ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Role of Nonhistone Circular
169, 678-685
(1975)
Chromosomal Dichroism
Spectra
CLAUDIO Department
of Physiology
Proteins
in Determining
of Chromatin
NICOLINI
and Biophysics, Temple Philadelphia, Pennsylvania
University 19240
Health
Sciences
Center,
AND
RENATO Department
of Pathology
and Fels Research Philadelphia, Received
BASERGA Institute, Temple Uniuersity Penns.yvluania 19140 January
School
of Medicine,
:30, 1975
Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250-300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because. when DNA is complexed with both histones and nonhistones. the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistoneDNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25-0.35 M NaC1, causes a decrease in the positive circular dichroism band in the 250-300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 M NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25-0.35 M NaCl have an opposite effect.
Circular dichroism is a useful technique for determining changes in the asymmetry of a macro molecule containing chromophores (1, 2). It has frequently been used, in the past few years, to study changes induced in the DNA molecule by its interaction with histone proteins (3-g). However, chromatin preparations contain, besides DNA and histones, other proteins that go under the collective name of nonhistone chromosomal proteins. These proteins have recently received a considerable amount of attention as possible regulators of gene expression in general, and cell proliferation in particular (10-15). The present communication is an attempt to study the role of nonhistone chromosomal proteins as a group, and of certain fractions of nonhistone proteins in determining the 678 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
shape of circular dichroism spectra of chromatin. All these studies were carried out on WI-38 human diploid fibroblasts grown in culture. MATERIALS
AND
METHODS
Cell culture. WI-38 human diploid fibroblasts were purchased from Flow Laboratory, Rockville. MD, and were used between the 25th and 27th passages. They were grown in Basal Eagle’s Medium (Associated Biomedical Systems, Buffalo, NY) supplemented with 10% fetal calf serum and antibiotics, as previously described (16). The cells were used 7 days after plating when they were confluent. Preparation of chromatin. Chromatin was prepared by the method of Marushige and Bonner (171, with the modifications introduced by Paul and Gilmour (18). The cell pellets were washed two times with 10 vol of 1% Triton X-100, 20 mM EDTA, and 80 rnM NaCl, pH 7.4. Clean nuclei were obtained by
679
CD OF CHROMATIN centrifugation through 0.25 M sucrose. The nuclear pe:!ct was washed twice with 10 vol of 0.15 M NaCl and 0.01 M Tris-HCl, pH 8, and allowed to swe!l in cold distilled water for 60 min in an ice bath. The nuclei were then lysed by gentle homogenization with a few strokes of a Teflon pestle and chromatin was purified by centrifugation through 1.7 M sucrose in an SW 39L Spinco rotor for 80 min at 37,000 rpm. The unsheared chromatin pellet was resuspended in water and dialyzed overnight against 0.01 M Tris, pH 8.0. This material was used after appropriate dilution with 0.01 M Tris, for the determination of circular dichroism spectra of chromatin. Dissociation of chromatin and separation of histones from nonhistone chromosomal proteins. Purified chromatin was dissociated in 6 M urea, 0.35 M guanidine-HCl, O.l”r P-mercaptoethanol, and 0.1 M sodium phosphate, pH 7, as described by Levy et al. (19). DNA was sedimented by centrifugation at 105,OObg at 4°C for 40 h using a 50 Ti rotor in a Spinco Model L 65B ultracentrifuge. Some of the proteins (, P. F., AN” SELIGY. v. L. (197%) Eur. J. Eiochem. 29, 426-432.
685
35. FASMAN, G. D.. SCHAFFHACWN, B.. GoLixhluw, L., AND ADIXK. A. (1970) Biochemistn 9, 2814-2821. 36. LIN, J. C., NI~OI,I~I, C.. AND BASEKW R. (1974) Biochemistn 13, 4127-4133. 37. BASERCA. R., BOMHIK, B., AND NICOLISI, C. (1975) in The Structure and Function of Chromatin. A Ciba Foundation Symposium, pp. 269-289. 38. NI~OI.IXI, C.. AND BASEK(:-\, R.. Chrm. Hid. Interactions, in press (1975). 39. GREEWIEIJ). N., 4x1) FAS~~IA?~, G. D. ( 19691 Biochemist? 8, 4108~4116. 40. EI.GIN, S. C. R.. ASI) BONSE‘K, J. (1972) Riochemistryv 11,77”~781. 41. WV, F. C.. EI.~;IX, S. C. R.. ANDHOOD, L. E. (1973) Biochemistn 12, 2792-X97. 42. E~.c;rs. S. C. R., ANI) Hoon, L. E. (19731 Biochemistc, 12, 4984-499 1. 43. STEIS, G.. CHAITIHI HI, S., AND BASER
OF BIOCHEMISTRY
AND
BIOPHYSICS
Role of Nonhistone Circular
169, 678-685
(1975)
Chromosomal Dichroism
Spectra
CLAUDIO Department
of Physiology
Proteins
in Determining
of Chromatin
NICOLINI
and Biophysics, Temple Philadelphia, Pennsylvania
University 19240
Health
Sciences
Center,
AND
RENATO Department
of Pathology
and Fels Research Philadelphia, Received
BASERGA Institute, Temple Uniuersity Penns.yvluania 19140 January
School
of Medicine,
:30, 1975
Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250-300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because. when DNA is complexed with both histones and nonhistones. the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistoneDNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25-0.35 M NaC1, causes a decrease in the positive circular dichroism band in the 250-300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 M NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25-0.35 M NaCl have an opposite effect.
Circular dichroism is a useful technique for determining changes in the asymmetry of a macro molecule containing chromophores (1, 2). It has frequently been used, in the past few years, to study changes induced in the DNA molecule by its interaction with histone proteins (3-g). However, chromatin preparations contain, besides DNA and histones, other proteins that go under the collective name of nonhistone chromosomal proteins. These proteins have recently received a considerable amount of attention as possible regulators of gene expression in general, and cell proliferation in particular (10-15). The present communication is an attempt to study the role of nonhistone chromosomal proteins as a group, and of certain fractions of nonhistone proteins in determining the 678 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
shape of circular dichroism spectra of chromatin. All these studies were carried out on WI-38 human diploid fibroblasts grown in culture. MATERIALS
AND
METHODS
Cell culture. WI-38 human diploid fibroblasts were purchased from Flow Laboratory, Rockville. MD, and were used between the 25th and 27th passages. They were grown in Basal Eagle’s Medium (Associated Biomedical Systems, Buffalo, NY) supplemented with 10% fetal calf serum and antibiotics, as previously described (16). The cells were used 7 days after plating when they were confluent. Preparation of chromatin. Chromatin was prepared by the method of Marushige and Bonner (171, with the modifications introduced by Paul and Gilmour (18). The cell pellets were washed two times with 10 vol of 1% Triton X-100, 20 mM EDTA, and 80 rnM NaCl, pH 7.4. Clean nuclei were obtained by
679
CD OF CHROMATIN centrifugation through 0.25 M sucrose. The nuclear pe:!ct was washed twice with 10 vol of 0.15 M NaCl and 0.01 M Tris-HCl, pH 8, and allowed to swe!l in cold distilled water for 60 min in an ice bath. The nuclei were then lysed by gentle homogenization with a few strokes of a Teflon pestle and chromatin was purified by centrifugation through 1.7 M sucrose in an SW 39L Spinco rotor for 80 min at 37,000 rpm. The unsheared chromatin pellet was resuspended in water and dialyzed overnight against 0.01 M Tris, pH 8.0. This material was used after appropriate dilution with 0.01 M Tris, for the determination of circular dichroism spectra of chromatin. Dissociation of chromatin and separation of histones from nonhistone chromosomal proteins. Purified chromatin was dissociated in 6 M urea, 0.35 M guanidine-HCl, O.l”r P-mercaptoethanol, and 0.1 M sodium phosphate, pH 7, as described by Levy et al. (19). DNA was sedimented by centrifugation at 105,OObg at 4°C for 40 h using a 50 Ti rotor in a Spinco Model L 65B ultracentrifuge. Some of the proteins (, P. F., AN” SELIGY. v. L. (197%) Eur. J. Eiochem. 29, 426-432.
685
35. FASMAN, G. D.. SCHAFFHACWN, B.. GoLixhluw, L., AND ADIXK. A. (1970) Biochemistn 9, 2814-2821. 36. LIN, J. C., NI~OI,I~I, C.. AND BASEKW R. (1974) Biochemistn 13, 4127-4133. 37. BASERCA. R., BOMHIK, B., AND NICOLISI, C. (1975) in The Structure and Function of Chromatin. A Ciba Foundation Symposium, pp. 269-289. 38. NI~OI.IXI, C.. AND BASEK(:-\, R.. Chrm. Hid. Interactions, in press (1975). 39. GREEWIEIJ). N., 4x1) FAS~~IA?~, G. D. ( 19691 Biochemist? 8, 4108~4116. 40. EI.GIN, S. C. R.. ASI) BONSE‘K, J. (1972) Riochemistryv 11,77”~781. 41. WV, F. C.. EI.~;IX, S. C. R.. ANDHOOD, L. E. (1973) Biochemistn 12, 2792-X97. 42. E~.c;rs. S. C. R., ANI) Hoon, L. E. (19731 Biochemistc, 12, 4984-499 1. 43. STEIS, G.. CHAITIHI HI, S., AND BASER
