modification of histones during spermiogenesis in trout: a molecular mechanism for altering histone...

Modification of Histones during Spermiogenesis in Trout: A Molecular Mechanism forAltering Histone Binding to DNAAuthor(s): Michael T. Sung and Gordon H. DixonSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 67, No. 3 (Nov. 15, 1970), pp. 1616-1623Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/60496 .

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Proceedings of the National Academy of Sciences Vol. 67, No. 3, pp. 1616-1623, November 1970

Modification of Histones during Spermiogenesis in Trout: A Molecular Mechanism for Altering

Histone Binding to DNA

Michael T. Sung and Gordon H. Dixon

DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER 8, CANADA

Communicated by Emil L. Smith, July 20, 1970

Abstract. At a late stage of spermatogenesis in rainbow-trout testis, the entire complement of histones is replaced by newly synthesized protamine and histones are extensively phosphorylated and acetylated. Tryptic digestion of purified histones labeled by incubation of testicular cells with [32P ]phosphate shows that phosphorylation occurs at a small number of seryl residues.

Histone I (lysine-rich) is phosphorylated in the sequence Lys-Ser(PO4)-Pro- Lys, which is located in the lysine-rich C-terminal region of the molecule. His- tones IIb1 (slightly lysine-rich) and IV (glycine, arginine-rich) give rise to the same phosphopeptide, Ac-Ser(PO4)-Gly-Arg, which comprises the amino terminus of each histone. Thermolysin digests of phosphohistones IIb1 and IV also released a phosphopeptide with composition corresponding to the first six residues of histone IV: Ac-Ser(PO4)-Gly-Arg-Gly-Lys-Gly. An a-helical model of the N-terminal region of histone IV shows that this region is a possible DNA-binding site. Phosphorylation at serine 1 together with e-amino acetylation at lysines 5, 8, 12, and 16 (observed in histone IV from trout testis) could profoundly modify ionic interactions and lead to an "unzipping" of histone IV from DNA

It is generally assumed that the high content of basic residues in the histones of eukaryote chromosomes provides the major basis for their strong binding to DNA by electrostatic interaction. Histones may be dissociated from DNA in vitro by exposure of chromatin to high ionic strengthsl12 or acidic pH,3 both of which weaken electrostatic bonds. However, these extreme conditions do not occur physiologically; the question therefore arises, are histones ever dissociated from DNA in vivo, and, if so, by what mechanisms?

Spermiogenesis, the process of sperm formation and maturation, provides an unequivocal biological answer to the first question. In the spermatid cells of salmonid fishes, the entire complement of histones is progressively dissociated from DNA and replaced by a newly-synthesized sperm-specific protein, protamine.46 Possible biochemical mechanisms for removal of histones from DNA include enzymatic modification of the histones,7'8 formation of complexes of anionic molecules with the histone,9 and perhaps also specific proteolysis of the histones in situ on the chromatin. The first possibility has been most explored and it now seems clearly established that three types of histone modification can occur: phosphorylation of the hydroxyl groups of seryl'0'-4 or

1616



VOL. 67, 1970 ALTERING HISTONE BINDING TO DNA 1617

threonyl'0 groups, acetylation of both a-"5-17 and e-amino groups'8'30 and methyl- ation of E-amino-'9'20 or guanidino-21 groups. During the replacement of histones by protamine in the testes of developing rainbow trout (Salmo gairdnerii), we have observed substantial phosphorylation'2l3 and acetylation32 of testis histones; we have characterized the sites of phosphorylation14 and partially characterized the sites of acetylation. In histone IV (the glycine, arginine- rich histone) whose sequence is known,'6'17'22 the sites of phosphorylation and acetylation are clustered in the N-terminal region of the molecule. We pro- pose here a molecular mechanism by which these modifications can alter the electrostatic binding of this portion of histone IV to DNA and lead to dissociation of the nucleohistone complex.

Materials and Methods. Suspensions of trout testis cells (1 vol of packed cells in 3 vol of Waymouth23-Tris medium) were allowed to incorporate carrier-free [32p]_

phosphate (75 uCi/ml) for 2 hr in phosphate-free Waymouth medium at 20?C. After the cells had been washed with cold medium, they were broken'2"83 in a Potter-Elvehjem homogenizer (5000 rpm, 30 sec) and chromatin was prepared.6 The washed chromatin! was extracted with 0.2 N HCl and the supernate, after centrifugation, was adjusted to pH 7.0 with NaOH and recentrifuged. The basic proteins in the supernate were then adsorbed onto a column of either CM-cellulosel2 or CM-Sephadex12 (2 X 15 cm) and the column was washed with 0.25 M lithium chloride to remove non-histone proteins and any contaminating inorganic ['2P]phosphate, and with water to remove salt. The histones were eluted with 0.2 N HCI. After lyophilization, the histone (plus protamine) fraction was further separated on a Bio-Gel P-10 column (2.5 X 170 cm), with 0.01 N HCl as eluent. After appropriate pooling, the major histone fractions were rechro- matographed under the same conditions. After the second chromatography, histones I, IIb,, HIb2, and IV were essentially free of each other as judged by electrophoresis on starch-urea-aluminum lactate gels.24 One or two tubes were selected from the peak of the appropriate ['2P]phosphohistone and lyophilized before digestion with porcine trypsin (1:50 by weight; Novo Industri) in 0.1 M ammonium bicarbonate, pH 8.0 for 2 hr at 38?C. 32P-labeled histones IIb, and IV were also digested with thermolysin2' (gift of Prof. T. Ando, University of Tokyo) at an enzyme: substrate ratio of 1:50 by weight in 0.1 M N-ethylmorpholine, 5 mM CaCl2, pH 8.0 for 8 hr at room temperature.

The ['2P]phosphopeptides resulting from digestion with trypsin and thermolysin were separated by high-voltage electrophoresis in pyridine-acetic acid-water 10:0.4:89.6, pH 6.5, and located by autoradiography.

Results and Discussion. Fig. 1 shows the elution profile of 32P-labeled histones and protamine from testes of naturally maturing rainbow trout collected in December, 1969. At this time, the number of spermatid cells in the testis was high and histones were being replaced by protamine. It may be seen that 32p is

associated with each histone fraction. Histone IV shows the highest 32P/A230 ratio, followed by histones IIb,, I, and IIb2. Histone III, the cysteine-con- taining histone, occurs in two forms, as the monomer (sulfhydryl) and dimer (disulfide), and incorporates very little 32p.

After tryptic digestion and electrophoresis of the [32P ]phosphopeptides, an autoradiograph (Fig. 2) shows the appearance of a single, acidic phosphopeptide (T) with the same mobility26 (MASP = -0.61) from histones JIbl and IV. We have reported previously'4 that this peptide comes from the N-terminus of histone IV and is the phosphorylated form, Ac-Ser(PO4)-Gly-Arg, of the known sequence Ac-Ser-Gly-Arg.15'-7 An identical phosphopeptide appears in histone IIb,; a finding consistent with Phillip's observation" of an N-acetylated, ninhydrin-



1618 BIOCHEMISTRY: SUNG AND DIXON PROC. N. A. S.

HISTONES -800 PROTAMINE 1600 1.5f

t I~~~~1 fb Ilb2 ib E t I 2 mm 600 ,e 1200 I

tR 1.0

0 800 40 60 80 100 120140U0 1 0

A ~~ 200 1 ~400

0.0~~~~~~~~~~~~~~~~~~ 0 20 40 60 80 100 120 140 160 180 200

TUBE NUMBER

FIG. 1. Bio-Gel P-10 chromatography of 32P-labeled basic chromosomal proteins from trout testis. The column was eluted with 0.01 N HCl and 6-ml fractions (flow rate 50 ml/hr) were collected. Absorbance at 230 nm was measured and the radioactivity of 0.2-ml aliquots in 10 ml of Bray's solution32 was determined in a scintillation couniiter.

negative peptide of identical composition in tryptic digests of f2a2 (IIb1) prepared by the Johns27 procedure. Two conclusions may be drawn concerning the in vivo phosphorylation of histones IV and IIb1. Only one seryl sequence is phosphorylated in each histone, and histone IV, the glycine, arginine-rich histone, shows the same N-terminal sequence as IIb1, a slightly lysine-rich histone.

To explore whether this identity of sequence extended further, 32P-labeled histones IV and IIb1 were digested with thermolysin25 rather than trypsin. Autoradiography of the ionophoretogram of the thermolysin digest (pH 6.5) showed a major phosphopeptide (,1ASP - -0.13), migrating close to the origin

r ~~~~~~ ~ ~ ~ ~ ~FIG. 2. Autoradiogram of a

high-voltaeinphrtcsepara- tion of 31P-labeled tryptic peptides

~;r K from histones I, 1Ib,, Ib2, and IV. High voltage ionophoresis was per- formed on Whatman 3 MM paper at pH 65 forl1hr at 62V/cm. The autoradiograph was obtained by exposure to Kodak Royal Blue x-ray film for 24 hr. The position of several markers of known mobilities- [2p] Pi Orange G (IAAsp = -0.92),

XCFF (UAsp = -0.38), and e dinitrophenyl-Lys (MAASp = 0)- is also shown. Mobilities of the phosphopeptides from individual histones are as follows (all as jA)

Histone I, Ti (-0.27) and T2 (+0.25); histones Ilb1 and IV, T (-0.61), histone I1b2, TI (-0.27)

~~~ ~~~ ~and T2 (- 0.073).




in both histones IIb1 and IV. These peptides had identical mobility at pH 3.6 and 1.9; their amino acid composition was Lyso.92 Argo.78 Ser 1.oo Gly3.15. This composition corresponds with that expected for the first six residues of histone IV16,17, thus thermolysin must have split the Gly-Gly bond* between positions 6 and 7 in the sequence Ac-Ser(1)-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu. It is clear, therefore, that the identity in N-terminal sequence of histones IV and IIb1 extends to at least six residues.

In contrast to the similarity of the phosphopeptides from histones IV and IIb1, quite distinct sites are phosphorylated on the lysine-rich histone I and the other slightly lysine-rich histone JIb2. For histone I, two major phosphopeptides, (Ti)1 and (T2)1, are seen in the tryptic digest. These peptides have been characterized and their sequences determined: (Ti)l is Ser(PO4)-Pro-Lys and (T2)1 is Lys-Ser(PO4)-Pro-Lys. It seems that a phosphoryl group on the seryl residue slows the rate of cleavage of the Lys-Ser(PO4) bond, thus accounting for the presence of (T2)I.

Bustin and Cole28 have shown that chemical cleavage of rabbit-thymus histone I with N-bromosuccinimide at its single tyrosyl residue bisects the molecule into an N-terminal portion rich in hydrophobic residues and a larger C-terminal portion rich in lysyl residues. Application of this technique to the 32P-labeled histone I isolated here shows that the Lys-Ser(PO4)-Pro-Lys sequence is in the C-terminal portion of the molecule. Two major conclusions may be drawn: first, a single seryl residue is phosphorylated in vitro in the C-terminal region of histone I; second, this sequence occurs in a region of the molecule which, be- cause of its high content of basic residues,28 is likely to be involved in DNA binding.

The phosphopeptides from histones JIb2 seen in Fig. 2 have not so far been characterized in detail. (Tl) Hb2 has a mobility similar to, but not identical with, that of (Ti)7, but (T2)IIb2 is different and may represent a third distinct phosphorylation site in testis histones.

What is the biological significance of these phosphorylation reactions at specific sites in the testis histones? As shown above, for IIb1 and IV the phosphorylation is in the N-terminal sequence, whereas for I the site appears nearer the C-terminal. For these histones, the site phosphorylated is in a portion of the molecule rich in basic residues. If, as seems likely, electrostatic bonds play an important role in DNA binding, the basic regions of the histone are likely to provide the binding sites. Thus phosphorylation occurs at a specific site within, or close to, a DNA-binding site.

What are the possible conformations of the N-terminal region of histone IV and the effect of enzymatic modification upon them? In Fig. 3, the first 18 residues of histone IV are arranged in the form of a helical wheel, a graphic representation29 of the view (from C- to N-terminal) along the axis of an a- helical polypeptide chain. The circumference of the circle represents the polypeptide backbone of the helix and the projections, the amino acid side chains. A striking feature of this representation is the cluster of cationic side chains provided by lysines 5, 8, 12, and 16 projecting in a limited 80? arc from one side of the a-helix.



1620 BIOCHEMISTRY: SUN\G AND DIXON PRoc. N. A. S.

Po=

Q-N-Ac-Ser ?-N-Ac -Lys

a-N-AcSer NH'-Lys

6-N-Ac-Lys 3L 8 Ala

AH'gLysu

is a folows'"72 fortheNirs 1reiusAcSr1)Gyrg-Lys(5-Gy4lyLylGy

E:N-Actys \/

NHL-Lys rH I t h 11 Gly

Gly 9 i i

< /~~~~~~~~~~~7 Gly

Gly 2 \X

/\ / \~~~~~~~~~~14

Gly 13 Glyr

Gly 1 7 lo Ar

Arg Leu

FIG. 3. The linear sequence of histone IV of both calf-thymus and pea-cotyledon chromatin is as follows16,17 22 for the first 18 residues; Ac-Ser(l)-Gly-Arg-Gly-Lys(5)-Gly-Gly-Lys-Gly- Leu(10)-Gly-Lys-Gly-Gly-Ala(15)-Lys-Arg-His-. In the figure, this N-terminal portion of the sequence is plotted in the form of a helical wheel, a convenient method proposed by Schiffer and Edmundson29 for representing the potential a-helical conformation of a polypeptide chain. On the outer periphery of the wheel the positions of known enzymatic modifications of the portion of the sequence of histone IV are shown. Phosphorylation of Ser-1 was reported by Dixon et al.14 E-Acetyl groups were found by DeLange et al. on Lys-16 in calf thymus histone IV1" and on either Lys-5 or -8, as well as Lys-16, in pea-cotyledon histone JV.22

During the sequence analysis of histone IV from calf thymus, the peptide that contains lysyl 16 was recovered both free and E-acetylated in roughly equal pro- portions.'6 In pea histone IV, in addition to the E-acetyl on lysyl 16, a second eacetyl group was present either on lysyl 5 or lysyl 8.22 In vivo, the arginine- rich histones III and IV of calf thymus nuclei'8'30 were observed to be acetylated upon the e-amino groups of lysyl residues. In the developing trout testis, during the phosphorylation period, extensive e-acetylation of histone IV also occurs at several lysyl residues in the N-terminal region (E. P. W. Candido and G. H. Dixon, unpublished). In fact, it is possible to demonstrate (by starch gel electrophoresis) nine modified species of histone IV in trout testis chromatin (Fig. 4). We have identified these (unpublished work of Candido, Sung, and Dixon) as mono-, di-, tri-, and tetraacetyl histone IV, together with monophosphohistone IV and four doubly modified derivatives (mono-, di-, tri-, and tetraacetyl histone IV, each with a phosphoryl group esterified to seryll. The acetylated bands have been shown to contain ['4C ]acetate by radioautography of dried gels (unpublished work of Candido, Sung, and Dixon) and the label can be recovered as E-['4C-acetyl]lysine by pronase digestion. The five slower-running bands disappear upon treatment with E. coli phosphatase and can be shown to contain [32P]phosphate by radioautography when the




FIG. 4. A series of modified histones IV Unsubstituted

present in naturally maturing trout testis. Al The histones were separated by chromatography on Bio-Gel P-10; the peak fractions corre- A2 sponding to histone IV (Fig. 1) were pooled, lyophilized, and applied to a starch-urea-aluminum A3 lactate gel24 as a 1% solution. Electrophoresis was at 8 V/cm for 16 hr at 4?C in a water- A4 cooled gel tray. Only that portion of the gel showing protein bands is included; the band of unsubstituted histone IV ran 12.4 cm from Pi Al the origin of the gel. The subscript numbers indicate the number of acetyl (A) and/or phos- --A phoryl (P) groups modified per histone IV molecule. P A

Pi A4

histone IV has been labeled in vivo with [32P ]Pi. Histone Ilb1, which shows the same N-terminal phosphorylated sequence as IV, is also acetylated at more than one site. Although the N-terminal sequence of IIb1 beyond position 6 is not known, it is possible that acetylation is occurring at lysyl residues homologous to those in histone IV.

In Fig. 3 (on the outer periphery of the wheel), the known sites of acetyla- tion16'22 and phosphorylation14 on histone IV are indicated through the extended projections. Upon full enzymatic modification of Ser-1 and Lys-5, -8, -12, and -16, as in trout testis histone IV, the net charge in this limited region of the molecule could change from +6 (4 Lys plus 2 Arg) to 0 (4 e-acetyl-Lys-, 2 Arg plus PO32-). Consequently, the strong electrostatic bonding of this region of histone IV to DNA would be almost eliminated.

When a three-dimensional molecular model of this region is constructed (CPK Models, Ealing Corp.), it can be seen (Fig. 5a) that the four lysine residues form a sweeping curve extending from the N-terminal Ac-Ser- residue along one side of the a-helix over a region of five turns. When this model is fitted to a model of the DNA double helix in the B-configuration (Fig. 5b), the a-helical histone fits neatly into the major groove. Alternative models of the polypeptide in a left-handed a-helix or a right-handed 7r-helix gave much less satisfactory fits. In the a-helical model, the four amino groups of the lysyl side chains at 5, 8, 12, and 16 and arginine at position 17 make close contacts with five consecutive phosphate groups on one DNA chain. The arginyl residue at position 3 binds to a phosphate on the other DNA strand. The occurrence of four glycyl residues at 2, 6, 9, and 13 on one side of the a-helix and four glycyl and one alanyl residues at 4, 7, 11, 14, and 15 on the other side enables this a-helical region of the histone to fit completely within the major groove. The bulky hydrophobic residue, Leu-10, is accommodated deep in the major groove where it can make hydrophobic contacts with two DNA base pairs. An interesting feature of this



1622 BIOCHEMISTRY: SUNG AND DIXON PROC. N. A. S.

00 16YS S Lys LYS 5 _rW 12

_ - L,YSIL 1)

FIG. 5. (a) A molecular model of the N-terminal region of histonie IV in the a-helical conformation. The side chains at positions 1, 5, 8, 12, and 16 are labeled. On the left side of the figure is a model of one turn of a DNA double helix in the B-conformation.

(b) The proposed binding of the N-terminal, a-helical region of histone IV in the major groove of DNA. The lysyl residues at positions 5, 8, 12, and 16, which are indicated by black circles on their e-amino hydrogens, make close contacts with four consecutive DNA phosphates (indicated by the letter P). The axis of the a-helix is indicated by the arrow at lower left.

model is that upon phosphorylation of the Ac-Ser- at the N-terminus of the histone, the phosphoryl group can readily form an electrostatic interaction with Lys-5, thus competing with and weakening the interaction of Lys-5 with a DNA phosphate. In fact, this competition would be in favor of the phosphomonoester group of the Ac-Ser(PO4)- since at pH 7.2 it would possess 1.7 negative charges compared with the single negative charge on the secondary phosphate ester of the DNA. It should be noted in the model that the serine hydroxyl group is situated so as to be easily accessible for phosphorylation by a histone kinase3' approaching the nucleohistone. In addition, it can be seen from Fig. 5b that although the e-amino groups of lysines 5, 8, 12, and 16 are complexed with DNA phosphates, they could be approached readily by a histone acetylase. Candido and Dixon have observed such an enzyme, bound to trout testis chromatin, which can transfer acetyl groups from acetyl CoA to histone IV.

The occurrence of this DNA-binding site at the N-terminus of two different histones and its modification in both cases by combined phosphorylation and acetylation could initiate a local "unzipping" of histones IV and lIb1, perhaps in preparation for the total removal of the histone and its replacement by newly synthesized protamine. Alternatively, the local, N-terminal, "unzipping" could provide a signal for a specific "histonase" to degrade the modified histone before its replacement by protamine.

Although the total replacement of histones by protamine probably provides an extreme example of the removal of histones from DNA, it seems possible that similar molecular mechanisms may operate where limited histone removal might be required. This could occur during gene activation, replication, cell differentiation, or prior to cell division.




This work was generously supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. M. S. is a Fellow of the Helen Hay Whitney Foundation. The authors wish to thank Peter Candido for communicating a great number of results prior to their publication, Don Wigle and Bengt Jergil for useful discussions, Michael Smith for critical reading of the manuscript, and Josef Durgo for assistance in preparing figures.

* Cleavage of Gly-Gly by thermolysin has not been reported previously. It is possible that this activity is that of a contaminating enzyme rather than that of thermolysin itself.

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