ARCHIVES

OF

BIOCHEMISTRY

AND

Histone-DNA

BIOPHYSXCS

172, 117-122 (1976)

interactions GERALD

in Erythrocyte

Chromatin

R. REECK’

Section of Developmental Biochemistry, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received June 6, 1975 The circular dichroism and thermal denaturation properties of chromatin isolated from duck erythrocytes have been carefully examined as has the chromatography of sonicated erythrocyte chromatin on ECTHAM-cellulose. The circular dichroism spectrum and thermal denaturation profile resemble much more closely those of chromatin from l.iver than has been previously reported by other workers. The chromatography of erythrocyte chromatin on ECTHAM-cellulose gave results that differ dramatically from those obtained from chromatography of fl-containing chromatins on this weak anion exchanger, in that no variations in histone content, circular dichroism spectra or thermal denaturation profiles were observed in the eluted material. Coupled with our earlier finding of no variation in relative content of individual histones (Reeck, G. R. et aE. (1974)Eur. J. Biochem. 49,407-414), we interpret the results of ECTHAM-cellulose chromatography of erythrocyte chromatin to indicate that f2c-depleted regions analogous to the fl-depleted regions found in fl-containing chromatins do not exist in duck erythrocyte chrcimatin.

Avian erythrocyte nuclei are essentially devoid of RNA synthesis (l), and chromatin isolated from these cells displays a much lower “template activity” in vitro than does chromatin from cells active in RNA synthesis (2). Those aspects of the nucleoprotein structure responsible for this complete restriction of transcription are not understood, but it is known that erythrocyte chromatin contains the specialized histone f2c (3). Because the amount of this histone increases during the maturation of erythrocytes in rough correlation with the condensation of chromatin and the gradual decrease in the level of RNA synthesis, it has been suggested (4) that histone t2c may somehow be responsible for the lack of RNA synthesis in the mature erythrocyte. Despite the presence of this specialized histone and the complete restriction of transcriptional activity in erythrocyte chromatin, Itzhaki and Cooper (5) found the accessibility to polylysine and to

DNase2 I of the DNA in this chromatin to be similar to that of the DNA in chromatin from cells active in RNA synthesis. In a further search for unique structural features of erythrocyte chromatin, I have examined its thermal denaturation and circular dichroism properties. I have also extended our study of the chromatography of sonicated erythrocyte chromatin on ECTHAM-cellulose, a well-characterized fractionation procedure that has been applied to a variety of chromatins lacking histone f2c (6-8) and, as we have previously reported (81, results in fractions having constant relative amounts of individual histones when applied to erythrocyte chromatin. METHODS New Zealand White rabbits were sacrificed by exsanguination, the livers removed, and nuclei prepared by the Triton X-100 procedure of Hymer and Kuff (9). Chromatin was purified by successive homogenization of the nuclei in 50, 10, 5 and 1 mM * Abbreviations used: DNase, deoxyribonuclease; ECTHAM-, epichlorohydrin-Tris(hydroxylmethyl)aminomethane.

‘ Present address: Department of Biochemistry, Kansas State University, Manhattan, Kan. 66506. 117 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Tris-Cl, pH 8.0, and centrifugation after each homogenization at 4OOOgfor 10 min. After the final centrifugation, the pellet was suspended in 1 mM Tris-Cl, pH 8.0, sheared for 90 s in a Virtis 23 homogenizer at 90 V, and centrifuged at 10,OOOgfor 30 min to remove unsheared material. One milliliter of the supernatant solution (A,,, “,,, = 10) was dialyzed for 16 h against 4 liters of 0.25 mre EDTA, pH 7.0. Erythrocytes were isolated from blood obtained by decapitation of mature ducks. The blood was collected in Alsever’s solution and then washed three times in 0.9% NaCl, collecting red cells after each wash by centrifugation at 4006g for 5 min and removing the buffy coat. Erythrocyte nuclei and chromatin were then prepared precisely as described above for rabbit liver. Gel electrophoresis of 0.4 N H,SO,-soluble proteins in urea gels at pH 2.7 gave a pattern very similar to that obtained by Panyim et al. (10) for duck erythrocyte histones. Thermal denaturation of the chromatin samples, which had been dialyzed against EDTA and adjusted to A,, nm = 1, was followed in an Acta III spectrophotometer. Absorbance at 260 nm was recorded every 2 min for each sample during a constant temperature rise of 0.25”C/min, controlled by a Neslab temperature programmer. The temperatures on the abscissa of Fig. 1 are the temperatures of the cuvette holder which were somewhat higher than the sample temperatures (see Ref. 6). Circular dichroism spectra were recorded on a Cary 61 spectropolarimeter on samples having A,,, values of about 1. Ellipticities are given as degrees. centimeters* (decimoles of phosphate)-‘. DNA concentration was estimated by absorbance measurements in 0.1% sodium dodecyl sulfate 0.01 M sodium phosphate, pH 7.2, assuming A& = 210. Chromatin, 350 A,, units, was subjected to chromatography on a column containing 10 g of ECTHAM-cellulose under conditions described premateviously (7). The recovery of 260-nm-absorbing rial was 91%. Protein content was determined by the Lowry procedure, with bovine serum albumin as standard, and DNA concentration was estimated by absorbance,measurements. The protein to DNA ratios are expressed as grams of protein per gram of DNA. k&o was determined on samples dialyzed against 0.25 mM EDTA, pH 7.0. The percentage of low melting sequences is defined as the percentage of the hyperchromicity at 100°C that was observed at 70°C on samples dialyzed against 0.25 mM EDTA, pH 7.0. RESULTS

In Fig. 1 are shown the thermal denaturation profiles of erythrocyte chromatin and rabbit liver chromatin, a representative of chromatins from cells active in RNA synthesis. Erythrocyte chromatin has a slightly greater hyperchromicity than

R. REECK

liver chromatin and a distinctive bump in the denaturation profile around 80°C which is absent from the liver chromatin profile. Below 7o”C, however, the profiles are very similar, a finding that is inconsistent with the results and interpretations of McConaughy and McCarthy (11). These workers reported that chicken erythrocyte chromatin contained a much smaller low melting component than liver chromatin and interpreted this to support the hypothesis that transcribed sequences are contained in low melting nucleoprotein. The data in Fig. 1 coupled with the lack of transcription in erythrocytes (1, 2) and the relatively high level of transcription (12) in liver demonstrate that there is no general correlation between the portion of the genome transcribed and the amount of low melting DNA in chromatin. Circular dichroism spectra from 250-300 nm of erythrocyte and liver chromatins are compared in Fig. 2. After dialysis against EDTA (Fig. 2A) the samples had similar maximum ellipticities. The shapes of the spectra are somewhat different, however, with the high wavelength portion of A ,:’

03 c

,,:...

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.:’

,,,........ / ’

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:

,...

B

,:’

Temperature

FIG. 1. Thermal denaturation profiles of rabbit liver chromatin (A) and duck erythrocyte chromatin (B). The increases in the absorbances of the two samples were followed simultaneously with an Ada III spectrophotometer as described in Methods. The thermal denaturation profiles of the samples were identical to each other in 1% sodium dodecyl sulfate, conditions in which protein-DNA interactions are destroyed.

HISTONE-DNA

250

270 290 WAVELENGTH

INTERACTIONS

310

FIG. 2. Circular dichroism spectra of rabbit liver and duck erythrocyte chromatins. The samples were prepared as described in Methods and dialyzed against 0.25 mM EDTA, pH 7.0 (A), or against 0.1 mM Tris-Cl, pH 8.0 (B). Circular dichroism spectra of the two chromatins in 1% sodium dodecyl sulfate were identical. Liver chromatin c-----j; erythrocyte chromatin (--).

the characteristic chromatin doublet being more pronounced in the erythrocyte chromatin. When dialyzed instead against Tris-Cl (Fig. 2B), the ellipticities of both samples were lower and the shapes of the spectra were more distinct from each other. The effect of dialysis against EDTA is not surprising in light of the work of Johnson et al. (13), who showed that extensive shearing of calf thymus glands in buffer containing EDTA resulted in increased ellipticity of chromatin isolated from the tissue. The distinctive shape of the erythrocyte chromatin spectrum has been pointed out by Williams et al. (14). These workers also claimed that this chromatin has a substantially lower maximum ellipticity than other chromatins, but the data in Fig. 2 demonstrate that when one compares erythrocyte and liver chromatins that are prepared in the same manner the maximum ellipticities are very similar. The differences between erythrocyte and rabbit liver chromatins in the shapes of their thermal deriaturation profiles and cir-

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cular dichroism spectra do not appear to be attributable to the presence of histone f2c in erythrocyte chromatin. Johnson et al. (13) carefully studied the thermal denaturation and circular dichroism properties of calf thymus chromatin, which does not contain histone flc. Their results, which I have confirmed in this laboratory, show that, in those features of the thermal denaturation profile and circular dichroism spectrum in which erythrocyte and liver chromatin differ, chromatin from calf thymus strongly resembles that from erythrocytes. That the arrangement of histones in erythrocyte chromatin differs from that in other chromatins is shown by an analysis of the chromatography of erythrocyte chromatin on ECTHAM-cellulose. This ion-exchange cellulose has been shown to fractionate chromatin from rabbit liver, kidney and brain, calf liver and thymus, and HeLa cells into similar spectra of nucleoprotein types that exhibit continuous variation in thermal denaturation and circular dichroism properties (6-8, 15). The basis of the fractionation appears to be histone/DNA ratio, which decreases across the elution profile (7, 8). Analyses of the chromatography of erythrocyte chromatin are shown in Fig. 3. There is no experimentally significant variation in protein/DNA ratio, ellipticity at 280 nm, or the percentage of low melting sequences. These results contrast sharply with the 30% decrease in hi&one/DNA ratio (7), the increase from virtually zero to 40% low melting sequences (6-81, and a 100% increase in ellipticity at 280 nm (15) which have been observed in this laboratory across the elution profiles of chromatins containing histone fl. Elsewhere (8), we have demonstrated that there is no change in the relative proportions of individual histones in the ECTHAM-cellulose fractions of erythrocyte chromatin, which contrasts to the preferential decrease in histone fl in the late eluted fractions of chromatins containing fl (6-S). DISCUSSION

Because of the extreme character of its functional state (total or near total repression), erythrocyte chromatin presents a

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10 -9 -8

=I -T j

FIG. 3. ECTHAM-cellulose chromatography of sonicated duck erythrocyte chromatin. The sample for chromatography had protein/DNA (P/DNA) = 1.20, k&lo = 4400, and percentage of low melting sequences (%LMS) = 22. Analysis of the chromatin bound during a “displacement chromatography,” which was performed as described by Reeck et al. (6) and which should theoretically have allowed examination of all but the most tightly bound 1% of the sample, gave values for P/DNA, [OlzsO, and %LMS similar to those for unchromatographed chromatin.

rather direct challenge to investigators interested in the relationship between the structure and function of chromatin. Despite the considerable effort that has been devoted to the investigation of erythrocyte chromatin, we have very little information on the structural basis for this repression apart from the chemical fact that a specialized histone, fit, appears during the maturation of the erythrocyte in rough correlation with chromatin condensation and repression (4). Prior to investigations of the chromatography of erythrocyk chromatin on ECTHAM-cellulose, there were only two reported differences in physical properties of erythrocyte chromatin and of chromatins from cells active in RNA synthesis: a difference in thermal denaturation profiles reported by McConaughy and McCarthy (11) and a difference in circular dichroism spectra reported by Williams et al. (14); and these two observations provided the only two indications of structural features unique to erythrocyte chromatin. In this paper I have presented data inconsistent with each of these reports that un-

R. REECK

dermine their seemingly significant insights into erythrocyte chromatin structure. In each case a straightforward explanation is apparent for the discrepancies between my results and those reported earlier . McConaughy and McCarthy (11) corn- . pared the thermal denaturation profiles of liver and erythrocyte chromatins prepared by two entirely different procedures. Of particular importance is the fact that in the preparation of liver chromatin, the sample was repeatedly homogenized in 0.075 M NaCYO.024 M EDTA, pH 8.0, whereas the erythrocyte sample was never exposed to EDTA but was instead homogenized in 0.05 M Tris-Cl. In their very systematic study, Johnson et al. (13) demonstrated that the extent to which chromatin is exposed to EDTA in a purification procedure affects the thermal denaturation profile of the purified chromatin: A more extended exposure and a harsher homogenization in the presence of EDTA results in greater content of low melting sequences. This provides a plausible resolution between McConaughy and McCarthy’s observation that their sample of liver chromatin contained more low melting sequences than did erythrocyte chromatin and my observation that erythrocyte and liver chromatins prepared using a single method for purification have virtually identical contents of low melting sequences. The differences between my conclusions and those of Williams et al. (14) on the circular dichroism spectrum of erythrocyte chromatin can be explained on the same basis. Williams et al. (14) isolated erythrocyte chromatin with a procedure in which no EDTA was used; they lysed cells with saponin and repeatedly washed the nuclei in 0.05 M Tris-Cl); but in each of the studies to which they referred (Ref. (1) and (37) in their paper) in which higher maximum ellipticities had been found for other chromatins, purification procedures were employed in which the samples were repeatedly homogenized in EDTA-containing solutions. Johnson et al. (13) clearly demonstrated that the greater the exposure to EDTA in an isolation procedure, the higher the maximum ellipticities ob-

HISTONE-DNA

INTERACTIONS

served with calf thymus chromatin. The results in Fig. 2 of this paper, in which the circular dichroism spectra are shown for chromatins purified without the use of EDTA but then dialyzed against either Tris-Cl or EDTA, are entirely consistent with the observations of Johnson et al. (13). The results in Fig. 2 furthermore demonstrate that erythrocyte and liver chromatins prepared by identical procedures have very similar maximum ellipticities. As was pointed out in Results, the small differences in thermal denaturation and circular dichroism properties which I have observed between erythrocyte and liver chromatins do not appear to be due to the presence of histone f2c in the erythrocyte chromatin since, in those respects in which erythrocyte and liver chromatins differ, the erythrocyte chromatin resembles flcontaining calf thymus chromatin. In this light, the results of chromatography of erythrocyte chromatin on ECTHAM-cellulose are of considerable interest in that they provide the only clue we currently have into hi&one-DNA interactions that might be unique to erythrocyte chromatin and that could be related to the unique functional properties of this nucleoprotein. The failure to observe variation across the ECTHAM-cellulose elution profile in relative content, of individual histones (B), total protein content, circular dichroism, and thermal denaturation is clearly not due to some experimental flaw since these observations have been made repeatedly in the course of these studies using a batch of ECTHAM-cellulose that both before and after the work on erythrocyte chromatin gave typical fractionation of H-containing chromatins. The constancy reported in this paper across the elution profile in total protein content, ellipticity at 280 nm and content of low melting sequences provides an entirely new line of evidence that supports the inference drawn from our work on fl-containing chromatins that the variations in physical properties in the chromatin fractions from ECTHAM-cellulose chromatography are in fact caused by changes in histone content and not by some hitherto unrecognized factor. We have interpreted the results of the

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fractionation of fl-containing chromatins to reflect an underlying similarity of histone organization consisting of extended @‘l-depleted) stretches of nucleoprotein on the order of 1000 base pairs long interspersed with condensed regions of the same approximate length which contain histone fl(8). Since duck erythrocyte chromatin contains very little histone fl (lo), it could not strictly have this same organizational pattern, but, as pointed out by Greenaway and Murray (16), histone f2c is similar to histone fl in molecular weight, amino acid composition, and NaCl concentrations required for its removal from DNA. Hence, one might imagine that erythrocyte chromatin has an organization comparable to other chromatins with histone f2c playing the role of histone fl. The results of ECTHAM-cellulose chromatography show that this is not the case, since histone f2c-depleted nucleoprotein fragments are not observed after chromatography on ECTHAM-cellulose. Recent work from several laboratories has led to the hypothesis that histones are bound to DNA as oligomeric complexes containing two or more types of histones (17, 18, 19). The electron micrographs of Olins et al. (17) have revealed essentially similar structures (V bodies) in chromatin from avian erythrocytes and rat liver. However, histone fl has not been found in any of the histone complexes observed to date (18, 19). Thus, the lysine-rich histones may be distributed on DNA independently of the histone complexes, and the results of ECTHAM-cellulose chromatography indicate that fl and t2c are not distributed in an identical manner. Despite the fact that erythrocyte chromatin is known to be inactive in transcription in vivo it bears a considerable resemblance to chromatin from cells active in RNA synthesis in the accessibility of its DNA to DNase I (5), thermal denaturation profile, and circular dichroism spectrum. Nonetheless, erythrocyte chromatin differs dramatically from these chromatins in its chromatographic behavior on ECTHAMcellulose. Simpson has recently demonstrated that the fl-depleted, late-eluted ECTHAM-cellulose fractions of calf thy-

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mus chromatin are enriched several fold in E. coli RNA polymerase binding sites compared to unfractionated chromatin (20). Using an entirely different procedure, Gottesfeld et al. (21) have isolated a nucleoprotein fraction of liver chromatin which is enriched several fold in transcribed DNA sequences. Histone fl, in contrast to other histones, is virtually absent from this transcriptionally active fraction. The results from these two fractionation procedures suggest that the distribution of histone fl and particularly the existence of fl-depleted regions play a role in determining which DNA sequences are transcribed. Chromatography of erythrocyte chromatin on ECTHAM-cellulose shows that histone f2c-depleted regions analogous to histone O-depleted regions of other chromatins do not exist in erythrocyte irhromatin and presents the possibility that this unique feature of the distribution of histones is related to the complete restriction of transcription in erythrocytes. ACKNOWLEDGMENTS I thank Dr. Robert T. Simpson for many stimulating discussions and I acknowledge the support and encouragement of the late Dr. Herbert A. Sober. REFERENCES 1. CAMERON, I. L., AND PRESCQTT, D. M. (1963) Exp. Cell Res. 30, 609-612. 2. SELIGY, V., AND MIYAGI, M. (lS6S)Exp. Cell Res. 58, 27-34. 3. HNILICA, L. S. (1972) The Structure and Biologi-

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

cal Functions of Histones, p. 30, C.R.C. Press, Cleveland, Ohio. BILLET, M. A., AND HINDLEY, J. (1972) Eur. J. Biochem. 28, 451-462. ITZHAKI, R. T., AND COOPER, H. K. (1973)5. Mol. Biol. 75, 119-128. REECK, G. R., SIMPSON, R. T., AND SOBER, H. A. (1972) Proc. Nat. Acad. Sci. USA 69, 23172321. SIMPSON, R. T., AND REECK, G. R. (1973) Biochemistry 12, 3853-3858. REECK, G. R., SIMPSON, R. T., AND SOBER, H. A. (1974) EUF. J. Biochem. 49.407-414. HYMER, W. C., AND KUFF, E. L. (1964) J. Histothem. Cytochem. 12,359-363. PANYMIN, S., BILEK, D., AND CHALKLEY, R. (1971) J. Biol. Chem. 246,4206-4215. MCCONAUGHY, B. L., AND MCCARTHY, B. J. (1972) Biochemistry 11, 998-1003. GROUSE, L., CHILTON, M. D., AND MCCARTHY, B. J. (1972) Biochemistry 11, 798-805. JOHNSON, R. S., CHAN, A., AND HANU)N, S. (1972) Biochemistry 11, 4347-4358. WILLIAMS, R. E., LURQUIN, P. F., AND SELIGY, V. L. (1972) EUF. J. Biochem. 29,426-432. POLACOW, I., AND SIMPSON, R. T. (1973) Biothem. Biophys. Res. Commun. 52,202-207. GREENAWAY, P. J., AND MURRAY, K. (1970) Nature New Biol. 229, 233-238. OLINS, A. L., CARLSON, R. D., AND OLINS, D. C. (1975) J. Cell Biol. 64.528-537. KORNBERG, R. D., AND THOMAS, J. 0. (1974) Science 184,865-868. D’ANNA, J. A., JR., AND ISENBERG, I. (1974) Biochemistry 13, 4992-4997. SI~SON, R. T. (1974) Proc. Nat. Acad. Sci. USA 71 2740-2743. G~TTESFELD, J. M., GARRARD, W. T., BAGI, G., WILSON, R. F., AND BONNER, J. (1974) PFOC. Nat. Acad. Sci. USA 71, 2193-2197.

Histone-DNA interactions in erythrocyte chromatin.

ARCHIVES OF BIOCHEMISTRY AND Histone-DNA BIOPHYSXCS 172, 117-122 (1976) interactions GERALD in Erythrocyte Chromatin R. REECK’ Section of D...
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