Chromatin structure

Inc. .I. Biochem.,

1976. Vol. 7, pp. 181 to 185. Pergnmon

Press. Printed m Great Britain

MINIREVIEW CHROMATIN HSUEH

STRUCTURE JEI LI

Division of Cell and Molecular Biology, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A. (Received 12 February 1976)

INTRODUCTION

Chromatin and its major proteins, histones, have been investigated in the last decade by many scientists including biologists, biochemists, and biophysicists. Extensive reviews on both chemistry and functions of histones and their interactions with DNA have been published (Bonner et al., 1968; Stellwagen & Cole, 1969; DeLange & Smith, 1970; Phillips, 1971; Hnilica, 1972; Huberman, 1973; Elgin & Weintraub, 1975). During the past few years great progress has been made in understanding histone-histone interactions, histone-DNA interactions and chromatin structure. Although the existence of histone subunits in chromatin has been accepted favorably by most scientists in the field, their views on the structure of these subunits vary greatly. The intent of this article is to review a limited number of papers which dealt directly with critical issues of chromatin structure. Since the main portion of DNA in chromatin is covered by histones and since most of the studies have been made on this portion, the term ‘chromatin structure’, as used in this article refers to the structure of histone-bound regions in chromatin. This review will be devoted to four major questions concerning chromatin structure: (a) DNA structure in the chromatin subunit; (b) histone assembly in the subunit, (c) interaction between histones and DNA in chromatin subunit; and (d) a beaded or a coiled chromatin subunit. DNA STRUCTURE

IN THE CHROMATIN

SUBUNIT

Crick & Klug (1975) suggested a kinky helix (Fig. 1) for DNA in the chromatin subunit: DNA within the subunit is composed of straight segments in the normal B conformation which are connected by kinks at which point one base pair is completely unstacked from the adjacent one. Such a kinky helix was shown to be compatible with the stereochemistry of DNA. The basic reasons used by these authors for the need of the hypothesis of a kinky helix are: (a) the basic unit of 200 base pairs is folded to form a bead with a diameter of about 6(rlOOA; (b) a rather large amount of energy is required to bend DNA

‘smoothly’ into such a small space; (c) nature tends to maintain the symmetry in B form DNA. As to point (a), the length of DNA within the subunit was estimated to vary greatly from 120 to 200 base pairs (Noll, 1974; Sollner-Webb & Felsenfeld, 1975; Axel, 1975). The length (200 base pairs) used by Crick & Klug (1975) was the upper limit reported in literature. The diameter of chromatin subunits viewed under electron microscopy also varies from 60 f 16 A (Olins & Olins, 1974) to 131 + 10 A (Oudet et al., 1975). Since formaldehyde fixation and dehydration tend to reduce the apparent size of the subunit, the larger value (130A) is probably closer to the true dimension in chromatin than the lower one (60 A). If the 130 A value is used as the diameter of a spherical subunit for calculation, the volume of the subunit is about 5 times that required for a DNA of 200 base pairs or about 8 times that required for 120 base pairs. Physically there is enough space within the subunit for DNA to bend ‘smoothly’. For point (b), so far there is no reasonable estimation of energy required to bend DNA ‘smoothly’. It was suggested to be rather high by the authors. It is noted, however, that the free energy of binding of histones to DNA in chromatin must be enormously large since no dissociation of histone from DNA is detectable at low ionic strength. If 80% of basic residues in histones are assumed to form ionic bonds with phosphates of DNA, there are about 170 ionic bonds formed between one octamer of histones and DNA within each subunit. Without including the I. Kinky Helix

U

II. Distorted Helix

Distorted E-DNA

(B-Cl

Fig. 1. Two different models of DNA structure in histonebound regions in chromatin. (I) A kinky helix (Crick & Klug, 1975). (II) A distorted helix (Li, 1975). 181

gmcd Iron1 other t>pcs 01‘ hondrng between histones and DNA, the ionic bonds alone skill provide a trcmend~)~ls aInouilt of encrgq for bending the DNA segment ‘smoothly’ within the subunit. For point {c). nature t&s allow DNA to maintain structures other than the normal B form. Depending upon external conditions (salts and humidity, for example) and binding molecules numerous examples of structural transitions from B to C or from B to A form. have been published. Intercalation of drugs into DNA is a good example. A single intercalation would extend the two adjacent base pairs from 3.4 B\ to about 6.X A and effect an unwinding of LIP to 36’ (Wang, 1974). Apparent]] the maintenance of s>nmetry in the B structure of DNA becomes less important if distorted structures formed in complexing with other molecules. or in transfer to a different environment. are thermodynamic~~liy more stable. So far there is no evidence to prove or disprove the hypothesis of a kinky helix. However. from the above discussion it seems unlikely that such evidence will be found. An alternative possibility for DNA structure within the subunit is that of a helix distorted from B to C form (Fig. I). According to the results of circular dichroism (CD), the extent of distortion was shown to be distributed non-uniformly within the subunit (Li et LI/,, 19’75) with an averaged structure close to the C form, Recently it was pointed out that the formation of about one ‘smooth’ turn or loop of the DNA within a chromatin s&unit is compatible with both base tilting and base rotation in the C conformation (Li. 1976). As far as the base rotation is concerned. this suggestion agrees with the order of magnitude of the unwinding angle per base pair of DNA within the subunit as measured by Germond rl r/l. (1975). The smoothly distorted helix shown in Fig. I is also in agreement with the drawings of DNA within the subunit presented by Van Holde c~f (I/. ( 1974) and Baldwin or ul. (1975). cncrp>

HISTONE

ASSEMBLY

IR

THE

St. Bt’NlT

An oxidized dimer of H3, (H3)z, was discovered by Fambrough & Bonner (1968) and Panyim rt ul. (1971). The histone H4 dimer. (H4)i, was first reported by Li et it. (1972) based upon kinetic and equilibrium studies of this histone in solution. A parallel dimer of H4 was further suggested as the basic subunit (Li, 1973). D’Anna & Isenberg (1974), utilizing the method developed in H4 (Li c’r ul.. 1972). studied other histones and reported a cross-complexing pattern among histones: strong interaction with H2A-H2B, H2B-H4, and H3--H4 and weaker interaction with H2ApH3. Kornberg & Thomas (1974) first reported a subunit of higher order. namely, a (H3)2 (H4), tetramer. Kornberg (1974) then extended the subunit to an octamer composed of two each of the four major histones, H2A. H2B. H3 and H4. Van Holde rt ifi. (1974) reported the existence of about X histones per nuclcase-resistant particle. These

authors further suggested :I protcm core composed of a linear array of X histones in contact through ~lydrophobic regions. A more detailed sequence of histone assembly was suggested by LI (1975): that histoncs HZA. HX H3 and H4 tirst form parallel dimers. (HZA)?. (H7B)L. (H3), and (114):: two tctramers (H3A)? (H2B), and (H3)? (H4), are then formed from the dimers through nonpolar interaction in the C-terminal regions: an octamcr is formed from these two tctramcrs which is capable of binding one HI molecule before complexing with DNA. Within the chromatin subunit the two tetramcrs can further interact bvith each other through non-polar interaction among the C-half molecules of these histones. The above assembly of histones was suggested as the most favorable subunit of histones for chromatin containing the five major histone classes in correct stoichiometry. Variation of this assembly is expected when a chromatin lacks one or more species of histones such as in yeast chromatin (Lohr & Van Holde. 1975) and in micronuclei isolated from TLrmk~mr~u pyrifbrmis (Gorovsky & Keevert. 1975). In agreement with the model of histone assembly suggested by Li (1975). Thomas & Kornberg (1975) recently reported four dimers (H2A):. (H2B),, (H3)> and fH4)11. using chemical cross-linking technique and electrophoresis of histones in chromatin. However, no detail with respect to the polarity of histones within the subunits was described in the latter report. Using chemical cross-linking techniques for histones. Martinson & McCarthy reported a close contact between histone H2B and H4. Hardison YI it/. (1975) also studied the frequency with which two histones are found adjacent to each other in chromatic. Their results suggest a higher frequency for H3-HZB, H3--H2A and H2A H2B and a lower frequency for H2B--H4. H3--H4 and H3 H3. Hyde & Walker (1Y75) reported the existence of a dimer of H3 and H4. and another ditner of H2A and H2B in chromatin. These results might suggest some tendency for those paired histones to be next to each other in chromatin. although it is still ditiicult to rule out the possibility of artifact when such cross-linking technique is employed. INTERACTION BETWEEN HISTONES AND IN THE CHROMATIN SUBLINIT

DNA

Kornberg (1974) suggested a model of contact beads with a diameter of about 100 8\; each bead contains 200 base pairs of DNA and one octamer of histones, with one HI molecule attached to the outside of each bead. No explicit description was presented with regard to the inter-relationship between histone subunit and DNA helix. Van Holde et (11.(1974) presented a particulate model of chromatin which consists of a protein core of 8 histones wound by DNA on the outside. Histone HI was considered to be linked to this particulate subunit. although no detail

183

Chromatin structure was given with regard to H 1 binding to DNA. A similar model was proposed by Baldwin et al. (1975) with a protein core complexed by DNA on the outside; histone Hl was suggested to be on the outside of the globular subunit and to play a cross-linking role between subunits in the same chromatin chain or in different chains. This model was suggested to explain their neutron scattering results of chromatin. The bases used for the construction of these models derived primarily from electron microscopy (Olins & Olins, 1974; Oudet et al., 1975; Griffith, 1975) and neutron scattering of chromatin (Baldwin et al., 1975), sedimentatioh and chemical composition of nucleaseresistant particles obtained from chromatin (Sahasrabuddhe & Van Holde, 1974) and histone-histone complexes @‘Anna & Isenberg, 1974; Kornberg & Thomas, 1974). When results from both thermal denaturation and circular dichroism (CD) studies of chromatin were considered as additional bases for the construction of chromatin structure, a more detailed model was proposed (Li, 1975). This model suggests the following key points: (a) Parallel histone dimers, (H2A)z, (H2B)2 (H3)2 and (H4),, are the basic subunits which bind DNA directly. (b) The more basic regions of these histones bind DNA primarily in the minor groove, while the less basic regions bind the major groove. (c) One histone Hl molecule binds about 3&40 base pairs which link the two nearest subunits of chromatin bound by histone octamers. (d) Formation of a histone core can be accomplished through hydrophobic interaction of the lessbasic regions of histones in the two tetramers, (H3)2 (H4), and (H2A)z (H2B),; however, the existence of this protein core is not the prior condition for the organization of histones and DNA within the chromatin subunit. Most recently Pardon et al. (1975) studied nucleaseresistant particles of chromatin by utilizing the technique of neutron scattering. They reported a radius of gyration of SOA for DNA and 30A for protein, suggesting two models to account for these findings: a spherical model with an inner protein core surrounded by an outer DNA shell, and a cylindrical model with an inner protein cylindrical core wound by two turns of DNA helix. Although there are some differences in the models suggested by Kornberg (1974), Van Holde et al. (1974), Baldwin et al. (1975) and Pardon et al. (1975), these models do have one thing in common, namely, a chromatin subunit composed of inner protein core (8 histones) wound by a DNA helix of 140-200 base pairs on the outside. The model suggested by Li (1975) also shows such a protein core formed by nonpolar interaction between the hydrophobic regions of the two tetramers under certain conditions; however, under those conditions, only about 50% of DNA within the subunit binds directly on the outside of

this core and another 50% is bound by the more basic regions of histones, which have less a-helical content (Li et al., 1975) and have less globular structure than the less basic regions of histones in the protein core. The latter model also suggests that the compact bead or particle is one of the major states but not the only state for the chromatin subunit, as implied in the other models. In addition, it gives a more detailed picture of histone-histone and histone-DNA interactions and explains more observable phenomena of chromatin than the others.

A BEADED

OR A COILED

CHROMATIN

SUBUNIT

A string of beads in chromatin has been observed from several laboratories using electron microscopy (Olins & Olins, 1974; Griffith, 1975; Germond et al., 1975; Oudet et al., 1975). Particulate structure of nuclease-resistant fragments in chromatin has also been demonstrated, using sedimentation (Sahasrabuddhe & Van Holde, 1974), electron microscopy (Oudet ef al., 1975; Finch et al., 1975), or neutron scattering (Pardon et a[., 1975). These experimental results suggest a beaded structure for chromatin subunits which is in agreement with the models of Kornberg (1974); Van Holde et al. (1974); Baldwin et al. (1975); and Pardon et al. (1975). Based upon experimental results in histone-histone interactions, histerone-DNA interactions and chromatin, it was deduced that chromatin structure could be in either a coiled state or a beaded state (Li, 1975). Whether a chromatin subunit assumes a coiled, a beaded or any intermediate state, will depend primarily upon environmental factors, such as pH, ionic strength, small molecules (water, for example) and histone modifications. The basic sequence of formation of chromatin subunits also seems to be different in these models. According to Li’s suggestion, after the synthesis of histones, a histone subunit is formed which is rather extended; the extended structure allows the histones to wind around the DNA helix and to interact with DNA in such a way that both the more basic and the less basic regions of each histone molecule bind DNA directly; the binding of each histone subunit to a stretch of DNA causes a localized structural distortion in DNA so that the whole chromatin subunit proceeds to a coiled but extended state; potentially this state can be transformed into a beaded and compact state by several means either naturally or artificially. On the other hand, the other models (Kornberg, 1974; Van Holde et al., 1974; Baldwin et al., 1975; Pardon et al., 1975) imply that beaded and compact histone cores are first formed in the cell; DNA helix then winds around these protein cores thus forming repeating units in chromatin. Li’s model suggests the winding of histones around the DNA helix, while the other models suggest the winding of DNA helix around histone cores.

1x4

Hs[

I H Jr

Although experimental data seem to imply the existence of beaded and compact structure for chromatin subunits. it was proposed by Li (Li. 1975, 1976) that such appearance of beaded structure could be induced artificially during experimental manipulation of chromatin by such techniques as formaldehyde fixation. dehydration and nuclease digestion. Theoretically this suggestion is quite plausible and has to be tested experimentally. With the question of a beaded

or a coiled state for chromatin subunit still unanswered. the following experiment is suggested to help clarify the issue: (a) preparation of chromatin from nuclei as gently as possible, ~itl~our any ,~~.YL(c;w~; (b) examination of a large number of chromatin molecules under the electron microscope; (c) measurement of the total length of both types of chromatin stretches. those showing regular beads and those without: and (d) calculation of the probability of seeing compact beads in a chromatin molecule. If the above experiment yields a probability of beaded structure greater than 0.8. the likelihood that such a beaded structure does exist in the subunits of native chromatin would be much higher than we can accept today. Structure of chromatin is an important. exciting and complex subject. After the early development by pioneers in the first 15 years. quite a few more scientists have brought their wisdom and scientific skills into this field recently. greatly extending our understanding of chromatin structure. This review presents some of the key questions and differences in current views on chromatin structure. Through the efforts of those already in the field and others who join them, the above questions probably will soon be solved. so that a more sound and more broadly based model of chromatin structure can be proposed in the near future. AcknoM:Icdyernr,lts~~Research ported in part by National PCM76-03268 and National GM 23079 and 23080.

for this report was supScience Foundation Grant Institutes of Health Grants

REFERENCES

AX~L R. (1975) Cleavage of DNA in nuclei and chromatin Biochemistry 14. staphylococcal nuclease. with 2921-2924. BALDWIN J. P., BOSELEY P. G., BRAI)BURY E. M. & 1st~ K. (1975) The subunit structures of the eukaryotic chromosome. Nature, Nrw Bid. 253. 245. 249. BONNER J., DAHMUS M. E., FAMBROUG~~ D., HLJANG R. C. C., MARUSHIC~ K. & TUAN D. Y. H. (1968) The biology of isolated chromatin. Scirncr 159. 47-56. CRICK F. H. C. & KLUG A. (1975) Kinky helix. Nururo, New Biol. 255. 530-533. D’ANNA J. A., JR. & ISENBERG I. (1974) A histone crosscomplexing pattern. Biochrmisrry 13. 4992-4997. DELANG~ R. J. & SMITH E. L. (1971) Histones: structure and function. Arln. Ret,. Biochrm. 40. 279-314. ELGIN S. C. R. & WEINTRACB H. (1975) Chromosomal proteins and chromatin structure. Arzn. Rrc,. Biochem. 44. 725-774.

I LI

FAMHROIIC;H D. M. 6i BOLLI K J. (1%X) Sequence humology and role of cysteine in plant and animal argtnlncrich histones. J. hid. Clam 243. JJ.31 -1339, FINCII J. T.. NOI.L M. & KOK~I~I KC, R. D. (lY75) Flcctron microscopy of defined lengths of chromatin. Proc. wtu Acad. SC;. c’..S.A. 72. 3.320 .3?32. J. E.. HIRT B.. Or I>I r P.. C‘KOSS-B~LLARI) M. & CFIAMHO~ P. (1975) Folding of the DNA double hrhx m chromatin-like structures from Simian virus 30. Pr,jc,.

GERMONI)

nrrtn Accltl. Sci. U.S.4.. 72, 1X4.%1847. GOROVSK~ M. A. & Kbf-vr KI .I. B. (1975) Subunit structure of :I naturally occurrmg chromatin lacking hlstoncs Fl and F3. Pm. nutn Acd SC,;. I~.S..-l. 72, 3536-3540. GRIFFITH J. D. (1975) Chromatin structure: deduced from a minichromosome. Scic,~c,c, 187. 1202 1203. HARI>II\SON R. C., EI~I~~I R M. E. & C‘IIAI.ICLIy R. (I Y7S) An approach to histone nearest neighbors in Extended chromatin. h’uclcic ncic/\ Rca. 2. 1751 1770. HNILICA L. S. (1972) T/Ic, Srrrrc~tuw um/ Bio/oqkc~I Fmctious of His/ones. Chemical Rubber Co. Press. Cleveland, OH, HURERMAN J. A. (1973) Structure of chromosome fibers and chromosomes. AM Rw. Bi~ch~n. 42, 355-378. HYIIF J. E. & WALKIR I. 0. (1975) Cobalcnt cross-IinkIng of histones in chromatin. FEBS /et/s 50. I50 153. KORNR[:R(; R. D. (19741 Chromatin ytructurc: a repeating unit of histones and DNA. Sc,ic,~cc, 184. X6X X71. KORNB~R(; R. D. & THOMAS J. D. (1974) Chromatin atructurc: oltgomers of the histones. .%ivwc 184. X65 X6X. LI H. J. (1973) Mechanism of conformatlonal changes in Histone IV. F&n Proc. .-lh\tr-~crs. NO. 2098. LI H. J. (1975) A model for chromatln structure. ,Vlrc./t,~c~ .4cid.s Rc\. 2. I275 1289. LI H. J. (19761 Chromatin structure: a model, in Mo/rcu/tr/ Biologic of The Martrmulitrrl Grwfic Appmvt~~.~--Iiv Rdtrtio~~.skip to Cunccr, Agir~g ud Mdictrl Gwt,tic.s. part A.

(Edited by TS‘O P. 0. P.) Elscvier Mcdla. North Holland (in press). LI H. J.. C‘FIA&;C; C., EVAC;ILINOI~Z. & Wt ISROI’F M. (1975) Circular dichroism of histone-bound regions in chromatin. Biopolyrnrrs 14. 21 l-226 LI H. J., CHANG C. & WEISKOPF M. (1973) Helix-coil transition in nucleoprotein
No~.t. M. (1974) Subunit .YcM. Bid.

structure

of chromatm

,!‘rrrurc,.

251. 249 251.

OLIM

A. L. & OLIUS D. E. (1974) Spheroid chromatin units (v bodies). S?icwc, 183. 330 332. O~I)~T P.. CROSS-BF~LLARDM. & CHAMIION P. (1975) Elcctron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell 4, 261-300. PA\lUlhI S.. SOMMI:RK. R. & CHAL KL~ Y R. (1971) Oxidation of the cysteine-containing histone F3. Detection of an evolutionary mutation In a conservative histone. Biochrmistr~~ 10. 391

I -39 17.

PARDON ?. F.. WORCESTI.K D. L.. W(X)LI y J. C.. TAT(~HFI.I. K.. VAI\: HOLF K. E. & RIC‘HARIIS B. M. (1974) Lowangle neutron scattering from chromatln subunit partlcIc\. \ I,( /Ci< lc.it/\ R<,\. 2. 7 I I,? 1176

185

Chromatin structure M. P. (1971) Histones and Nucleohistones. Press, London. SAHASRABUDDHE C. G. & VAN HOLDE K. E. (1974) The effect of trypsin on nuclease-resistant chromatin fragments J. hiol. Chem. 249. 152-I 56. SHAW B. R., HERMAN T. M., KOVACIC R. T., BEAUDREAU G. S. & VAN HOLDE K. E. (1976) Analysis of subunit organization in chicken erythrocyte chromatin. Proc. PHILLIPS D. Plenum

natn Acad. Sci. U.S.A. 73, 505-509.

SOLLNER-WEBB B. & FELSENFELD G. (1975) A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease. Biochemistry 14. 29 1552920. STELLWAGEN R. H. & COLE R. D. (1969) Chromosomal proteins. Ann. Rev. Biochem. 38, 951-990. THOMASJ. D. & KORNBERGR. D. (1975) An octamer of histones in chromatin and free in solution. Proc. natn Acad. Sci. U.S.A. 72. 262G2630.

VAN HOLDEK. E., SAHASRABUDDHE C. G. & SHAW B. R. (1974) A model for particulate structure in chromatin. Nucleic Acids Res. I. 1579-1586.

VARSHAVSKY A. J., BAKAYEVV. V. & GEORCIEVG. P. (1976) Heterogeneity of chromatin subunits in vitro and location of histone Hl. Nucleic Acids Res. 3, 477492. WANG J. C. (1974) The degree of unwinding of the DNA helix by ethidium. J. molec. Biol. 89. 783-801.

NOTE ADDED IN PROOF Very recently Varshavsky et al. (1976) and Shaw et al. (1976) showed two types of the monomers of nucleaseresistant fragments: one type containing all histones and a DNA segment of 18(r200 base pairs and the other type a DNA segment of l&l70 base pairs with all histones minus Hl (or Hl and H5). These results suggest one histone Hl per chromatin subunit: 14&170 base pairs of DNA bound by one histone subunit and 3&40 base pairs by one histone Hl, in full agreement with the earlier conclusion derived primarily from thermal denaturation studies (Li et al., 1973; Li, 1975).