiates chromosome condensation, (iv) Phosphor-ylation of HI at Ser-37 leads to a partial or complete re-lease of the globular moiety from DNA (Glotov et ai,1977); however, it does not affect the cross-linking ofDNA by HI (Glotov et ai, 1978). Phosphorylation at clus-tered -Ser-Pro-X-Lys/Arg- motifs in the amino-terminaldomain of sperm-specific HI is expected to weaken orabolish electrostatic interaction of this motif with DNA(Hill et ai, 1990). (v) Phosphorylated HI binds preferen-tially to AT-rich DNA both at low (20 mM) and intermedi-ate (50 mM) NaCl concentrations while preferential bind-ing of unmodified HI occurs only at 50 mM NaCl(Marekov and Beltchev, 1981). Thus, phosphorylation ofhistone HI may bring about subtle changes in the interac-tion of HI with the underlying DNA, thus exerting an ef-fect on the structure and function of defined chromatin re-

gions.Hl-DNA complexes are a useful system for studying the

role of HI in chromatin. However, many problems re-

main, mainly concerned with the cooperativity of interac-tion, with the preference of binding to differently super-coiled DNA and, most importantly, to the possibility of se-

quence-specific interactions.

ACKNOWLEDGMENTS

The authors thank Reviewer II and the Editor for theirgreat help in improving the initial manuscript. Mrs. E.Nickolova (Institute of Molecular Biology) is also ack-nowledged for supplying some of the literature.

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Address reprint requests to:

Prof. J. ZlatanovaInstitute of Genetics

Bulgarian Academy of Sciences1113 Sofia, Bulgaria

Received for publication August 8, 1990, and in revised formFebruary 21, 1991.