ANALYTICAL

BIOCHJ?.MISTRY

200,

339-341

(19%)

Western Blotting and lmmunochemical Detection of Histones Electrophoretically Resolved on Acid-UreaTriton- and Sodium Dodecyl Sulfate-Polyacrylamide Gels Genevieve P. Delcuve and James R. Davie’ Department of Biochemistry and Molecular Biology, Faculty of Medicine, University Winnipeg, Manitoba, Canada R3E OW3

Received

August

15, 1991

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mu acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mu Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mu 3-(cyclohexylamino)1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated. o isez Academic press, h.

Chromatin typically contains five classes of histones: H2A, H2B, H3, H4, and Hl. Four of these histones (H2A, H2B, H3, and H4) are involved in the formation of the nucleosome, the basic repeating structural unit of chromatin. Histone Hl is associated with the linker DNA that joins the nucleosomes, and this histoneparticipates in the condensation of the chromatin fiber (1,2). Each histone class is susceptible to a variety of postsynthetic modifications (e.g., acetylation, methylation, ubiquitination, and/or phosphorylation) and with the exception of histone H4, the histone classes have several variants that differ in primary amino acid sequence (l-6). Several recent reports demonstrate the importance of histone modifications and histone variants in modulating the structure and function of chromatin. For example, deletion of the Drosophila gene encoding for the histone variant HBAvD, which is similar to mammalian and avian histone variant HBA.Z, is lethal 1 To whom

of Manitoba,

correspondence

should

0003-2697192 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

be addressed.

(7). Genetic analysis of histone H4 acetylation has also shown that acetylation of histone H4 has an essential role in chromosome dynamics (8). Recent reports have demonstrated that acetylated and ubiquitinated histones, particularly ubiquitinated histone H2B, are complexed to transcriptionally active DNA (4,9). The electrophoretic resolution of histones on acetic acid-urea-Triton (AUT)2-polyacrylamide gels is the method of choice for separating histone variants and modified histone species (2-6). In previous studies, we have used an anti-ubiquitin antibody in Western blotting experiments to detect ubiquitinated histones that were resolved by two-dimensional (AUT into SDS) gel electrophoresis (4,6,10-12). The detection of ubiquitinated histones and determination of their relative abundance in several histone samples would be greatly facilitated by the immunochemical detection of ubiquitinated histones transferred directly from AUT gels to nitrocellulose filters. Here, we present a method that allows the efficient transfer of histones from AUT-polyacrylamide gels to nitrocellulose filters. The immunochemical detection of ubiquitinated histone species on these Western blots is demonstrated. MATERIALS

AND

METHODS

Preparation of h&ones. Histones were isolated from the nuclei of chicken erythrocytes and T-47D-5 human breast cancer cells as described by Delcuve and Davie (11) and Davie and Murphy (4). Chicken erythrocyte histones from transcriptionally active gene-enriched, 0.15 M NaCl-soluble oligonucleosomes (chromatin fraction F,,) were prepared as previously described (11). ’ Abbreviations sodium dodecyl fonic acid.

used: AUT, acetic acid/urea/Triton X-100; sulfate; Caps, 3-(cyclohexylamino)-l-propanesul-

SDS,

339 Inc. reserved.

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DELCUVE

AND DAVIE

Polyacrylumide gel electrophoresis. SDS- or AUT [acetic acid16.7 M ureaY0.375% (w/v) Triton X-lOO]15% polyacrylamide minislab gels were prepared as described (12,15). Two-dimensional gel electrophoresis (AUT into SDS) was performed as described by Davie (15). Transfer of histones from A UT or SDS gels and immunochemical detection. AUT minislab gels were washed

for 2 X 30 min in 100 ml of 50 mM acetic acid, 0.5% SDS, and for 30 min in 100 ml of modified buffer 0 of O’Farrell (16) (5% /I-mercaptoethanol, 2.3% SDS, and pH 6.8). SDS minislab gels were 62.5 mM Tris-HCl, washed for 30 min in the modified buffer 0. Transfers to nitrocellulose membranes (0.45~Frn pore size; Schleicher & Schuell, BA85) positioned at the anode side of the gel were performed with a Bio-Rad TransBlot apparatus. Following the addition of transfer buffer [25 mM Caps, pH 10, and 20% (v/v) methanol], protein transfer was carried out at 70 V for 2 h or at 30 V overnight with cooling to 4°C. Following transfer, the gel was stained with 0.25% Coomassie blue G-250 in 45% methanol and 9% acetic acid. The proteins that were transferred onto the nitrocellulose filter were visualized by staining the nitrocellulose filter with india ink (17). The procedure described by Bio-Rad (18) was used for immunochemical detection of ubiquitinated proteins. The first antibody solution contained affinitypurified anti-ubiquitin IgG (12) and the second antibody solution contained goat anti-rabbit IgG conjugated to alkaline phosphatase (Stratagene). To detect the alkaline phosphatase antibodies, two 9 X 6-cm filters were placed in a 20-ml solution containing 0.3 mg/ml nitro blue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3pH 9.5,lOO InM indolyl phosphate in 100 InM Tris-HCl, NaCl, 5 mM MgCl,. The color development time was approximately 5 to 10 min. The reaction was terminated pH 2.9,l by the washing the blot with 20 InM Tris-HCl, mM EDTA. RESULTS

AND

DISCUSSION

Waterborg and Harrington (19) have described a method for transferring histones from AUT gels to nitrocellulose. This procedure includes washing the AUT gel with 50 mM acetic acid, 0.5% SDS (2 X 30 min) to displace the Triton X-100 followed by two 30-min washes in transfer buffer [25 mM Tris, 192 mM glycine, 20% (v/v) methanol, and 0.1% SDS]. We used this protocol to transfer histones from AUT minislab gels to nitrocellulose filters. Inspection of the india ink-stained nitrocellulose filters and the Coomassie blue-stained AUT gels following transfer demonstrated that transfer of the histones from the gels was incomplete (not shown). In an attempt to increase the efficiency of transfer of histones from AUT gels to nitrocellulose, we modified this procedure as follows. First, the minislab

a

b

c

abc

a bc

Electrophoretic transfer of histones from AUT minislab gels. (A) Histones (9, 18, and 36 pg in lanes a, b, and c, respectively) isolated from T-47D-5 human breast cancer cells were electrophoretitally resolved on AUT minislab gels. The gel was stained with Coomassie blue. (B) The Coomassie blue-stained AUT minislab gel pattern of histones remaining after transfer (30 V overnight) to nitrocellulose. (C) The india ink-stained nitrocellulose pattern of histones transferred from the AUT minislab gel in B. A,, A,, and A, correspond to the un-, mono-, and diacetylated species of histone H4, respectively. The ubiquitin adduct of histone H2A is denoted uH2A. FIG.

1.

AUT gel was washed with acetic acid-SDS followed by a pH 6.8,2.3% SDS, wash in buffer 0 (62.5 mM Tris-HCl, 5% /3-mercaptoethanol). Second, the transfer buffer was changed to an alkaline transfer buffer [25 mM Caps, pH 10, 20% (v/v) methanol]. Since Szewczyk and Kozloff (20) reported that alkaline transfer buffers increased the efficiency of transfer of strongly basic proteins from SDS gels to nitrocellulose, we reasoned that this transfer buffer would improve the transfer of histones from AUT gels that had been treated with SDS. Figure 1 shows the Coomassie blue-stained AUT gel pattern of the electrophoretically resolved histones, the Coomassie blue-stained gel after transfer, and the india inkstained nitrocellulose filter. Most of the histones were efficiently transferred. The efficiency of elution was poorest for histone Hl. Figure 1C demonstrates that the histone variants of histone H2A (H2A, H2A.Z) and of histone H3 (H3.1, H3.2, H3.3) and the modified histone species (e.g., ubiquitinated histone H2A, acetylated histone H4) were transferred. Densitometric tracings of the gel patterns before and after transfer demonstrated that greater than 90% of the histones H2A, H2B, H3, and H4 and approximately 80% of histone Hl were eluted from the AUT gel. Our principal objective was to transfer histones from AUT gels to nitrocellulose such that the relative levels of ubiquitinated histones in several histone samples could be ascertained by immunochemical detection with an anti-ubiquitin antibody. Two sources of histones, which differed in their levels of ubiquitinated histones, were electrophoretically separated on an AUT minislab gel. The histones were transferred to nitrocellulose, and the ubiquitinated histones were immunochemically de-

IMMUNOCHEMICAL

A

DETECTION

OF HISTONES

B u2H2A uH2A uH2A.

Z

uH2B

a

b cd

abed

FIG. 2. Immunochemical detection of ubiquitinated histones transferred from AUT minislab gels to nitrocellulose. (A) Histones (15 pg, lane a) isolated from 150 mM NaCl-soluble oligonucleosomes of chicken erythrocytes and histones (9, 18, and 36 pg in lanes b, c, and d, respectively) extracted from T-47D-5 human breast cancer cell nuclei were electrophoretically resolved on an AUT-polyacrylamide minislab gel. The gel was stained with Coomassie blue. (B) The nitrocellulose filter containing the histones transferred (30 V overnight) from the AUT gel was immunochemically stained for ubiquitin with an anti-ubiquitin IgG and alkaline phosphatase-conjugated goat antirabbit antibody as described under Materials and Methods. The ubiquitin adducts of histones H2A, HSA.Z, and H2B are denoted uH2A, uH2A.Z, and uH2B, respectively. The diubiquitinated species of H2A, which has been characterized by Nickel and Davie (21), is labeled uxH2A.

tected with anti-ubiquitin IgG and goat anti-rabbit antibody conjugated to alkaline phosphatase. Figure 2 demonstrates that the ubiquitinated species of histones H2A, HBA.Z, and H2B and the multiubiquitinated form of histone H2A could be visualized. Furthermore, the different amounts of the ubiquitinated histones in the samples were readily seen. In agreement with our previous observations, the chicken erythroid histones isolated from transcriptionally active gene-enriched oligonucleosomes had relatively high levels of ubiquitinated histone H2B [Fig. 2, lane a (ll)], while the amount of ubiquitinated histone H2B from human breast cancer cells was considerably lower [Fig. 2, lanes b to d (4)]. The immunochemically stained gel was photographed. Densitometric tracings of the negative revealed that the intensities of the immunochemical-stained bands corresponding to the ubiquitinated histone species of histones H2A or H2B were proportional to the amount of T-47D-5 human breast cancer histones loaded on to the AUT gel shown in Fig. 2 (lanes b to d). These three lanes contained 9, 18, and 36 pg of protein and the corresponding values from the scan (in arbitrary units) were 2.5, 5.2, and 9.6 for ubiquitinated histone H2A and 0.7, 1.7, and 3.9 for ubiquitinated histone H2B, respectively. We also used this alkaline transfer buffer to transfer histones from SDS to nitrocellulose. In contrast to the observations of Szewczyk and Kozloff (20), we found

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that washing the SDS minislab gel in 62.5 InM TrisHCl, pH 6.8, 2.3% SDS for 30 min improved the efficiency of elution of the histones from the SDS gel (not shown). In summary, we have demonstrated that the approach described here efficiently transfers histones from AUT (or SDS) minislab gels to nitrocelhlose filters. Application of this technique to detect by an immunochemical staining procedure a modified histone has been shown. This procedure should aid in the characterization and chromosomal localization of modified histones and histone variants. ACKNOWLEDGMENTS This project was supported by a grant from the National Cancer Institute of Canada. We thank Darcy Salo for technical assistance and Dr. Leigh Murphy for the T-47D-5 human breast cancer cells. J. R. Davie is a Scientist of the Medical Research Council of Canada.

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