Cross-linking of histone proteins to DNA by UV illumination of chromatin stained with Hoechst 33342[para]

Photochemistry and Photobiology, Jun 2003 by Davis, Sara K, Bardeen, Christopher J

ABSTRACT

The photochemical effects of near-UV light on chromatin labeled with the vital DNA dye Hoechst 33342 (H33342) are studied. Several types of experiments demonstrate that illumination at both 365 and 410 nm results in significant cross-linking of proteins with the DNA. Fluorescence microscopy of dye-stained Xenopus XTC-2 nuclei shows that UV illumination has effects similar to chemical fixation by formaldehyde. At 365 nm a dose of ~70 J/cm^sup 2^ results in 50% of the DNA being cross-linked, as measured by chloroform-sodium dodecyl sulfate extraction. At 410 nm the efficiency of cross-linking was smaller by a factor of 3. Gel electrophoresis of the cross-linked proteins shows them to be predominantly core histones. The implications of these results for experiments on live cells stained with H33342, for example, fluorescence microscopy of nuclear dynamics or cell sorting, are discussed.

INTRODUCTION

Interest in DNA-containing chromatin has increased rapidly, because it now appears that chromatin structure and dynamics play an important role in biological processes like gene transcription (1-3). The ability to image DNA using fluorescence microscopy is critical for studying the structure and dynamics of chromatin in the nuclei of live cells. This ability is made possible by the different fluorescent dye molecules that can bind DNA selectively and with large affinity (4). Although there are many such molecules available for labeling DNA in vitro, the inability of most of these molecules to pass through live-cell membranes has limited the number of live-cell DNA stains to a relatively small subset of molecules. Of these, one of the most widely used is 2,5'-bi-1H-benzimidazole,2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-tri-chloride, commonly known as Hoechst 33342 (H33342), whose structure is shown in the inset of Fig. 1. H33342 absorbs in the near-UV region and has a broad emission centered at around 500 nm (5). It binds to the minor groove of DNA, with larger affinity for the AT base pairs, and this binding enhances its fluorescence quantum yield by 10-40 times (5-7). Although it is known that H33342 and related dyes can interfere with the action of nuclear proteins on DNA, e.g. topoisomerase (8), it does not prevent cell proliferation or growth (9). Because of this, it is commonly used for cell-sorting applications where the stained cells are later used for other biological purposes, for example, sex selection (10). But although H33342 in its electronic ground state is relatively benign, it is an open question whether the excited-state photochemistry of H33342 can produce photochemical changes in biological systems. A previous study found no evidence of direct DNA damage when cells stained with H33342 were exposed to low levels of light during a cell-sorting experiment (11). On the other hand, Hoechst dyes are known to produce reactive species like free radicals on photoexcitation (12), and H33342 has been shown to cause cytotoxicity at high concentrations and extended exposures (13).

In this article, we investigate the photochemistry of H33342 in both isolated cell nuclei and dilute chromatin in buffered solution. On excitation with near-UV light, H33342 can initiate the formation of cross-links between nuclear proteins and DNA. This cross-linking can be observed in a number of ways, most dramatically through the apparent fixation of nuclei stained with H33342 and exposed to 365 nm light. Significant cross-linking occurs at UV exposures of ~70 J/cm^sup 2^, and gel electrophoresis reveals that the cross-linked proteins are predominantly core histones. The implications of this UV-induced protein-DNA cross-linking for live-cell studies using fluorescence microscopy or flow cytometry will be discussed.

MATERIALS AND METHODS

Nuclear isolation. Xenopus XTC-2 cells were grown in phenol red-free 70% Dulbecco's Modified Eagle's Medium with F-12 nutrient mixture (DME-F12) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY) at room temperature. For nuclear isolation, cells were resuspended in cell media and then rinsed with ice-cold 70% phosphate-buffered saline (BioWhittaker, Walkersville, MD). Subsequent steps were done on ice. After centrifugation, the cell pellet was loosened, and mammalian cell lysis reagent (Pierce, Rockford, IL) was added. The sample was agitated for 30 s, and then the cell membranes were disrupted by resuspending the solution twice with a glass pipette. An excess of cell media was added, and then the nuclei were centrifuged once and resuspended in regular cell media. To examine salt-dependent morphology changes, nuclei were treated (fixed or UV exposed) in regular cell media and then resuspended in an excess of media with 2 M NaCl. After incubating for about 30 min, this solution was spread on a coverslip and imaged using a fluorescence microscope with a charge-coupled device camera.

Chromatin isolation. After nuclear isolation, chromatin was isolated using a slightly modified method of Bhorjee and Pederson (14). Briefly, nuclei were rinsed three times, resuspended in 10 mM NaCl, 1.5 mM MgCl^sub 2^ and 10 mM Tris-HCl (pH 7.0) and then disrupted by sonication with three 10 s pulses at 20 kHz and 20 W (Sonics and Materials VCX 600, Danbury, CT). The resulting solution was layered over 30% sucrose in NaCl-Tris buffer (10 mM NaCl and 2.5 mM Tris-HCl, pH 7.2) and centrifuged at 4500 g for 15 min (Sorvall RC-5 centrifuge, SS-34 rotor, Newtown, CT). The top layer was removed, and aliquots of 5.5 mL were layered over 15.5 mL 60% sucrose in NaCl-Tris buffer with 24 mM ethylenediaminetetraacetic acid in nitrocellulose tubes. The top two-thirds of the tubes were gently mixed, and then the samples were centrifuged for 90 min at 131 000 g (Beckman L7-65 centrifuge, 60Ti rotor, Fullerton, CA). The pellet was resuspended in and then dialyzed overnight against NaCl-Tris buffer. The absorbance ratio at 260 and 240 nm of the resulting chromatin solution was 1.3.