Global modulation of chromatin dynamics mediated by dephosphorylation of linker histone H1 is necessary for erythroid differentiation

Abstract

Differentiation of metazoan cells involves dramatic changes in gene expression patterns and proliferative capacity driven primarily by epigenetic mechanisms. Here we used in vivo photobleaching techniques and biochemical assays to investigate the contribution of alterations in chromatin dynamics to the differentiation of murine erythroleukemia (MEL) cells, a model system for erythroid development. As MEL cells differentiate the binding affinity of all linker histone variants increases, indicative of an overall decrease in chromatin flexibility. Changes in H1⁰ binding properties depend on phosphorylation at one or more of three cyclin-dependent kinase sites. The presence of constructs mimicking constitutively phosphorylated H1 results in a significant inhibition in the acquisition of commitment to terminal cell division and the expression of erythroid-specific properties. These data indicate that the progressive loss of cdk activity associated with MEL cell differentiation leads to the accumulation of dephosphorylated linker histones and restricted chromatin flexibility and that these are necessary events in the progression of erythroid differentiation. We present additional data indicating that the presence of phosphorylated H1 has a dominant effect on the binding behavior of other linker histones and propose a model for the role of linker histone phosphorylation in which these modifications act within the context of assembled chromatin.

The development of metazoan organisms requires the sequential maturation of pluripotent stem cells through lineage-committed, self-renewing cell lines to the differentiated, usually nondividing cells that comprise most of the adult somatic tissues. The differentiation process involves progressive dramatic changes in cell morphology, gene expression patterns, and proliferative capacity (1, 2). The observation that fully differentiated adult nuclei can be reprogrammed, albeit inefficiently, to direct the complete development of viable adult organisms indicates that the differentiation process is driven primarily by reversible epigenetic modifications of the genome (3).

Eukaryotic nuclear DNA exists in situ as the heterogeneous nucleoprotein complex termed chromatin. The reversible covalent modification of specific amino acid residues in the core histones has been proposed to constitute an informational code that confers functional diversity to chromatin (4). Although the details of how this code is read remain unclear, it most likely represents a major epigenetic mechanism to regulate a variety of chromatin-related functions (5, 6). Evidence for the involvement of the linker histones in this code is beginning to emerge (7, 8).

Recently, imaging methods have been developed to measure the binding properties of chromatin proteins in the nuclei of living cells using photobleaching techniques such as fluorescence recovery after photobleaching (FRAP). These approaches show that, although, for the most part, the core histones remain statically bound to chromatin (9), the linker histones display dynamic binding characterized by transient residence at a given chromosomal location followed by dissociation, movement to another location and rebinding (10, 11). Global or localized modulation of the dynamic properties of chromatin could represent an effective mechanism to regulate accessibility of the underlying DNA to specific regulatory proteins (12).

We used photobleaching and biochemical techniques to show that the chromatin of undifferentiated ES cells displays hyperdynamic binding properties indicative of a substantial pool of loosely bound architectural proteins, including core and linker histones (13). This chromatin plasticity is critical for the maintenance of pluripotency in ES cells as experimental manipulation of the hyperdynamic pool of histones was shown to either restrict or accelerate differentiation. Interestingly, this type of hyperdynamic chromatin was not observed in several undifferentiated but lineage-committed cell lines. However, this does not preclude the possibility that other chromatin transitions might be functionally involved in later stage differentiation decisions.

Murine erythroleukemia (MEL) cells are a well established model system for studying terminal cell differentiation (14, 15). These cell lines are Friend virus-transformed erythroid precursors that display rapid proliferation and are tumorogenic when injected into mice. Treatment of these cells with chemical inducers causes them to reinitiate erythroid differentiation, withdraw from the cell cycle, and accumulate erythrocyte-specific gene products. Significant structural changes in chromatin conformation are associated with the terminal differentiation of most cell types, although it is not clear to what degree these changes in chromatin structure are the cause or the effect of the multiple processes that contribute to establishing the differentiated phenotype. Here we have applied photobleaching and biochemical techniques to assay the dynamic behavior of H1 as a measure of global chromatin stability during differentiation. We have characterized a differentiation-dependent change in global chromatin dynamics and provided evidence for its involvement in the commitment to terminal differentiation. We also present results suggesting that this chromatin transition is effected by global dephosphorylation of linker histones and suggest a molecular mechanism by which this is mediated.

Results and Discussion

Dynamics of Linker Histone: Chromatin Interactions Change During MEL Differentiation.

To measure chromatin dynamics in living MEL cells we created stable cell lines expressing low levels of fusion proteins consisting of the enhanced GFP linked to the C terminus of individual H1 variants. We used FRAP to measure the in vivo binding properties of the H1-GFP constructs in cycling control cells and in terminally differentiated, nondividing cultures. The latter were obtained after 4 days of treatment with the chemical inducer hexamethylene bisacetamide (HMBA). A small region of the nucleus was photobleached, and fluorescence recovery was subsequently monitored for several minutes (Fig. 1A–D and Fig. 6, which is published as supporting information on the PNAS web site). An estimate of the binding affinity of this mobile fraction is represented by the T₅₀ value, derived from fitting of the recovery curve to a single exponential function (Fig. 1E). For H1⁰-GFP the average T₅₀ of the mobile fraction in differentiated cells was 69 ± 12 s compared with 39 ± 6 s observed in untreated controls (Fig. 1 A and E) (P < 0.01). Similar differences in exchange rates were observed for each of the other H1 variants. Thus, as MEL cells terminally differentiate they exhibit stronger interactions between linker histones and chromatin manifested by a slower exchange rate.

Fig. 1.

Chromatin binding properties of H1-GFP variants in untreated and differentiated MEL cells. (A–D) FRAP recovery curves of the indicated constructs from control MEL cultures (Untreated) and parallel cultures treated with 5 mM HMBA for 4 days (Differentiated). The FRAP recovery curve is a plot of the normalized fluorescence recovery versus time and represents the average from at least five individual cells. (E) The average T₅₀ ± SD derived from FRAP assays of cells expressing the indicated H1-GFP variants.

We focused further studies on the H1⁰ variant partly because we have accumulated the most information on the behavior of this isotype (11, 16, 17) but also because there is evidence that the expression pattern of this variant changes considerably during MEL differentiation (18). We previously showed that low levels of expression of H1⁰-GFP do not compromise the in vivo binding properties of endogenous or exogenous linker histones or the growth characteristics of stably transfected fibroblasts or ES cells (11, 13). To confirm that this level of expression does not affect the growth or differentiation properties of the transfected MEL cell lines we compared their behavior to that of nontransfected control cells (Fig. 7, which is published as supporting information on the PNAS web site). In none of these assays did we detect a significant difference between cell lines expressing H1⁰-GFP and control cell lines. Thus, we conclude that expression of H1⁰-GFP at low levels does not perturb the differentiation process and that the observed alterations in H1–chromatin binding properties represent a differentiation-dependent physiological response.

Alterations in H1⁰-Chromatin Binding Properties Are Linked to the Commitment to Terminal Differentiation.

Attainment of the fully differentiated phenotype is a multistep process that occurs over the course of several days (14). We investigated the temporal relationship between the change in H1⁰-GFP recovery rates and the progression of the differentiation program. FRAP experiments were performed at various times after the induction of an unsynchronized population of MEL cells (Fig. 2A). Aliquots from the same culture were assayed for commitment to terminal cell division and for the accumulation of hemoglobin. Individual MEL cells become committed, defined as the ability to complete the differentiation program upon removal of the inducer, in a stochastic manner beginning ≈12 h after initial treatment with the inducer and by 48 h most of the population is committed (Fig. 2A). Detection of cells staining positive for hemoglobin accumulation with the benzidine reagent lags ≈24 h after commitment and approaches 100% by 4 days of treatment. A major change in the chromatin binding properties of H1⁰-GFP occurs between 48 and 60 h after induction. In this experimental protocol, this transition appears to occur after most cells are committed to differentiate but well before complete cell cycle arrest or overt expression of the erythrocyte-specific gene products suggesting that the chromatin modulation might be dependent on events that are also necessary for the establishment of commitment.

Fig. 2.

Changes in H1⁰–chromatin interactions are linked to commitment. (A) Time course study of H1⁰-GFP recovery in HMBA-induced MEL cells. T₅₀ ± SD values of H1⁰-GFP recovery (○) in HMBA-induced MEL cells at different time points is represented along with the percentage of committed cells (●) and cells staining positive for Hb with the benzidine reagent (■). (B) Average T₅₀ ± SD values of H1⁰-GFP recovery in DEX-sensitive cultures treated for 60 h with HMBA, DEX, or HMBA and DEX compared with those of untreated cells.

Some MEL cell sublines can be synchronized with respect to the differentiation process by treatment with HMBA and the synthetic hormone dexamethasone (DEX) (19, 20). To apply this technique to this study we had to first isolate and characterize DEX-responsive sublines of our H1⁰-GFP-expressing MEL cells. Cultures of these sublines treated with HMBA and DEX for 60 h are poised in late precommitment stage as displayed by their rapid commitment following DEX removal (Fig. 8, which is published as supporting information on the PNAS web site). FRAP assays revealed that cultures treated in this way exhibit H1⁰-GFP recovery rates nearly identical to that of cultures treated with HMBA alone (Fig. 2B). This suggests that, in this protocol, changes in chromatin dynamics are associated with events in the commitment process and occur before completion of the program necessary for irreversible continuation of differentiation. Cultures treated with DEX alone displayed recovery kinetics identical to that of uninduced cultures. As HMBA and DEX treatments have similar effects on DNA replication (20) this indicates that cell cycle exit alone does not result in major changes in chromatin dynamics. We conclude that alterations in global chromatin dynamics may represent an important aspect of the commitment phase of MEL cell differentiation.

It is important to contrast the chromatin changes we observe during MEL cell differentiation to those seen in ES cells. In pluripotent ES cells, we detected a distinct fraction of mobile linker histones that recovered very rapidly following photobleaching, indicative of a distinct soluble or loosely bound pool, a conclusion that was verified by biochemical analyses (13). In MEL cells we do not observe such a rapidly recovering subpopulation of the mobile pool. Instead we have characterized what we believe is a differentiation-specific transition in global chromatin dynamics that is reflected in quantitative alterations in the binding properties of the bulk of the mobile pool of linker histones. This transition occurred well before cells enter the G₀ phase or accumulate a large scale of histochemically detectable heterochromatin suggesting that it may contribute to, rather than be a consequence of, the terminal stages of the differentiation process.

Dephosphorylation of H1 Is Required for Differentiation-Dependent Changes in Chromatin Binding.

Recent studies have described a sequential down-regulation of specific cyclin-dependent kinase (cdk) activities throughout MEL cell differentiation and demonstrated that interruption of this down-regulation by forced expression of exogenous kinases can block differentiation (21, 22). H1 histones are reversibly phosphorylated, primarily at cdk consensus sites, in response to a variety of physiological signals (23–25) and results from several studies have provided evidence that phosphorylation of linker histones has a significant effect on chromatin binding properties (26–28). Although the cdk substrates critical to MEL cell differentiation have not been identified, we considered the possibility of links between the down-regulation of cdk activity, H1 phosphorylation status and the differentiation-dependent change in H1–chromatin interactions.

There are three cdk consensus sequences in the C-terminal tail of histone H1⁰ and these sites are reversibly phosphorylated in most cell types (25). We introduced mutations into the codons for the Thr residues of each of these sites to create constructs H1⁰Ala-GFP and H1⁰Glu-GFP, designed to mimic a constitutively hypophosphorylated and constitutively hyperphosphorylated state, respectively. Stable cell lines expressing low levels of these constructs were isolated and the in vivo binding properties were measured by FRAP in fully differentiated and untreated cells (Fig. 3 A–C). In untreated cells, the H1⁰Ala-GFP construct recovered significantly slower (T₅₀ = 55 s, P < 0.02) and the H1⁰Glu-GFP construct significantly faster (T₅₀ = 28 s, P < 0.06) than the wild-type H1⁰-GFP (T₅₀ = 39 s). Upon differentiation, the H1⁰Ala-GFP construct displayed a further increase in chromatin binding affinity to a value nearly identical to that of the wild-type construct under these conditions (Fig. 3 A and C). Strikingly, upon differentiation, the H1⁰Glu-GFP construct displayed no significant change in residence time and the measured T₅₀ was nearly identical to that measured in untreated cells (Fig. 3 B and C). This suggests that dephosphorylation at one or more of the targeted cdk consensus sites may be critical for differentiation-dependent changes in linker histone binding.

Fig. 3.

The state of H1 phosphorylation affects linker histone–chromatin interactions. (A and B) FRAP recovery curves of H1⁰Ala-GFP and H1⁰Glu-GFP from at least 10 individual untreated or differentiated MEL cells. (C) T₅₀ ± SD values derived from FRAP assays. (D–F) Elution profiles of the chromatin-bound H1⁰-GFP, H1⁰Ala-GFP, or H1⁰Glu-GFP at different KCl concentrations as a percentage of the values obtained from unwashed nuclei from proliferating or differentiated MEL cells. The amount of chromatin-bound H1⁰-GFP, H1⁰Ala-GFP, or H1⁰Glu-GFP was determined by fluorimetry as described in Materials and Methods.

Expression of these constructs as GFP fusions allowed us to use fluorescence spectroscopy to quantitatively assay the binding properties of individual constructs expressed at low levels. We prepared nuclei from untreated and fully differentiated cultures of MEL cells expressing the phosphorylation constructs, as well as the wild-type control, and measured the extraction of the labeled H1 as a function of salt concentration (Fig. 3 D–F). Not surprisingly, we found that the removal of wild-type H1⁰-GFP required higher salt concentrations in differentiated relative to untreated cultures. Extraction of H1⁰Ala-GFP from untreated cells required much higher salt concentrations than did the wild-type H1⁰-GFP under the same conditions, displaying a profile that resembled that of H1⁰-GFP in differentiated cells. The H1⁰Glu-GFP construct displayed a reciprocal behavior. Extraction of H1⁰Glu-GFP from differentiated cells required much lower salt concentrations than did the wild-type construct and displayed a profile that resembled that of H1⁰-GFP in untreated cells. These results are completely consistent with the FRAP data. Collectively, these data suggest that dephosphorylation of the linker histones at one or more of the mutated cdk sites may be a necessary, albeit perhaps not sufficient, condition for the observed changes in H1–chromatin interactions during terminal differentiation.

Moderate H1⁰Glu Overexpression Inhibits MEL Cell Differentiation.

In the previous set of experiments, the level of expression of the H1⁰-GFP reporters was purposely kept low and all lines displayed differentiation properties identical to nontransfected control MEL cells. We next asked whether global dephosphorylation of H1 is necessary for the MEL cell differentiation pathway. We transfected MEL cells with vectors driving the expression of H1⁰ constructs bearing the Ala or Glu mutations described above, as well as the wild-type sequence, but lacking the GFP tag sequences. These vectors are identical in design to those used previously to create 3T3 lines with altered H1 variant composition (29). Large perturpations of the relative amount of individual isotypes or of the total stoichiometry of linker histones can result in significant affects on cell physiology (29, 30). For initial experiments we therefore carefully selected matched stable cell lines and culture conditions in which the level of the exogenous H1⁰ was ≈20% of the total linker histone composition and there was no significant increase in total linker histone stoichiometry (Fig. 9, which is published as supporting information on the PNAS web site). We then measured the progression of these cell lines through differentiation upon induction with HMBA by measuring the accumulation of hemoglobin-positive cells (Fig. 4A). At this level of expression of the wild-type H1⁰ or the H1⁰Ala mutant there was no differences in the rate of terminal differentiation relative to that of nontransfected control cells. However, cell lines expressing H1⁰Glu displayed a significantly slower accumulation of hemoglobin-positive cells. In control experiments, in which this same cell line was grown in the absence of zinc and therefore expressed low or negligible levels of H1⁰Glu, a normal entry into the differentiation pathway was observed (Fig. 4A). We then measured the effect of expression of these proteins on the commitment step of terminal differentiation and observed a marked inhibition specifically in the H1⁰Glu cell line (Fig. 4B). Relative to wild-type cells, HMBA-treated cultures overproducing H1⁰Glu maintained a larger nuclear volume (Fig. 4C) and displayed a lower accumulation of heterochromatin (Fig. 4D and Fig. 10, which is published as supporting information on the PNAS web site). Thus, by several parameters, forced expression of a modest amount of H1⁰ bearing phosph-mimetic mutations in putative cdk sites dramatically impairs MEL cell differentiation.

Fig. 4.

Dephosphorylation of H1 is necessary for the differentiation program. (A and B) Wild-type MEL cells and cell lines expressing exogenous H1⁰, H1⁰Ala, or H1⁰Glu were treated with 5 mM HMBA. The percentage of hemoglobin-positive cells (A) and committed cells (B) was determined. (C and D) Comparison of the relative nuclear volume and amount of condensed chromatin of HMBA-induced wild-type MEL cells and those overexpressing H1⁰Glu.

H1⁰Glu Has a Dominant Effect on Chromatin Structure.

We were surprised that such a modest expression of H1⁰Glu had a dramatic effect on the progression of differentiation. It seems unlikely that commitment to differentiation would require an essentially complete transition in global chromatin dynamics or that specific events, such as the repression of critical genes, would be hypersensitive to the presence of limited amounts of open chromatin. We then considered the possibility that the expression of small amounts of phosphorylated H1 might influence the structure and dynamic behavior of regions of chromatin considerably larger than the localized nucleosomal binding interface. To investigate, we introduced low levels of GFP-tagged H1 probes into the H1⁰Glu expressing cell line. We initially assayed the binding properties of H1⁰-GFP in differentiating cells to ask whether the expression of H1⁰Glu would block the transition of H1⁰-GFP to tighter binding (data not shown). The results were equivocal in that we observed a heterogeneous population of cells with either fast exchanging (T₅₀ ≈ 40 s) or slow exchanging (T₅₀ ≈ 75 s) H1⁰-GFP, a result that could be explained by the fact that H1⁰Glu expression confers a slower, more asynchronous pattern of differentiation. We then asked whether H1⁰Glu expression could influence the binding properties of other probes in undifferentiated cells. In undifferentiated wild-type cells, H1⁰Ala-GFP exchanges significantly slower than does H1⁰-GFP, displaying a T₅₀ of 55 s (Fig. 5A). However, in untreated cells expressing H1⁰Glu, H1⁰Ala-GFP exchanges significantly more rapidly (T₅₀ = 34 s, P < 0.01) (Fig. 5A), To investigate further, we used the H1⁰cc-GFP construct, which contains a tandem duplication of the C-terminal domain of H1⁰. We previously showed that, in ES cells, this construct displays much tighter binding and a significantly slower exchange rate in FRAP assays (13). We confirmed that this behavior is also observed in MEL cells (Fig. 5B). Interestingly, when this probe was introduced into MEL cells expressing H1⁰Glu, FRAP recovery of H1⁰cc-GFP was significantly faster than that observed in wild-type cells (Fig. 5B). These results suggest the intriguing possibility that binding of small amounts of phosphorylated H1 might have a dominant effect on the dynamic properties of other chromatin proteins.

Fig. 5.

Expression of H1⁰Glu affects the mobility of other linker histones. Shown are FRAP recovery curves of H1⁰Ala-GFP (A) and H1⁰cc-GFP (B) in the wild-type MEL cells and those overexpressing H1⁰Glu. Undifferentiated MEL cell lines overexpressing H1⁰Glu were transiently transfected with vectors expressing H1⁰Ala-GFP or H1⁰cc-GFP using Lipofectamine as per the manufacturer's instructions. FRAP assays were conducted 24 h after transfection.

An Alternative Mechanism for the Role of H1 Phosphorylation.

In most models for the role of linker histone phosphorylation in modulating chromatin structure and function it is proposed that the phosphorylation of H1 stimulates the loss of this protein from localized regions thereby allowing access to positive transcription factors or architectural proteins (24, 31, 32) or by promoting chromatin relaxation mediated by chromatin remodeling complexes (33). Some of the results presented here suggest an additional or alternate mechanism of action.

The residence time of H1⁰Glu-GFP, although considerably shorter than that of H1⁰Ala-GFP, is much longer than that measured for nonbinding mutants (16). The FRAP data revealed no evidence of a significant fraction of free or loosely bound protein, as was observed in ES cells, and the vast majority of the linker histone population, including that of the H1⁰Glu mutant, remains bound to chromatin at any time. Finally, we found that expression of even modest amounts of H1⁰Glu has a dramatic effect on the binding behavior of other linker histones conferring on them a faster exchange rate. Thus the H1⁰Glu protein is behaving in a dominant fashion. It is difficult to envision how this dominant effect could be due solely to the lower binding affinity of this protein and hence it's relative absence from chromatin. These results could be explained if a single molecule of H1⁰Glu, in the bound state, can influence the structure of larger chromatin domains. We propose that, at least in the system we are studying here, the functional effect of H1 phosphorylation lies not in the relative residence times of phosphorylated versus unphosphorylated protein, but rather in qualitative differences in the consequences of binding of these proteins upon global chromatin structure. We suggest that phosphorylation of H1, which may occur in a targeted fashion within the context of assembled chromatin (34) does not promote the immediate release of H1, but rather results in a local alteration of chromatin structure to a more relaxed form. In metazoan linker histones, both the globular and C-terminal domains contribute to chromatin binding (11, 35), with the latter contributing primarily to the formation of an apposed stem structure believed to facilitate the condensation of nucleosome arrays through interactions with the linker DNA between adjacent nucleosomes (36). Phosphorylation of specific sites within the C terminus might be expected to, at least partially, disrupt this association, thereby relaxing chromatin structure. This would not necessarily result in release of the linker histone, which would still be bound to the nucleosome through interactions involving the globular domain, although it certainly could influence the inherent residence time as has been observed. The main difference between this model and those previously proposed is that the phosphorylated H1 functions in the bound state rather than by being removed. Furthermore, this model confers on the process of H1 phosphorylation many of the properties conducive to its function in a regulated epigenetic manner. Most notably, loosening of chromatin by the initial action a targeted kinase, could promote subsequent modifications, including additional phosphorylations or other modifications, and lead to a localized spreading of an altered chromatin structure.

Materials and Methods

Cells.

DS19 MEL cells were propagated as a liquid suspension culture at 37°C under 5% CO₂ in DMEM supplemented with 10% FBS. All stable MEL cell lines were induced to differentiate by treatment with 5 mM HMBA (Sigma, St. Louis, MO) in the regular growth medium. Induced cells were diluted every 24 h in fresh medium supplemented with HMBA to maintain a cell density of <2 × 10⁵ cells per milliliter. Differentiation of MEL cells, detected by scoring for the cells that stained intensely with benzidine staining solution, and commitment assays were performed as described (20).

Plasmids.

MTH1⁰neo, MTH1⁰GFPneo, MTH1cGFPneo, and MTH1⁰ccGFPneo have been described (11, 13, 29). MTH1bGFPneo and pMTH1dGFPneo were constructed by inserting the coding region for the respective histone variant into MTH1⁰GFPneo. MTH1⁰Alaneo and MTH1⁰Gluneo were constructed by three rounds of site-directed mutagenesis of MTH1⁰neo using a QuikChange kit (Stratagene, La Jolla, CA). In all constructs, transcription of the expressed H1 construct is driven by the heavy metal-inducible mouse metallothionein I gene (29).

Construction of Stable Cell Lines.

Stable MEL cell lines expressing H1-GFP variants or overexpressing H1⁰, H1⁰Ala, or H1⁰Glu were established from exponentially growing DS19 MEL cells transfected with the vectors expressing the respective H1 variant using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. Single-cell isolates of the transfected MEL cells were selected in growth medium containing 800 μg/ml G418 sulfate. To identify and isolate MEL cell lines overexpressing wild-type or mutant H1⁰, total chromatin-bound histones were extracted and resolved on HPLC as previously described (29). Isolation of DEX-sensitive sublines was performed as described (20).

Determination of Nuclei Size and Heterochromatin.

Approximately 1 × 10⁵ MEL cells were fixed in 90% methanol/PBS and stored at −20°C. Subsequently the cells were washed with PBS and incubated for 5 min in PBS containing Hoechst 33258 (3 μg/ml) at room temperature. Stained nuclei were photographed by using a Nikon Labophot-2 epifluorescence microscope equipped with a Sensys digital camera. The area of the nuclei and the intensity of Hoechst staining were determined by using Metamorph imaging software.

Photobleaching Recovery Studies in Live Cells.

FRAP analysis of all H1-GFP variants in MEL cells was performed by using the Zeiss 510 LSM META. All of the images were collected by using the 488-nm Ar laser line (30-mW output, detection 500–575 nm) with a Plan-Apochromat ×63/1.4 Oil DIC objective lens. A heat-regulated stage (Heating insert P) and an objective heater (Carl Zeiss Microimaging, Inc., Thornwood, CA) were used to maintain the specimen at a physiological temperature of 37°C. Cell lines used for FRAP experiments were plated and observed on Lab-Tek II chambered coverglass (Nalge–Nunc, Rochester, NY) coated with 0.2 mg/ml polyd-lysine in regular growth medium. For all of the FRAP experiments three prebleach scans of the cells were acquired at 1% laser intensity in the linear rage of detection, followed by the bleaching of a random 2-μm heterochromatic spot in the nucleus at 100% laser intensity. The fluorescence recovery was monitored for 250 s after the bleaching step by scanning the cell at 3-s intervals at 1% laser intensity. The raw fluorescence recovery data were normalized to obtain the relative fluorescence intensity as described (11). The fluorescence recovery data were obtained by plotting the relative fluorescence versus time. To obtain the T₅₀ of H1-GFP recovery, the fluorescence recovery data were fitted to a single exponential equation as described (37).

Fluorimetry of H1⁰-GFP, H1⁰Ala-GFP, and H1⁰Glu-GFP.

Nuclei were isolated from ≈1 × 10⁷ MEL cells as described (17). The isolated nuclei were aliquoted into seven fractions that were washed with high-salt buffer (5 mM MgCl₂/10 mM Tris·HCl, pH 7.5/0.5 mM PMSF) with 0–600 mM KCl. The washed nuclei were spun down and resuspended in high salt buffer with 1 M KCl to extract the chromatin-bound proteins. The level of H1-GFP from the resulting fraction was measured by using an AMINCO-BROWNMAN Series 2 Spectrometer at an absorbance wavelength of 488 nm and an emission of 510 nm. The percentage of H1-GFP bound to chromatin after wash with high-salt buffers of varying KCl concentrations was derived as a percentage of the amount extracted from the unwashed nuclei.

Abbreviations

FRAP

fluorescence recovery after photobleaching

MEL

murine erythroleukemia

HMBA

hexamethylene bisacetamide

DEX

dexamethasone.