A. DTNB Prevents Histone Dephosphorylation in Isolated Mitotic Chromosome Clusters
For an initial test of the ability of DTNB to block histone dephosphoryation, HeLa cells were grown in RPMI 1640 medium, synchronized in S-phase by thymidine treatment, released from the thymidine block and arrested in prometaphase with nocodazole. The cells were lysed and “chromosome clusters” (Paulson 1980, 1982) were prepared in the presence or absence of 5 mM DTNB. Histones were extracted with 0.2 M H2SO4 either immediately or after storage for 2 hr at room temperature. Finally, the samples were analyzed on acid-urea gels, which separate largely based on charge and can distinguish phosphorylated (mitotic) and unphosphorylated (interphase) histone H1.
The results in Fig. 1 show clearly that 5 mM DTNB prevents histone H1 dephosphorylation. Since the culture from which the chromosome clusters were prepared had a mitotic index of 96%, nearly all the H1 is in the slower migrating phosphorylated form (H1P) at T = 0, though a small amount of unphosphorylated H1 is also present. For chromosomes isolated in the presence of DTNB, the proportion of H1P is unchanged after storage for 2 hr at room temperature (Fig. 1, lane 4). In the absence of DTNB, however, H1 is completely dephosphorylated within 2 hr (Fig. 1, lane 3).
Since phosphorylated and unphosphorylated histone H3 are not well resolved in acid-urea gels (Fig. 1), a separate experiment was performed using different detection methods to test whether DTNB prevents H3 dephosphorylation and to see how rapidly H1 and H3 are dephosphorylated following cell lysis. Chromosome clusters were isolated either with or without DTNB and incubated at room temperature. Samples were taken at various times for analysis by SDS-PAGE.
Figure 2 shows the results of this experiment. In the upper panel, the gel was stained with ProQ Diamond, a fluorescent stain specific for phosphoproteins. The same gel was subsequently stained with Coomassie Blue to confirm equal loading of all samples (lower panel). The upper panel (right side) shows clearly that DTNB prevents dephosphorylation of H3 for at least 3 hr and confirms that DTNB prevents dephosphorylation of H1. The left side of the upper panel shows that in the absence of DTNB both H1 and H3 are significantly dephosphorylated within 30 min.
B. Effect of DTNB Concentration:
To determine how much DTNB is necessary to protect histones from dephosphorylation, metaphase-arrested cells were lysed at 2 × 106 cells/mL in isolation buffer containing various concentrations of DTNB, then incubated at room temperature for 2 hr before extracting histones. The acid-urea gel in Fig. 3(a) indicates that 0.05 mM DTNB is sufficient to prevent dephosphorylation.
This is approximately the concentration of sulfhydryl groups in the lysate. DTNB consists of two 5-thio-2-nitrobenzoic acid (TNB) molecules joined by a disulfide bond. When it reacts with a free sulfhydryl group, one TNB is released and its absorbance at 412 nm (A412) can be used to determine the concentration of sulfhydryl groups in the sample (Ellman 1959; Riddles et al. 1983).
The graph in Fig. 3(b) shows the A412 of the supernatants after pelleting the chromosomes for the samples in Fig. 3(a). Since DTNB itself absorbs at 412 nm, the absorbance value for each sample was corrected by subtracting the A412 of the lysis solution (without cells) that was used for that sample. For high concentrations of DTNB the A412 levels off at about 0.37. To relate A412 to sulfhydryl concentration, we prepared a standard curve using known concentrations of cysteine. Based on that standard curve, the absorbance of 0.37 corresponds to 0.035 mM sulfhydryl groups. This agrees reasonably well with the value (0.026 mM) calculated using a molar extinction coefficient ε = 14,150 M− 1cm− 1 as reported by Riddles et al. (1983).
Figure 3(c)-(e) show the effects of DTNB concentration for both H1 and H3. In this case, cells were lysed at 5 × 106 cells/mL and incubated 2 hr at 37°C. Histone phosphorylation was detected using ProQ Diamond stain (Fig. 3(c)) or by western blotting with anti-phosphohistone H1 (Fig. 3(d)) or anti-phosphohistone H3 (Fig. 3(e)) antibodies. The results with these detection methods differ somewhat, but they are in general agreement with Fig. 3(a). In this case, the concentration of sulfhydryl groups in the lysates was determined to be 0.090 mM which reflects that the cells were lysed at 5 × 106/mL as compared to 2 × 106 cells/mL for Fig. 3(a)-(b).
C. Reversibility of DTNB
Figure 4 shows that after chromosomes have been isolated with DTNB, phosphatase inhibition can be reversed by treatment with dithiothreitol (DTT) or 2-mercaptoethanol. Histones extracted immediately after isolation of chromosome clusters (T = 0) are shown in lane 1. Chromosome cluster samples in lanes 2 and 3 were prepared either without (lane 2) or with DTNB (lane 3) and incubated at 37°C for 80 min before extracting histones. For the samples in lanes 4–6, chromosome clusters were isolated with DTNB, then pelleted and washed twice with RB before resuspending in RB containing 20 mM dithiothreitol (lane 4), RB containing 50 mM β-mercaptoethanol (lane 5), or RB alone (lane 6). These samples were also incubated at 37°C for 80 min before extracting histones.
Histone H1 phosphorylation was analyzed using acid-urea gels (Fig. 4 (a)) and both H1 and H3 phosphorylation were observed by staining SDS gels with ProQ Diamond phosphoprotein stain (Fig. 4 (b)). H3 phosphorylation was also observed by western blotting (Fig. 4 (c)). Histones in DTNB-treated chromosomes remain phosphorylated even after washing with resuspension buffer (Fig. 4, lane 6) but they are dephosphorylated following treatment with a reducing agent, dithiothreitol (lane 4) or β-mercaptoethanol (lane 5).
D. Isolation of Individual Aqueous Chromosomes with DTNB
All the experiments described above employed chromosome clusters (Paulson 1982) to explore the effects of DTNB on histone phosphorylation during isolation of mitotic chromosomes. Although chromosome clusters are very useful for biochemical studies, isolation of individual metaphase chromosomes is sometimes essential, for example in studies of chromosome structure and morphology.
To test the compatibility of DTNB with isolation of individual metaphase chromosomes we used the aqueous method of Lewis and Laemmli (1982). Inclusion of 5 mM DTNB in the isolation buffers preserves histone phosphorylation (Fig. 5 (a), lane 2) and the isolated chromosomes display normal morphology (Fig. 5 (c)).
Interestingly, chromosomes isolated with DTNB (Fig. 5 (c)) appear to have less cellular debris associated with them than when DTNB is not used (Fig. 5 (b)). They also consistently show a “cleaner” protein pattern on gels, with fewer bands and less background in the gel pattern. This is evident with acid-extracted samples on acid-urea gels (compare lanes 1 and 2 or lanes 3 and 4 in Fig. 1) but also holds true when total chromosomal proteins are analyzed on SDS gels (Fig. 6). Calyculin A (Fig. 6, lane 4) does not have the same effect, so this is not simply a consequence of inhibiting protein phosphatases. We suggest that DTNB somehow helps prevent the artefactual association of cytoplasmic proteins and cytoskeletal debris (cf., Fig. 5 (b)) with chromosomes.