Low Angle X-ray HeLa Metaphase Phosphorylation Diffraction Studies of Chromosomes: Effects of Histone and Chromosome Isolation Procedure

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of pchloromercuriphenylsulfonate (CIHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of CIHgPhSO3 can also profoundly affect the pattern. The goal of structural studies of chromatin is first to understand the static structure of the chromatin fiber, and then to understand how that fiber structure may change during gene activation, organismal development, chromosome replication and repair, and throughout the cell cycle. In this study we asked the question: Can changes in bulk chromatin structure during the mitotic cell cycle be detected by low angle x-ray diffraction? More specifically, can we detect differences between interphase nuclei and metaphase chromosomes? Comparison of interphase and metaphase chromatin structures is the obvious first experiment for two reasons. First, metaphase is one of the few points in the cell cycle at which cells can be efficiently arrested or synchronized; second, and more importantly, mitotic metaphase is the stage of the cell cycle at which detectable differences would be most likely to occur, since at this stage the cells and nuclei undergo several dramatic structural and biochemical transformations. These include breakdown of the nuclear envelope, shut-off of transcription, condensation of chromosomes into their familiar 1132 compact form, and extensive phosphorylation of histones HI and H3 and other chromatin proteins. Our current understanding of the structure of mctaphase chromosomes is summarized by the radial loop model (24), which states that the 25-30-nm thick chromatin fiber is folded into loops and that the bases of these loops are anchored at the axis of the chromatid by nonhistone proteins (reviewed in reference 27). The underlying structural organization of interphase chromosomes is probably very similar, since interphase chromatin is organized into supcrcoiled domains about the same size as the loops in metaphasc chromosomes (4, 9, 17), and since there is evidence for similar mctalloprotein interactions in the higher-order structure of both interphase and metaphasc chromosomes (21-23). A number of previous studies have compared interphasc and mctaphasc chromatin fibers by different techniques. Morphological studies in the electron microscope, using surface-spreading or thin-sectioning, show both interphase and metaphasc chromosomes to consist mainly of 20--30-nm fibers (I, 2, 12, 24). Careful measurements by Golomb and Bahr (3, 15) showed THE JOURNAL Of CELL BIOLOGY VOLUME 96 APRIL 1983 1132-1137 © The Rockefeller University Press • 002%9525/83/04/1132/06 $1.00 on O cber 0, 2017 jcb.rress.org D ow nladed fom b o t h in te rphase and metaphase fibers to be 20 n m in d iameter in surface spread, cr i t ical-point-dried preparat ions, a l though metaphase fibers appea r to be 15-22% thicker after colchicine arrest (3, 14). Repor ts of 50-nm fibers in metaphase appear to be due to coiling of the 20--30-nm fiber on itself in the presence o f hexylene glycol (2, 11, 24). Nuclease digest ion studies o f ch romat in have shown that nucleosomes exist in metaphase chromosomes and that the ch roma t in repeat remains the same between in terphase and metaphase (8, 16, 36, 37). In the electron microscope, nucleosomes can be observed to be packed into 20--30-nm fibers (29-31) bo th in in terphase and metaphase , bu t dehydra ted electron microscope specimens do not show enough regulari ty to allow compar i son of the in ternal s tructure of the fibers. In one study, in te rphase ch romat in and metaphase chromosomes were compared by x-ray diffract ion and bo th were found to give diffract ion peaks at 6.0, 3.8, 2.7, and 2.1 n m (25). Thus, despite the d ramat ic changes in chromosomes between in terphase and metaphase , no differences in ch romat in f iber s t ructure have yet been observed, e i ther by x-ray diffraction, e lectron microscopy, or nuclease digestion. A serious difficulty wi th previous studies o f metaphase chroma t in was pointed out by D ' A n n a et al. (10), who found that his tones H 1 and H3 become dephosphory la ted dur ing isolation of me taphase ch romosomes by convent iona l procedures. One might expect that , i f s t ructural differences exist be tween interphase and metaphase chromat in , they might result f rom the phosphory la t ion of H I and H3; but none of the early studies took account of historic phosphoryla t ion , and the chromosomes studied were very likely dephosphoryla ted . To make a more mean ingfu l compar ison o f in terphase and metaphase chromat in , we looked for methods to prevent histone dephosphoryla t ion . We found tha t certain sulfhydryl reagents, such as p -ch loromercur iphenyl sulfonate (C1HgPhSO3), effectively inhib i t the phosphohis tone phospha tase present in the chromosomes, and they also inact ivate the endogenous proteases in the ch romosome prepara t ions (26). In the present study, we improved on previous x-ray studies in three respects. First, we studied metaphase chromosomes conta in ing phosphory la ted histones. Second, we directly compared in terphase nuclei with metaphase chromosomes under the same ionic condit ions. Third, we used an improved x-ray camera wi th which all of the reflections characterist ic of chrom a t i n in vivo (at 30-40, I 1.0, 6.0, 3.8, 2.7, and 2.2 rim) can be observed (20). In part icular , we were interes ted in observing the 30 -40 -nm a n d 11.0-nm reflections, since they are p robab ly the most sensitive indicators o f the side-by-side packing and in terna l s t ructure of the fibers, and had not yet been observed f rom metaphase chromosomes. W e show, first, tha t in terphase and metaphase fibers differ only in the i r packing, not in thei r in ternal structure, and that phosphory la t ion does not detectably affect the f iber structure. Second, we show tha t some of the isolation methods and some o f the sulfhydryl reagents used can alter the x-ray pat terns and therefore should be used wi th caut ion in s tructural studies of me taphase ch romat in fibers. MATERIALS A N D M E T H O D S Chemicals and Buffer Solutions: The basic isolation buffer (IB) for HeLa aqueous chromosomes, chromosome clusters, and interphase nuclei consisted of l0 mM HEPES, pH 7.3, l0 mM NaC1, and 5 mM MgC12. Colcemid, thymidine, and monosodinm C1HgPhSOa were obtained from Sigma Chemical Co. (St. Louis, MO). Methylmercury (II) hydroxide (l M aqueous solution) was obtained from Ventron (Danvers, MA), and 5,5'-dithiobis(2-introbenzoic acid) (Nbs2) was obtained from British Drug Houses, Ltd. (Poole, Dorset, England). Tissue culture media and components were obtained from Flow Laboratories, Inc. (McLean, VA), Isolation of Metaphase Chromosomes and Interphase Nuclei: Reka $3 ceils were grown and arrested in metaphase as described in the previous paper, which accompanies this (20). Aqueous chromosomes were isolated either as individual chromosomes by the method of Marsden and Laemmli (24) (except that modified isolation buffers were used) or as chromosome clusters by the method of Paulson (26) from cultures which had been arrested to 90-95% in metaphase. In both methods, cells were lysed in IB plus 0.5 M sucrose, 0.5 mM CaC12, and 0.1% Nonidet P-40 (NP-40), and the chromosomes were subsequently resuspended and washed in IB plus 0.5 mM CaCIs and 0.1% NP-40. No differences in the x-ray patterns were observed between the two methods, nor did the additional use of 0.05% sodium deoxycholate in the isolation buffers make any difference in the diffraction patterns. Hexylene glycol chromosomes were isolated by a modification of the method of Wray and Stubblefield (38) as described by Paulson (26). The isolation buffer consisted of 0.1 mM PIPES, pH 6.7, 1 M hexylene glycol (2-methylpentan-2,4diol), and 0.5 mM CaC12. Polyamine chromosomes were isolated by a modification of the method of Lewis and Laemmli (23) as described by Paulson (28). HeLa interphase nuclei were isolated as described in the accompanying paper (20). For x-ray experiments involving sulfhydryl reagents to prevent dephosphorylation, chromosomes were isolated as chromosome clusters as previously described (26) with 5 mM C1HgPhSOa, 1 mM methyl mercury, or 5 mM Nbs~ present in all solutions. In the experiments using 0.015 mM CIHgPhSO3, the volumes of solutions used were increased to ensure that the phosphatase in the chromosomes was completely inactivated. The cells were suspended to no >4 x l0 s cells/ml in the lysis solution (with 0.015 mM CIHgPhSOa) and after pelleting, the chromosome clusters were washed twice in 40 ml of IB containing 0.5 mM CaC12, 0.1% NP-40, and 0.015 mM C1HgPhSO3. To separate the possible structural effects of phosphorylation from the effects of the suLfhydryl reagents themselves, chromosomes were isolated as chromosome clusters, allowed to become dephosphorylated by incubation for 3 h at 4°C, and finally treated with CIHgPhSOa by pelleting and resuspending in IB + 0.5 mM CaCI2 + 0.1% NP-40 + 5 mM C1HgPhSO3. In another experiment, interphase nuclei were isolated and then finally resuspended and washed in IB + 0.5 mM CaC12 + 0.1% NP-40 + 5 mM C1HgPhSO3. Handling of X-ray Specimens and Analysis of Diffraction Pat terns: Our previous paper (20) describes the handling of the x-ray specimens and the analysis and presentation of the diffraction data. All figures are presented as log s21 vs. s, with arbitrary vertical positioning of the curves to prevent overlap. All of the experiments reported here were done with fresh material. It should be noted that the x-ray diffraction pattern of chromosomes or nuclei changes with time if the material is stored in the absence of inhibitors for several days after isolation, even at 4°C. These changes are presumably due to proteolytic or nucleolytic degradation, since C1HgPhSOa, Nbs2, and methyl mercury, which are known to prevent such degradation (26), completely prevent the time-dependent changes in the x-ray diffraction patterns. Specimens were monitored for proteolysis before and after the x-ray exposure by running 15% SDS polyacrylamide gels according to LaemmLi and Favre (18), and in all cases there was little or no proteolysis. Specimens were also monitored for phosphorylation or dephosphorylation of histone H l by extracting a sample of chromosomes (or the contents of a specimen capillary after the x-ray exposure) with 0.2 M H2SO4 and analyzing the acid-extractable proteins on acid/urea gels (26).