Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1304-1308, April 1975
Histone-Histone Propinquity by Aldehyde Fixation of Chromatin (chromatin structure/chromosome/formaldehyde/glutaraldehyde/DNA)
ROGER CHALKLEY AND CONNIE HUNTER Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242
Communicated by Hewson Swift, December 16, 1974 Histones have been fixed within the chroABSTRACT matin complex using either formaldehyde or glutaraldehyde. Evidence is presented which argues that in short time periods formaldehyde fixation leads to the formation of reversible covalent bonds between histone and DNA. On the other hand, fixation of chromatin with glutaraldehyde leads initially to the formation of polymers of Fl histone, and at a later stage to multiple small oligomers of the remaining histones. These oligomers then increase in size until they become too large to detect by polyacrylamide gel electrophoresis. Exclusive formation of histone dimers or tetramers was not observed. The simplest model for histone distribution on DNA which encompasses these observations is one in which histones are organized as a fairly extensive linear overlapping array.
Considerable interest is now being focused on the structure of chromatin. A critical role in the development of our understanding of chromatin structure will most likely come from increased knowledge of the way in which histone molecules are organized with respect to one another in their interaction with DNA. The development of bifunctional crosslinking reagents (1) and their application to ribosomal material (2, 3) has already been described. Histones can be fixed within the chromatin structure by reaction with formaldehyde (4-6, 13), and it appears likely that at least short-term exposure to this agent gives a substantial degree of covalent histone-DNA interactions (6). Olins and Wright (7) have utilized glutaraldehyde to explore histone-histone proximity in avian erythrocyte nuclei, where they analyzed in detail the fixation of the lysine-rich histones (F1 and F2c). They showed that it is possible to demonstrate the formation and isolation of polymers rich in the lysine-rich histones. We have extended their studies to isolated chromatin and have also observed the formation of polymers of the other histone fractions. The differences in nature of histone fixation by formaldehyde and glutaraldehyde have been analyzed.
Fixation by Aldehydes. The nucleohistone was adjusted to
A260 about 10, and to the required ionic strength with a stock solution of triethanolamine- HCl, pH 7.0. The fixatives used were 2% formaldehyde or 0.6% glutaraldehyde. Stock solutions were prepared by adding 2.0 ml of 38% formaldehyde or 1.5 ml of 50% glutaraldehyde (purchased from Fischer Scientific) to 5.5 ml of H20. The pH was adjusted to 7.0 + 0.2 with 0.1 MI NaOH. Immediately before use, the glutaraldehyde was diluted 3-fold with water. The fixatives were then added to the nueleohistone solution (0.2 ml of diluted fixative per 1.0 ml of nucleohistone) at 4°. The fixation reactions were terminated by adding a 1/10 volume of 2 Ml H2SO4. After centrifugation (14,000 rpm/75 min) the supernatant (extracted histones and soluble polymers) was dialyzed against 2 liters of 0.2 M H2SO4 (4-6 hr minimum) and, finally, the histones were precipitated by dialysis against ethanol (6-12 hr). The histones were collected by centrifugation and were dissolved in either 0.9 M acetic acid, 20% sucrose, 0.5 M 2-mereaptoethanol for acid-urea electrophoresis or in 0.1% sodium dodecyl sulfate, 0.01 M glycine, 4 M urea, pH 10.5, for high pH sodium dodecyl sulfate electrophoresis. Fractionation of Histones and Histone Polymers. Histones were fractionated into three groups, F1, F2b, and (F2A + F3), by the modified procedures of Johns (8). Electrophoresis of Histones in low pH-urea (9) or in high pHsodium dodecyl sulfate systems (10) followed described methods. After the gels were destained, they scanned with a Beckman Acta III microdensitometer. RESULTS
Fixation of Histones by Formaldehyde and Glutaraldehyde. A comparison of the nature and rates of fixation of nucleoprotein by formaldehyde and glutaraldehyde is shown in Fig. 1. At the ionic strength used (5 X 10-4), formaldehyde fixation of histones to the nucleoprotein proceeds quite rapidly so that about 80% of the original histone content is not extractable in acid after 30 min. As shown previously, histone becomes wholly bound after an additional 90 min (6). All histone fractions are bound at essentially the same rates. In contrast, the rate of fixation of different histone fractions varies during glutaraldehyde treatment. As shown in Fig. 1, the lysine-rich (Fl) histone is fixed (and therefore not extracted into acid) most rapidly, in accord with similar observations made by Olins and Wright, who studied fixation in isolated nuclei (7). In fact, most of the F1 histone has been fixed before significant inroads have been made into the other
MATERIALS AND METHODS
Isolation of Nucleohistone. Calf thymus was collected from the slaughterhouse, carried to the laboratory ice, and rapidly frozen. Samples of tissue (2-3 g) were homogenized, and nucleohistone was isolated by standard procedures (9). Nucleohistone is operationally defined as chromatin that has been vigorously sheared and centrifuged. Control fixation studies with unsheared chromatin revealed an identical pattern of fixation. The nucleohistone was stored at 40 in water before use. In general, most experiments were performed within an hour of preparation; however, storage in H20 totally prevents proteolysis, and identical results are obtained material stored for as long as 48 hr. on
on
1304
Proc. Nat. Acad. Sci. USA 72 (1975)
Histone
Propinquity
1305
TABLE 1. Reversibility offixation by formaldehyde or glutaraldehyde % Reversal
Substrate Nucleohistone Free histone
Formaldehyde 90-100
Histone-Histone Propinquity by Aldehyde Fixation of Chromatin (chromatin structure/chromosome/formaldehyde/glutaraldehyde/DNA)
ROGER CHALKLEY AND CONNIE HUNTER Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242
Communicated by Hewson Swift, December 16, 1974 Histones have been fixed within the chroABSTRACT matin complex using either formaldehyde or glutaraldehyde. Evidence is presented which argues that in short time periods formaldehyde fixation leads to the formation of reversible covalent bonds between histone and DNA. On the other hand, fixation of chromatin with glutaraldehyde leads initially to the formation of polymers of Fl histone, and at a later stage to multiple small oligomers of the remaining histones. These oligomers then increase in size until they become too large to detect by polyacrylamide gel electrophoresis. Exclusive formation of histone dimers or tetramers was not observed. The simplest model for histone distribution on DNA which encompasses these observations is one in which histones are organized as a fairly extensive linear overlapping array.
Considerable interest is now being focused on the structure of chromatin. A critical role in the development of our understanding of chromatin structure will most likely come from increased knowledge of the way in which histone molecules are organized with respect to one another in their interaction with DNA. The development of bifunctional crosslinking reagents (1) and their application to ribosomal material (2, 3) has already been described. Histones can be fixed within the chromatin structure by reaction with formaldehyde (4-6, 13), and it appears likely that at least short-term exposure to this agent gives a substantial degree of covalent histone-DNA interactions (6). Olins and Wright (7) have utilized glutaraldehyde to explore histone-histone proximity in avian erythrocyte nuclei, where they analyzed in detail the fixation of the lysine-rich histones (F1 and F2c). They showed that it is possible to demonstrate the formation and isolation of polymers rich in the lysine-rich histones. We have extended their studies to isolated chromatin and have also observed the formation of polymers of the other histone fractions. The differences in nature of histone fixation by formaldehyde and glutaraldehyde have been analyzed.
Fixation by Aldehydes. The nucleohistone was adjusted to
A260 about 10, and to the required ionic strength with a stock solution of triethanolamine- HCl, pH 7.0. The fixatives used were 2% formaldehyde or 0.6% glutaraldehyde. Stock solutions were prepared by adding 2.0 ml of 38% formaldehyde or 1.5 ml of 50% glutaraldehyde (purchased from Fischer Scientific) to 5.5 ml of H20. The pH was adjusted to 7.0 + 0.2 with 0.1 MI NaOH. Immediately before use, the glutaraldehyde was diluted 3-fold with water. The fixatives were then added to the nueleohistone solution (0.2 ml of diluted fixative per 1.0 ml of nucleohistone) at 4°. The fixation reactions were terminated by adding a 1/10 volume of 2 Ml H2SO4. After centrifugation (14,000 rpm/75 min) the supernatant (extracted histones and soluble polymers) was dialyzed against 2 liters of 0.2 M H2SO4 (4-6 hr minimum) and, finally, the histones were precipitated by dialysis against ethanol (6-12 hr). The histones were collected by centrifugation and were dissolved in either 0.9 M acetic acid, 20% sucrose, 0.5 M 2-mereaptoethanol for acid-urea electrophoresis or in 0.1% sodium dodecyl sulfate, 0.01 M glycine, 4 M urea, pH 10.5, for high pH sodium dodecyl sulfate electrophoresis. Fractionation of Histones and Histone Polymers. Histones were fractionated into three groups, F1, F2b, and (F2A + F3), by the modified procedures of Johns (8). Electrophoresis of Histones in low pH-urea (9) or in high pHsodium dodecyl sulfate systems (10) followed described methods. After the gels were destained, they scanned with a Beckman Acta III microdensitometer. RESULTS
Fixation of Histones by Formaldehyde and Glutaraldehyde. A comparison of the nature and rates of fixation of nucleoprotein by formaldehyde and glutaraldehyde is shown in Fig. 1. At the ionic strength used (5 X 10-4), formaldehyde fixation of histones to the nucleoprotein proceeds quite rapidly so that about 80% of the original histone content is not extractable in acid after 30 min. As shown previously, histone becomes wholly bound after an additional 90 min (6). All histone fractions are bound at essentially the same rates. In contrast, the rate of fixation of different histone fractions varies during glutaraldehyde treatment. As shown in Fig. 1, the lysine-rich (Fl) histone is fixed (and therefore not extracted into acid) most rapidly, in accord with similar observations made by Olins and Wright, who studied fixation in isolated nuclei (7). In fact, most of the F1 histone has been fixed before significant inroads have been made into the other
MATERIALS AND METHODS
Isolation of Nucleohistone. Calf thymus was collected from the slaughterhouse, carried to the laboratory ice, and rapidly frozen. Samples of tissue (2-3 g) were homogenized, and nucleohistone was isolated by standard procedures (9). Nucleohistone is operationally defined as chromatin that has been vigorously sheared and centrifuged. Control fixation studies with unsheared chromatin revealed an identical pattern of fixation. The nucleohistone was stored at 40 in water before use. In general, most experiments were performed within an hour of preparation; however, storage in H20 totally prevents proteolysis, and identical results are obtained material stored for as long as 48 hr. on
on
1304
Proc. Nat. Acad. Sci. USA 72 (1975)
Histone
Propinquity
1305
TABLE 1. Reversibility offixation by formaldehyde or glutaraldehyde % Reversal
Substrate Nucleohistone Free histone
Formaldehyde 90-100
