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Molecular Architecture of the Chromatin Fiber A. WORCEL Department of Biochemical Sciences, Princeton University, Princeton, New Jersey 08540

1973; Olins and Olins 1974) composed of three nucleosome cores separated by I)NA spacers 60 bp long. Each nucleesome core contains 140 bp of DNA coiled around the two heterotypic histone tetramers (Weintraub et al. 1975) in a left-handed, negative, noninterwound supercoil of about 90 bp per turn as proposed by Sobell et al. (1976). The histone tetramers alternate in their polarity (shown here by the two alternating shades of gray shadowing) all along the chromatin fiber as postulated (Weintraub et al. 1976; Alberts et al. 1977). The two protein tetramers interact isologously (Monod et al. 1965) in each nucleosome, and the whole nucleosome (protein plus DNA) is organized around a twofold axis of symmetry which intersects the "front" and "rear" of the particle. The nucleosome cores of Figure l a are oriented to show (from lel~ to right) the rear, s/de, and front views of each particle. It is possible to define the nucleosome orientation because there is twice as much DNA along the front contacts (where the twofold axis intersects the particle between the two DNA fibers) than along the rear contacts (where the twofold axis intersects the I)NA helix). This is because the front contacts bring together the two terminal regions of the 140-bp coil so that they lie parallel to each other (see Figs. la, 2a, and 3a) (Weintraub et al. 1976). Note that the two parallel DNA fibers have been placed close to each other at the front of the particle. Nucleosome cores with a compressed, low-pitch DNA supercoil are required to allow for the higher-order I)NA coiling in the 200-300-A fiber (the "wedged" front DNA contacts will face the hole of the solenoids; see below and Fig. 3). Such a compressed DNA coil is consistent with the structure that Finch and Klug have proposed (this volume) based on their studies on nucleosome core crystals. Figure lb demonstrates that it is possible to generate a uniform 100-A fiber from such a beaded string by successive right-handed rotations of the nucleosomes (as seen from the direction of helix propagation). After a rotation of one-half turn per nucleosome, the entire DNA will follow a continuous lefthanded supercoil of 90 bp per turn and about 47-A average pitch (Sobell et al. 1976), and the internucleosome spacer DNA will be coiled in the groove made up by the two contiguous nucleosomes. Each ring denoting the center of a spacer will be facing what we will refer to as the rear of the 100-~ fiber

The D N A helix is coiled in the nucleohistone fiber in a nonrandom, precise manner. At one end of the coiling spectrum we have the nucleosome and at the other end the metaphase chromosome, with higher-order D N A coils bridging the gap between these two levels of D N A organization. Since the interactions between the D N A and the histones are not DNA-sequence-sl~ific, the molecular assembly of the chromatin fiber must be a function solely of the general, stereochemical, histone-histone and histone-DNA associations.Using a set of symmetrical histone-histone and histone~DNA interactions, as well as a large body of available data on chromatin, we have recently constructed a space-fillingmodel for the higher-order coiling of D N A in chromatin (Worcel and Benyajati 1977). In this paper I will review the mode[ and expand on the proposed role ofhistone H I in generating symmetrical and flexible 2(D-300-A fibers. ! will also discuss briefly some of the biological implications of the proposed nucleohistone structure. The 100-~, Fiber (Nucleofilament or "Thin" Chromafin Fiber) The basic unit of chromatin, the nu body (Olins and Olins 1974) or nucleosome (Oudet et al. 1975), is a roughly spherical particle of about 100-/k diameter (Baldwin et al. 1975; Pardon et al. 1975) which contains 140 base pairs (bp) of coiled DNA (Axel 1975; Sollner-Webb and Felsenfeld 1975; Shaw et al. 1976) and two each of the four intranucleosome histories H2A, H2B, ti3, and H4 (Kornberg 1974) organized, in all likelihood, around a dyad axis of symmetry (Weintraub et al. 1976). The internucleosome spacer DNA, with a variable length between 10 and 70 bp (Compton et al. 1976a; Noll 1976; Morris 1976; Lohr et al. 1977), is probably also coiled symmetrically between the two adjacent nucleosomes (Alberts et al. 1977), generating a jointed, flexible chain (Kornberg 1974). Electron microscopic (EM) studies of eukaryotic nuclei have indeed revealed such a general unit fiber, the "thin" 100-/k chromatin fiber (for reviews of the prenucleosome EM chromatin literature, see Wolfe 1969; Solari 1974; RIS 1975). To analyze the many possible paths of DNA coiling in chromatin, I have built scale models starting with the symmetrical nucleosome core. Figure l a shows a space-filling model of a "beaded string" (Woodcock 313

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Figure I. Space-~ling model of a trinucleo6ome with a 200-bp repeat. Each nucleosome core (diameter I00 A; Baldwin et al. 1975; Pardon et al. 1976) contains 140 bp of D N A (diameter 20 A; Watson and Crick 1954) coiled in a lei~-handed noninterwound supercoil of 90-bp circumference (this value is used for the D N A length calibration).The marks at the center of the 60-bp internueleosorne D N A are spaced 200 bp apart. The alternating light and dark gray hemispheres represent the half-nucleosornesof alternating polarities(Weintraub et al. 1976). (a) Beaded string.The nucleosomes are oriented to show (leftto right)their rear,side,and front faces (see text).The 60-bp spacers between the nacleosornes are fully extended. (b)The 100-A fiber.The 60-bp spacers are coiled between the neighboring, interacting nucleosornes. The D N A follows the path of a left-handed supercoil of 90 bp per turn and 47-Ik average pitch.There are 2.2 negative turns of the D N A supercoil per nucleosorne repeat (from center to center of spacer DNA). As can be seen from both the rear and front views, the fiber is symmetrical; the intra- and internucleosornalaxes of symmetry pass through the D N A coil at the rear and between the D N A coilsat the front of the fiber.The two arrows point to the sites on the D N A supercoil which will be cross-linkedby one histone HI molecule (see Figs. 3 and 4) according to the models shown in Figs. 3 and 5.

(made up by the rear faces of the juxtaposed nucleosomes), and the two rings will be separated by a little over two turns of the left-handed D N A supercoil (--2.2 turns: 200-bp repeat/--90 bp per turn). Note that because of the alternating polarity of the heterotypic tetramers on the D N A fiber (Weintraub et al. 1976), the contacts between the histone tetramers, beth within a nucleosome and also between nucleosomes, will be isologous (Monod et al. 1965). Thus, the twofold symmetry of the nucleosome core will extend throughout the 100-1k fiber. It is possible to construct uniform 100-A fibers containing different repeat lengths (spanning 150210 bp; C o m p t o n et al. 1976a; Nol11976; Morris 1976; Lohr et al. 1977; Todd and Garrard 1977) by keeping the D N A supercoil in Figure Ib constant and sliding

the nucleosomes along it. This sliding will change beth the n u m b e r of D N A superhelical turns (r) (Bauer and Vinograd 1968) per nucleosome repeat and the length of the repeat. Figure 2a shows a 100-/~ fiber with a 160-bp repeat constructed in this manner. The marks at the center of the 20-bp internucleosome D N A are spaced 160 bp apart, with less than two turns of the D N A supercoil between them (--1.8 turns: 160-bp repeat/--90 bp per turn). Figure 2b shows a fiber with a 210-bp repeat constructed as above. The two marks at the center of the 70-bp internucleosome D N A are spaced 210 bp apart, with more than two turns of the D N A supercoil between them (--2.3 turns: 210-bp repeat/--90 bp per turn). The internucleosome contacts between the isolo-

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A MODEL FOR CHROMATIN

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Figure 2. Space-filling models of symmetrical 100-A fibers with different nucleosome repeat lengths. The repeat length was adjusted by sliding the DNA through the nucleosome cores shown in Fig. 1. Only the rear view of the fibers is shown. The photographs were taken parallel to and centered at the twofold axis of the middle nucleosome. Like the fiber shown in Fig. lb, these fibers are symmetrical (as can be easily ascertained by turning the figure upside down). (a) The 160-bp nucleosome repeat, regular DNA supercoil of 90 bp per turn. The marks at the center of the 20-bp internucleosome DNA are spaced 160 bp apart, with 1.8 negative turns of the DNA supercoil between them. (b)The 210-bp nucleceome repeat, regular DNA superceil of 90 bp per turn. The marks at the center of the 70-bp internucleosome DNA are spaced 210 bp apart, with 2.3 negative turns of the DNA supercoil between them. (c)The 210-bp nucleoeome repeat with a nonregular DNA supercoil. The repeat length is that of the fiber in b, and internucleosome contacts, the same as the fiber in a (1.8 negative turns per nucleceome repeat).

gous side faces will change as the nucleosomes rotate vis-a-vis each other as they slide along the DNA supercoil. In the model previously described (Weintraub et al. 1976), these surfaces belong mostly to histones H2A and H2B; we would thus expect that variations in at least these histones should occur in order to accommodate different interacting surfaces (see below). As shown in Figures lb and 2a,b, the azimuthal rotation of the nucleosomes will rotate the internucleosomal twofold axis. This rotation will, in turn, lead to changes in the diameter of the "thick" fiber in the models of Figures 3 and 5 and Table 1 (see below). I suggest that a common le~-handed DNA supercoil (of about 90 bp per turn and an average pitch of about 47/k; Worcel and Benyajati 1977) is present in chromatin from different species displaying different nucleosome repeat lengths. Perhaps the strongest argument for such a universal DNA supercoil comes from the DNase-I digestion studies (Noll 1974). Both rat liver, with a 195-bp repeat, and yeast, with a 150-bp repeat, show the characteristic ~'lad-

der" of single-strand fragments at 10-base intervals which extends to fragments of up to 300 bases in length (Noll 1974; Lohr et al. 1977, and this volume). This strikingly regular pattern indicates that (1) the path of the spacer DNA must be similar to the path of the core DNA (because the regular 10-base periodicity extends beyond the core DNA length of 140 bases) and (2) the DNA coil must follow a similar path in both yeast and rat-liver chromatin. This path could well be (but does not have to be; see Camerini-Otero et al., this volume) the Sobell et al. (1976) DNA supercoil kinked every 10 bp. The models shown in Figures lb and 2a,b can adequately explain the variation in repeat length observed when comparing chromatins from different species having different histones H2A and H2B (Mohberg and Rusch 1969; Felden et al. 1976; Goff 1976; Compton et al. 1976a; Lohr et al. 1977; Spiker and Isenberg 1977). However, it should be pointed out that it is possible to change the repeat length without changing the internucleosome contacts by simply looping out the spacer DNA. The resulting

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fiber will still be symmetrical, but the DNA supercoil will no longer have a uniform diameter (Fig. 2c). Such a looping~ut of the spacer DNA may perhaps take place when the repeat length changes in a given chromatin because of proteins (other than H1) that bind to the spacer DNA. For instance, this may well be the case for the maturing chick erythroid cells, which show parallel increases in repeat length (from 190 to 212 bp) and histone H5 content (from 0.2 to 1 molecule per nucleosome) during terminal red-cell differentiation (H. Weintraub, pers. comm.). Coiling the 100-~k Fiber into a Thick Fiber The 100-A nucleofilament can compact further into a 200-300-A chromatin fiber (Wolfe 1969; Solari 1974). Most of the inactive interphase chromatin is, in all likelihood, in this 200-300-A fiber conformation (H. Ris, pers. comm.). This thick fiber is most stable and evident at metaphase (DuPraw 1970) but can also be observed after spreading interphase chromatin in the presence of trace amounts of divalent cations or 0.15 M NaC1 (Solari 1974). Electron microscopic analysis of chromatin in 0.2 mM Mg +* led Finch and Klug (1976) to suggest that the 200300-A chromatin fiber may be the result of a coiling of the 100-A nucleofilament into a 110-A pitch helix (solenoid). To test this idea and hoping to define the actual path of the DNA helix through such a solenoid, I constructed a 5000-bp version of the 200-bprepeat fiber shown in Figure 1. The specific model of the thick chromatin fiber shown in Figure 3 was built on four principal assumptions: (1) symmetrical nucleosome cores of 140bp DNA; (2) a uniform, left-handed DNA supercoil of 90 bp per turn and 47-,~ average pitch; (3) a basic symmetry principle for any helix, i.e., within a given "patch" (Yamamoto and Alberts 1976) of thick fiber, each nucleosome makes the same contacts with its neighbors as any other nucleosome; and (4) the internucleosome spacer DNA must face the outside of the thick fiber and thus always will be accessible. This arrangement of the spacer DNA is the only one which is consistent with the kinetics of staphylococcal nuclease digestion of interphase nuclei and metaphase chromosomes (Bostock et al. 1976; Compton et al. 1976b; Hozier and Kaus 1976; Vogt and Braun 1976; Wigler and Axel 1976). These four simple and reasonable assumptions turn out to define unequivocally the path of the DNA helix through the thick fiber. In the resulting model shown in Figure 3, the outside of the thick fiber is made up by the rear face of each nucleosome alternating with the fully exposed spacer DNA. The spacer DNA will first be cleaved by staphylococcal nuclease (both at mitosis and interphase) at a position near the rings on the DNA, generating DNA fragments which are multiples of 200 bp, as observed. The enzyme will further

Figure 3. Space-filling model of a thick chromatin fiber with a 200-bp repeat. Nucleosomes and DNA coil (DNA helix, superhelix, and super-superhelix) are to scale (see legend to Fig. 1). For clarity, only one of the two halfnucleesomes is shadowed. The rings around the DNA fiber mark the center of the 60-bp internucleosome DNA. The lower (first) nucleosome is in the beaded-string conformation. The next two (second and third) nucleesomes are in the 100-A fiber conformation, with the spacer DNA coiled between the two nucleosomes as in Fig. 1. The fourth nucleosome has a dark (COOH) H1 terminal bound to it; the other, white (NH,) terminal is not bound to the 100-A fiber and dangles free next to the second nucleesome. Each H1 histone molecule is represented by five regions in the model: a central, more hydrophobic (Rail and Cole 1971; Elgin and Weintraub 1975) region (this hydrophobic globular region of H1 is represented by a spring and, for clarity,is not to scale);two subterminal regions (with heter-

ologousbonding domains), and two terminal regions (which bind to the D N A supercoil). Differential shading (white and dark) stresses the asymmetric nature of the two ends (NH, and COOH) of H1. Both ends of H1 cover about 10 bp each (like the D N A contacts of the intranucleosome histones; see Weintraub et al. 1976) of spacer DNA, leaving 40 bp of exposed internucleosome DNA. The arrows point

to the HI-termini interactions with the 100-A fiber. The 200-300-A fiber is stabilized in the model by H1-H1 heterologous interactions (Monod et al. 1965) between NH,- and COOH-subterminal regions of H1 which face each other between adjacent helical turns of the nucleofilament. One such interaction is circled in this figure.

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A MODEL FOR CHROMATIN nibble the exposed spacer DNA, gradually shortening these DNA fragments (Lohr et al. 1977). The coiling of the nucleoiilament into a tightly packed thick fiber will cause a uniform bending of the DNA supercoil into a "toruslike" structure. As shown in Figure 3, the contiguous turns of the DNA supercoil will be close to each other on the inside and far apart from each other on the outside of the thick fiber. This arrangement of the DNA is consistent with recent electron microscopic observations which point to a higher DNA density near the hole of the solenoid (Davies, pers. comm.). Note that the outermost sites on the DNA super-superhelix (see Fig. 3) contain both the intranucleosomal and the internucleosomal centers of symmetry, which are separated from each other by 100 bp. The external location of those two sites could explain the peculiar DNase-II digestion pattern of chromatin: a 100-bp periodicity (which is not seen in Hi-depleted chromatin) is observed at 0.15 M NaC1 or 1 mM CaCls (Altenburger et al. 1976), conditions which are known to favor the 200-300-/~ fiber conformation (Solari 1974). The 100-/~ fibers with nucleosome repeat lengths other t h a n 200 bp (of the kind shown in Fig. 2a,b) will also coil into tightly packed, 100-/k-pitch supersuperhelices, but both the handedness and the number of nucleosomes per helical turn (n) of the supersuperhelix will vary. The reason for this variation is that a helix can only be generated by an azimuthal rotation of each residue accompanied by a translation along the helix axis (Pauling et al. 1951). In theory, a value of r/nucleosome repeat of exactly 2.0 (corresponding to a repeat length of 180 bp) will result in an azimuthal angle of 0 ~ between nucleosomes and no translation along the super-superhelix axis; in other words, after bending the 100-/~ fiber,

317

such nucleosemes will lie fiat on a surface making a ring. To pack nucleosomes into helices, a value of r/nucleosome above or below 2.0 is needed. Values of r/nucleosome above 2 will result in lefthanded super-superhelices of the kind shown in Figure 3, whereas values of r/nucleosome below 2,0 will give rise to right-handed super-superhelices. It can be shown, both in theory and with actual space-filling models, that the fraction of a turn above or below 2.0 will be equal to 1/n, where n is the number of nucleesomes per turn in the super-superhelix (Table 1). To give an example, an additional one-sixth of a turn (1/6 • 90 bp per turn---15 bp) will be required in order to build a left-handed supersuperhelix with six nucleosomes per turn (this is the solenoid observed by Finch and Klug [1976] in rat-liver chromatin). The calculated repeat length, both in the nucleofilament and in the thick fiber, will be 2 • 90 bp + 15 bp, or 195 bp (which is, incidentally, the repeat length in rat-liver chromatin; Compton et al. 1976a), and the r / r e p e a t will be --2.2 (195 bp/--90 bp). Space-filling models (not shown) of the supersuperhelices defined in Table I satisfy the four general principles stated previously. They all have (1) 140-bp symmetrical nucleesome cores, (2) a uniform left-handed DNA superhelix of 90 bp per turn, (3) symmetrical nucleosome-nucleosome interactions, and (4) spacer DNA on the outside. Since both the diameter and the packing ratio of the super-superhelices are a function of n, the number of nucleosomes per turn, they should both vary with the nucleosome repeat length (Table 1). Packing ratios around 40 (DuPraw 1970; Finch and Klug 1976) and fiber diameters ranging between 200 and 300 A (Wolfe 1969; Solari 1974; Davies and Haynes 1975) have been reported in the literature.

Table 1. Predicted Changes in the Thick Chromatin Fiber Resulting from Changes in the Nucleosome

Repeat r/Nucleosome (rl--90) --2.3

Nucleosome repeat (r) 2 X9Obp +InlX 90 bp

Nucleosomes/turn (n)

--2.1

210 2O3 198 195 193 191

--3 --4 --5 --6 --7 --8

--2.0

180

rings, no helices

--2.2

Fiber all.meter (~)

DNA packing ratio

205 235 265 295 325 355

20 25 31 36 42 47

325 295 265 235 205 175

42 36 31 25 20 14

2 • 90 bp --tllx_.90 bp --1.9

169

--1.8

167 165 162 158 150

--1.7

+8 +7 +6 +5 +4 +3

For a detailed explanation and quantitation of the various helical parameters in this table, see Worce| and Benyajati (1977).

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WORCEL ant DNA fragment, i.e., at least 10 extra base pairs of DNA at each end of the 140-bp coil are protected by H1 (Whitlock and Simpson 1976; Varshavsky et al. 1976; Noll and Kornberg 1977). Note that, because of this location of the H1 termini on the DNA supercoil (and independently of any more specific assignments for the Hl-100-A-fiber interactions), the NH2-terminal region of a given H1 molecule will be in proximity to the COOH-terminal region of another H1 molecule located in the next adjacent helical turn of the nucleofilament. Thus, "head-to-tail" ("heterologous"; Monod et al. 1965) H1-H1 interactions m a y stabilize the thick chromatin fiber (one such interaction is circled in Fig. 3). Heterologous associations would be expected to give rise to large helical homopolymers (Monod et al. 1965) and thus could be responsible, in this case, for the observed H1 cross-linked homopolymers in nuclei and chromatin (Olins and Wright 1973; Bonner and Pollard 1975; Chalkley and Hunter 1975; Thomas and Kornberg 1975; Gaubatz et al., this volume). According to the coordinate system introduced previously (see Fig. 2 in Weintraub et al. 1976), each H1 in the model goes from base pair +70 to +80 in nucleosome nl to base pair --70 to --80 in nucleosome ns (see arrows in Figs. I and 3), and the central region of H1 covers the front trypsin-sensitive (Weintraub et al. 1976) contacts of the middle nucleosome n2. The proposed H1 location is shown in greater detail in Figure 4a. As suggested previously (Worcel and Benyajati 1977), instead of their pointing all the same way as in Figure 3, the H1 molecules should alternate in their polarity along the 100-A fiber (notice that the dinucleosome is the unit of

Although repeat lengths for various chromatins have now been determined accurately, we do not yet have reliable data on the actual diameter of the corresponding chromatin fibers at metaphase. Nonetheless, based on symmetry considerations, it is possible that the number of nucleosomes per turn of the super-superhelix, and thus the diameter of the 200-300-A fiber, may turn out to be a function of the chromatin repeat length as indicated in Table 1. The Location o f H i s t o n e H 1 in the Chromatin Fiber It is known that histone H1, located outside the nucleosome core (at a ratio of about one H1 per nucleosome; Kornberg 1974), is somehow involved in the higher-order compacting of chromatin (Littau et al. 1965; Mirsky et al. 1968; Bradbury et al. 1973a), with the phosphorylation of H1 perhaps triggering mitotic chromosome condensation (Bradbury et al. 1973b; Gurley et al. 1974, 1975; Balhorn et al. 1975). Thus, H1 must bind to the 100-A fiber (which probably is the native form of the Hi-depleted chromatin; Worcel and Benyajati 1977), bending the nucleofilamerit into a more compact shape. This HI-induced coiling of the 100-/k fiber (H1 is represented in the model by a spring; see legend to Fig. 3) m a y very well cause the initial helical packing of the nucleosomes. I have placed the two basic H1 terminal regions (Elgin and Weintraub 1975; D. Cole, pers. comm.) on the DNA supercoil just outside the nucleosome core. This assignment of the histone H1 termini is consistent with recent evidence indicating that nucleosomes carrying H1 have a longer nuclease-resist-

Figure 4. Schematic representation of the proposed interactions between histone H1 and the 100-A fiber. The front of the 100A fiber is shown (see Fig. lb, front view). The continuous DNA supercoil is drawn through the two upper-lei~ nucleosomes; for clarity, only the spacer DNA is shown on the other nucleesomes. Note the interactions between H1 and the DNA supercoil. The "heavy" half of H1 in this figure represents the COOH-terminal half of the molecule, which binds strongly to the DNA supercoil (Singer and Singer 1976). Using the coordinate system introduced previously (see Fig. 2 of Weintraub et al. 1976), each H1 in the model goes from base pair +70 to +80 in nucleosome nl to base pair --70 to --80 in nucleosome ns (see arrows in Figs. 1 and 3), whereas the central region of H1 covers the front, trypsin-sensitive (Weintraub et al. 1976) contacts of the middle nucleosome n2. Note that, instead

0

o

0

0

0

o

0

0

o

L

u

of pointing all the same way as in Fig. 3, the H 1 molecules here alternate in their polarity along the 100-A fiber. (a) Continuous H I lattice.This H 1 lattice can generate a continuous solenoid (of the type shown in Fig. 3) by helically coiling the 100-A nucleofilament and then stabilizing the resulting solenoid through H1-H1 head-to-tail interactions as shown. (b) Discontinuous H 1 lattice.Ten HI-containing nucleosomes of the type shown in a alternate with one nucleosome which carries a nonhistone protein instead of H1. This discontinuous H 1 lattice will generate the discontinuous solenoid shown in Fig. 5.

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A MODEL FOR CHROMATIN symmetry of the HI-containing 100-A fiber in this model). I have chosen this particular H1 assignment for the following reasons: Both the NH2 and the COOH termini of i l l are basic (Elgin and Weintraub 1975), and they probably both bind to the negatively charged DNA phosphates, cross-linking the DNA supercoil and thus preventing its relaxation by the nicking-closing enzyme (Bina-Stein and Singer 1977). The NH2 and COOH termini of H1 bind to the DNA supercoil just outside the nucleosome core (see above), although the central region of H1 also recognizes and binds (over the front middle nucleosome contacts in this model) to the DNA supercoil (Fig. 4a). It is known that H1 binds more tightly to supercoiled DNA than to relaxed DNA (Vogel and Singer 1975, 1976; BSttger et al. 1976), and the central region of H1 (amino acids 72-106) appears to be responsible for this discrimination (Singer and Singer 1976). In the model, H1 causes a bending (coiling) of the 100-/~ fiber, in keeping with the observation that H1 containing oligonucleesomes 2-10 units long are more compacted than the same length of Hi-depleted oligonucleosomes (Noll and Kornberg 1977). The H1 span in the coiled 100-A fiber is about 80 A (measured from H1 NH2 to COOH termini in the scale model of Fig. 3), a value which is consistent with the measured Stokes radius of H1 of about 40 A (Weintraub et al. 1975). The Hi-induced coiling of the 100-/~ fiber places the 40-bp spacer DNA (bracketed by the H1 termini) on the convex side (see Fig. lb, rear view) of the 100-A fiber; this side will constitute the outside of the 200-300-A fiber, whereas the more hydrophobic central region of H1 fits in the hole of the solenoid (see also Finch and Klug 1976). This arrangement could explain beth the preferential trypsin sensitivity of i l l (Weintraub and Van Lente 1974) and the kinetics of staphylococcal nuclease digestions of interphase nuclei and metaphase chromosomes. As shown in Figure 4a, the alternating polarity of the H1 molecules preserves the overall twofold symmetry of the 100-A fiber. In addition, because of the tighter binding of the COOH-terminal half of H1 to the DNA supercoil (Bradbury et al. 1975; Singer and Singer 1976), this symmetric arrangement of the H1 molecules can explain the puzzling enrichment for di- and tetranucleosomes among the products of DNase-II digestion of chromatin (Altenburger et al. 1976). Although the DNA is cleaved with a 100-bp periodicity under these conditious, H1 molecules can hold together chains of evennumbered nucleosomes having internal doublestrand breaks (Altenburger et al. 1976). As can be deduced by a careful analysis of Figure 4a, only the DNase-II cuts in the spacer DNA flanked by the weakly bound H1 NH2 termini will break the 100A fiber; DNase-II cuts in the spacer DNA flanked by the strongly bound H1 COOH termini will not break the oligonucleosome chain. It is likely that

319

staphylococcal nuclease is able to release mononucleosomes because, unlike DNase II, it further nibbles away at the spacer DNA and displaces the H1 termini (Varshavsky et al. 1976; Lohr et al. 1977; Noll and Kornberg 1977).

A Flexible 200-300-A Fiber (Discontinuous Solenoids, Minisolenoids, or "Superbeads") I have suggested that the thick chromatin fiber may not have a uniform diameter. According to Table 1, local variations 5n the nucleosome repeat length (see, e.g., Todd and Garrard 1977) should result in local variations in the diameter of the solenoids. The next question to ask is, What could be the average length of a given uniform solenoid? I suggest here that solenoids may have as few as two helical turns of the nucleofilament. Moreover, such minisolenoids, with 6-16 nucleosomes arranged in two helical turns of 3-8 nucleosomes per turn, may well be the most common forms of solenoids. There are two main reasons for proposing such minisolenoids. The first is that uniform solenoids are rigid structures, whereas the 200-300-A fiber must be flexible in order to bend into higher-order coils (see, e.g., Sedat and Manuelidis, this volume). Although the proposed variation in diameter (see Table 1) may give some flexibility to the solenoids, a periodic discontinuity seems to be required in order to produce a truly flexible 200-300-A fiber. The second and obviously more compelling reason is that Renz et al. (1977), Kiryanov et al. (1976), Franke (this volume), and Olins (this volume) have recently presented evidence which suggests that the 200-300/~ fiber may indeed be discontinuous and made up of adjacent 200-300-~ superbeads. Uniform 100-A fibers of the type schematically shown in Figure 4a will generate a continuous solenoid. In order to generate discontinuous solenoids, discontinuities must exist along the 100-A fiber itselfi For instance, a nonhistone protein binding to the 100-A fiber with a given periodicity could generate such a discontinuity. Busch et al. (this volume) have recently characterized a protein, A24, which they feel may have a role in a symmetrical supercoiling of inactive chromatin. Protein A24 is composed of a "backbone" of histone H2A linked to "ubiquitin" (a peptide containing about 80 amino acids) by a Gly-Gly isopeptide linkage on lysine 119 (Goldknopf and Busch 1977). Protein A24 is present in nucleosomes in onetenth the amount of histone H2A (Busch et al., this volume). If protein A24 is present in the two heterotypic tetramers of a given nucleosome (which seems likely in order to preserve the symmetry of the nucleosome) and if the A24-containing nucleosomes are periodically spaced along the inactive chromatin, the resulting 100-A fiber should look as schematically drawn in Figure 4b. The two arms protruding from the A24-containing nucleosomes in the figure

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represent the extra 80 amino acids of ubiquitin cross-llnked to H2A. This large bulk of pretein (160 amino acids, almost as large as H1 itself) should impede the binding of i l l to that nucleosome, generating a periodic discontinuity in the proposed H1 lattice. Figure 5 shows a space-filllng model of the discontinuous solenoid generated by coiling such a 10()-/~ fiber. The figure shows one nucleosome in a beadson-a-string conformation followed by two superbeads. The nucleosomes in each superbead are arranged in two helical turns of 110-,/~ pitch and five nucleosomes per turn. The non-Hi-containing nucleosomes between superbeads (these are the nucleosomes cont~inlng A24 or some other suitable nonhistone protein) act as flexible joints; therefore, the minisolenoids can be either stacked on one another, generating longer solenoids, or unstacked as in Figure 5.

The discontinuous solenoid shown is a minor variant of the general solenoid model proposed previously (compare Fig. 4b with 4a and Fig. 5 with Fig. 3). It therefore satisfies all of the previous considerations (Worcel and Benyajati 1977; see also above). It also has the following additional attractive features: 1. It generates a flexible 200-300-A fiber. 2. It exposes the H1 molecules at both ends of the unstacked minisolenoids (whereas only the H1 termini were able to reach the outside of the continuous solenoid of Fig. 3), which is more consistent with the preferential trypsin sensitivity of H1 (Weintraub and Van Lente 1974). 3. It generates unstacked minisolenoids of about 250 A in length and 200-300 A in diRmeter (depending on the number of nucleosomes per helical turn; see Table 1). This is in good agreement with

Figure 5. Space-filling model of a discontinuous solenoid with a 200-bp repeat. Nucleosomes and DNA coil are to scale (see legends to Figs. 1 and 3). This photograph shows a nucleosome in a beads-on-a-string conformation followed by two minisolenoids (superbeads). The heavy arrows point to the non-HI-containing nucleosomes between the minisolenoids. Each minisolenoid has ten nucleosomes arranged in two, lei~-handed, negative (see Table 1) helical turns of 110-/~ pitch and five nucleosomes per turn. For a detailed plan of the Hl-100-A-fiber interaction in this model, see Fig. 4b. The internucleosome spacers are flanked alternately by the COOH (black) and NH2 (white) termini of H1 along the bent 100-A nucleofilament, and the H1 termini interact head to taft between the turns of the nucleofilament, as before, stabilizing the mlnisolenoids. As before (see Fig. 3), the internucleosomal (heavy rings) and intranucleosomal (thin rings) centers of symmetry (see Fig. lb, rear view) are the most-exposed sites on the DNA supercoil.

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T h e proposed model for n u c l e o h i s t o n e h a s profound s t r u c t u r a l a n d f u n c t i o n a l implications. Indep e n d e n t l y o f e a c h other, D N A supercoiling a n d cryst a l l i n e histone-histone interactions will each have

possiblenucleohistone structures.The factthat each order of D N A coiling must allow, or better yet dictate, a higher order of coiling further restrictsthe possible molecular conformations. I have stressed all along the regular, crystalline nature of the nucleohistone fiber.However, chromatin is certainly not a crystal in the c o m m o n sense of the term. Its subunits are not exactly identical, nor are they that regularly packed. As suggested above, there are probably discontinuitiesin the solenoids, as well as variations in both the length and the diameter of the minisolenoids. These variations probably have important structural implications (they could dictate the higher-order coiling of the thick chromatin fiber) as well as functional ones (they could differentiallyaffect gene activity; see Alberts et al. 1977). Table 2 summarizes the prolx~ed structural features in chromatin. The lei~panel liststhe invariant features c o m m o n to all chromatins, and the right panel liststhe possible variations within that comm o n general plan. Each panel is further subdivided into three sections representing the three orders of D N A coiling discussed in this paper. Each order of D N A coiling has its own universal structural element: (1) the nucleosome core, (2) the 100-A nucleofilament, and (3) the thick chromatin fiber.Each of these defined structures, in turn, has strict rules and demands upon the kind of higherorder structures it can generate. Structural variations at one level of organization will therefore necessarily affect the next higher level as shown. W h a t could be the nature and origin of the variations in chromatin structure? I believe that variations in the basic subunit, the nucleosome, are responsible for the proposed (and observed) changes in the higher-order structures.Modified (acetylated, phosphorylated, etc.) and variant inner histones, modified (phosphorylated, polyADPribosylated, etc.)

well-defined geometry rules and properties. Their combination restrictsthat geometry and generates new properties, drasticallyreducing the number of

a n d v a r i a n t H1 histones, a n d specific n o n h i s t o n e p r o t e i n s m a y a l t e r t h e basic c h r o m a t i n subunit. T h e a l t e r e d s u b u n i t s will modify t h e 100-A fiber, a n d

the dimensions of the superbeads which can be seen with the electron microscope (Kiryanov et al. 1976; Renz et al. 1977, and thisvolume; Franke et al.,this volume; Olins, this volume). 4. The numerous and precise Hl-nucleosome interactions within each minisolenoid could explain the observed cooperativityin H I binding to oligonucleosomes. Renz et al. (1977) have shown that H 1 binds tighter to longer chains of nucleosomes than to chains containing fewer nucleosomes, with a plateau in cooperativityreached at a chain length of about 8. 5. It is consistent with the generation of m o n o m e r superboads of about 40S by mild nuclease digestion of nuclei and with the fact that these discrete nucleohistone particles do not possess a D N A molecule of discrete length (Renz et al.,this volume). As shown in Figure 5, the internucleosome spacer D N A is as exposed in the minisolenoids as itisbetween the minisolenoids. Staphylococcal nuclease (cleaving randomly the fully exposed internucleosome spacer D N A ) could generate superbeads al~r cleaving the spacer D N A between minisolenoids; such superbeads would have, in all likelihood, some internal double stranded D N A breaks. Superbeads having fully exposed interuucleesome spacer D N A are also consistent with the well-known kinetics of D N A fragmentation by staphylococcal nuclease, which show no transient accumulation of superbeadequivalent D N A fragments at early digestion times. CONCLUSIONS AND IMPLICATIONS

Table 2. Postulated General Structural Features in Chromatin

Constant: possibly reflecting common requirements for mitosis and cell cycle traverse

Nucleosome core: 140 bp of D N A coiledin a 90-bp/turn left-handedsuperhelixaround two symmetricallypaired histone tetramers (H2A-H3-H4-H2B) 100-A Nucleofilament: uniform left-handed DNA superhelix of 90 bp/turn and 47-A pitch; internucleosome spacer DNA coiled between the symmetrical outer surfaces of adjacent nucleosomes

Variable: possiblyreflecting differential gene activation mechanisms

Variant and modifiedintranucleosomehistones Variant and modifiedHI histones Specific,DNA- and/or nucleosome-bound nonhistone proteins Internucleosome contacts~ azimuthal angle between nucleosomes * D N A superhelicalturns per nucleosome repeat (--1.7 to --2.3)-intornucleosornespacer D N A length (10-70bp) - repeat length (150-210bp) Handedness (+ or --)and number ofnucleosomesper helical turn of the super-superhelix(3-8)* diameter of the thick ~F

Thick chromatin fiber: tightly packed, ll0-A-pitch DNA super-superhelices stabilized by histone H1-H1 interactions between adjacent helical turns of the nucleofilament; internucleesome spacer DNA located on the outside

fiber (20(0300 .~) ~ packing ratio (20X to 50)0

The arrowsin the right(Variable)panelrepresentpostulatedcause~effectrelationshipsbetweenchromatinstructuralchanges,both withinand betweenordersofDNA coilin~

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this, in t u r n , will affect th e t h i c k fiber, as indicated by t h e arrows in Table 2. (A full discussion of t h e

changes in the nucleofilament and in the thick fiber was given above.) W h a t could be the function of such structural changes? As previously discussed (see Alberts et al. 1977), gene regulation in eukaryotes could operate via such changes in chromatin structure. The following is just one representative example. It is likely that active genes have either an extended nucleohistone or a beaded-string conformation (of the kind shown in the lower part of Fig. 3), whereas inactive heterochromatin is in the form of compacted thick chromatin fibers (of the kind shown in the upper part of Fig. 3). O n e m a y speculate, for instance, that a nonhistone protein which could disrupt the interactions that lock the nucleosomes into solenoids could generate a more open, active chromatin state. In this regard, it is relevant to mention the finding (Levy W . et al.,this volume) that two high-mobility-group proteins ( H M G proteins) (Goodwin and Johns 1973) are preferentially released w h e n active genes are degraded by DNaseI digestion a la Weintraub and Groudine (1976). H M G proteins bind tightly to specific H 1 subfractions in solution (Smerdon and Eisenberg 1976). By thus binding to H 1 and, presumably, breaking the H 1 interactions in the solenoids, the H M G proteins could maintain a given patch of chromatin in an open, active conformation. Moreover, such modified chromatin states could also be inherited (Tsanev and Sendov 1971), given the conservative nature of histone segregation (Weintraub et al., this volume). M a n y of the exciting n e w findings reported in this volume are clearly consistent with this view ofchromatin. It is also clear that a detailed knowledge of chromatin structure will help us understand, in molecular terms, the maintenance of the diverse, differentiated state as well as the mechanism for the generation of such a diversity. Acknowledgments

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COMMENTS/QUESTIONS

Question by: D. LOHR Oregon State University

Can your model incorporate r e p e a t sizes which v a r y between adjacent nucleosomes and, i f not, w h a t is the m i n i m u m n u m b e r of correlated nucleosome repeats t h a t will build a good model?

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Response by: A. WORCEL Princeton University

The repeat lengths between adjacent nucleosomes should not vary randomly ifnucleosomes are to be packed helically.I would estimate that two turns, i.e.,eight nucleosomes in a four-nucleosomes-per-turn minisolenoid, would be the shortest coil that could be packaged this way. Bill Garrard (J. Biol. Chem. [1977 in press])has evidence which suggests that repeat lengths do vary in a tandem manner in oligonucleesomes. According to our model, such tandem variation in the nucleosome repeat length will generate changes in the diameter of the "thick" chromatin fiberas well as discontinuitiesin the fiber(minisolenoids or superbeads) which could have important structural and/or functional implications.

Comment by: S. BRAM Institut Pasteur

I think that your nucleosome model is very interesting and is rather similar in m a n y respects to a model I proposed in 1975 (Bram, Biochimie 57: 1301). Our electron micrographs of freeze-etchedchromatin show an external coilof about 100 base pairs around a central 80-A diameter core. Such a coilingagrees well with our neutron-scattering data on chromatin and on nucleosomes.

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Molecular Architecture of the Chromatin Fiber A. Worcel Cold Spring Harb Symp Quant Biol 1978 42: 313-324 Access the most recent version at doi:10.1101/SQB.1978.042.01.033

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