Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.
THE packaging of DNA into chromatin is critical for the organization and regulation of eukaryotic genes. The basic unit of chromatin structure—the nucleosome—can be packaged in 30-nm fibers and increasingly compact structures. Higher-order chromatin structure influences many aspects of gene expression, including transcription factor binding, enhancer–promoter interactions, and the organization of chromatin into functional domains. Histone H1 and related linker histones are important determinants of higher-order chromatin structure. These abundant, basic proteins share a common structure consisting of a globular winged helix DNA-binding domain flanked by a short N-terminal segment and a C-terminal domain of ∼100 amino acids (Brown 2003). The winged helix domain of H1 binds the nucleosome near the site of DNA entry and exit; the flanking domains interact with core and linker DNA to promote the formation and packaging of 30-nm fibers in vitro (Robinson and Rhodes 2006; Maier et al. 2008).
In vitro studies suggest that nucleosomal arrays have an intrinsic propensity to fold into 30-nm fibers that are stabilized by association of H1 (Carruthers et al. 1998). However, the function of H1 in vivo is not well understood. In lower eukaryotes, proteins related to H1 play surprisingly subtle roles in chromosome organization and gene expression (Godde and Ura 2008). In higher eukaryotes, the study of H1 function has been complicated by the presence of multiple, functionally redundant H1 subtypes (Khochbin 2001). H1 expression has been partially reduced in nematodes, frogs, and mice (Godde and Ura 2008). A partial reduction in H1 levels has limited effects on gene expression in mice, but leads to the formation of nucleosome arrays that are less compact than normal (Fan et al. 2005). The immunodepletion of H1 from Xenopus extracts results in the assembly of elongated metaphase chromosomes that fail to align and segregate properly (Maresca et al. 2005). These findings suggest that H1 plays an important role in chromosome organization. Since it has not been possible to completely eliminate H1 in any higher eukaryote, its function in vivo remains a topic of considerable debate.
The association of H1 with chromatin is highly dynamic. In both Tetrahymena and mammals, H1 is rapidly exchanged between chromatin fibers (Lever et al. 2000; Misteli et al. 2000; Dou et al. 2002; Catez et al. 2006). The dissociation of H1 from chromatin is thought to disrupt 30-nm fibers and provide an opportunity for transcription factors or other regulatory proteins to access DNA. The association of H1 with chromatin is influenced by H1 phosphorylation, core histone acetylation, and competition with other chromatin-binding proteins (Catez et al. 2006). However, little is known about either the mechanism of H1 exchange or how this process is regulated in vivo.
One of the best candidates for a factor that regulates H1 assembly is Drosophila ISWI. ISWI is the ATPase subunit of multiple chromatin-remodeling complexes—including CHRAC, NURF, and ACF—that slide nucleosomes and alter the spacing of nucleosome arrays (Bouazoune and Brehm 2006). ACF also promotes the assembly of chromatin containing H1 in vitro (Lusser et al. 2005). Although ISWI is not required for H1 expression in vivo, the loss of ISWI function leads to the decondensation of mitotic and polytene chromosomes accompanied by the loss of H1 (Corona et al. 2007). On the basis of these observations, we proposed that ISWI regulates chromosome structure by promoting H1 assembly (Corona et al. 2007). To test this hypothesis and clarify the function of histone H1 in vivo, we investigated phenotypes resulting from the loss of H1 in Drosophila.
MATERIALS AND METHODS
Drosophila stocks and crosses:
Flies were raised on cornmeal, agar, yeast, and molasses medium, supplemented with methyl paraben and propionic acid. The GAL4 system (Brand et al. 1994) was used to drive the expression of His1-RNAi and ISWIK159R. da-GAL4 is expressed broadly at all stages of development (Gerber et al. 2004). For viability studies, UAS-His1-dsRNA males were crossed to da-GAL4 or Df(1)w67c2 females and the progeny were scored for survival to adulthood. All crosses were carried out at 29° unless otherwise indicated.
Generation of transgenic strains bearing UAS-His1-dsRNA transgenes:
The Drosophila His1 coding region was amplified from Canton-S genomic DNA by PCR using the primers 5′-CGAATTCGACAGTTGAGAAGAAAGTGGTCC-3′ and 5′-GGGTGGCCATCTTGGCCGTAGTCTTCGCT-3′ or 5′-CCGCTCGAGACAGTTGAGAAGAAAGTGG-3′ and 5′-GGGTGGCCTAGATGGCCGTAGTCTTCGCTT-3′. The resulting PCR products were digested with SfiI and ligated to form an inverted repeat flanked by EcoRI and XhoI sites. The inverted repeats were cleaved with EcoRI and XhoI and subcloned into pUAST. BLAST searches revealed that the His1 fragment in this construct is not sufficiently related to other regions of the Drosophila genome to generate off-target effects. Transformants were generated by P-element-mediated transformation using the Df(1)w67c2 strain. Homozygous viable transformants used in the study include UAS-His1-dsRNA-8-4 and UAS-His1-dsRNA-13-1 on the X chromosome and UAS-His1-dsRNA-10-3 on chromosome 3.
Generation of H1-Flag-CFP transgenic strains:
The coding sequence for Drosophila His1 was amplified by PCR from a cDNA clone using the primers 5′-GCTATGCTATGCGGCCGCATGTCTGATTCTGCAGTT-3′ and 5′-CATACCGGTCTTGTCGTCGTCGTCCTTGTAGTCCTTTTTGGCAGCCGTAG-3′. The sequence of CFP was amplified by PCR using the primers 5′- GCTATGCTATGCGGCCGCACCGGTATGGTGAGCAAGGGCGA-3′ and 5′-CACTAGTTACTTGTACAGCTCGTCCATG-3′. The PCR products were cloned in the pCR2.1-TA Topo vector (Invitrogen). The H1 insert was digested with SpeI and NotI and subcloned into pBS-SK. The CFP fragment was digested with AgeI and SpeI and cloned into pBS-dH1 using the same restriction sites. The H1–flag–CFP fusion was digested with NotI and SpeI and subcloned downstream of a constitutively expressed α-tubulin promoter in pCaSpeR4 (generously provided by Konrad Basler). The construct was sequenced and used to generate a y w strain bearing a homozygous viable insertion on the third chromosome by P-element-mediated transformation.
Analysis of polytene chromosome structure:
Salivary glands of third instar larvae were dissected in 0.7% NaCl and fixed in 1.85% formaldehyde/45% acetic acid as previously described (Corona et al. 2007). To analyze the effect of H1 knockdown on chromosome structure, da-GAL4 females were mated to P[w+, UAS-His1-dsRNA-8-4]/Y males. To analyze the effect of ISWIK159R expression on chromosome structure, H2AvD-GFP females were mated to w; P[w+, eyGAL4], P[w+, UAS-ISWIK159R-HA-6His]11-4/TM3 males at 18°. Chromosome preparations were analyzed using a Zeiss Axioskop 2 plus fluorescent microscope equipped with an Axioplan HRm CCD camera and Axiovision 4.2 software (Zeiss). For DNA quantification, images were captured using identical exposure times. Chromosome boundaries were identified and the sum pixel intensity within chromosomes was calculated using Volocity software (Release 4.2.1; http://www.improvision.com). Antibodies used in this study are affinity-purifed rabbit anti-ISWI(Tsukiyama et al. 1995), rabbit anti-H3K9me3 (Abcam, ab8898), and rabbit anti-H4Ac(tetra) (Active Motif, 39179).
Electrophoresis and protein blotting:
To analyze the effect of H1 knockdown on nucleosome repeat length, da-GAL4 females were mated to P[w+, UAS-His1-dsRNA-8-4]/Y males. Salivary gland protein extracts were prepared from third-instar larvae and analyzed by protein blotting as described previously (Corona et al. 2007) using affinity-purified rabbit antibodies against ISWI (Tsukiyama et al. 1995) and rabbit polyclonal antibodies against H1(Ner and Travers 1994) and H3 (Abcam, ab1791).
Analysis of salivary gland chromatin by micrococcal nuclease digestion:
Salivary gland chromatin was extracted from P[w+, UAS-His1-dsRNA-8-4]/+; da-GAL4/+ or control da-GAL4/+ third instar larvae and partially digested with micrococcal nuclease as described previously (Corona et al. 2007). Images were obtained using a GelDoc camera and Quantity One software (Bio-Rad Laboratories). Separate experiments were carried out at least three times and gave highly reproducible results.
Confocal microscopy and FRAP analysis:
For live analysis of polytene chromosome phenotypes resulting from the loss of ISWI or H1 function, or ATP depletion, one or two representative nuclei were chosen to be analyzed per gland. In general, the appearance of nuclei within a single salivary gland was very reproducible. Thus, the imaged nuclei are representative of a much larger number of nuclei observed in several glands. Nuclei at the tip of the gland were analyzed whenever possible to ensure consistent results. To analyze the effect of H1 knockdown on chromosome structure in living cells, da-GAL4, H2AvD-GFP females were mated to P[w+, UAS-His1-dsRNA-8-4]/Y males. To analyze the effect of ISWIK159R expression on chromosome structure in living cells, H2AvD-GFP females were mated to P[w+, eyGAL4], P[w+, UAS-ISWIK159R-HA-6His]11-4/TM3 males at 18°. Live polytene chromosome nuclei were imaged using an inverted microscope (DM IRB, Leica Microsystems) equipped with a laser confocal imaging system (TCS SP2, Leica Microsystems). Three dimensional reconstruction and volume calculations of 0.5-μm sections of polytene nuclei were performed by Volocity software (release 4.2.1, http://www.improvision.com). The change in chromatin compaction was established by calculating the ratio of volume to DNA. The ratio of control samples was normalized to one. FRAP analysis of salivary gland nuclei was carried out using an inverted microscope (DM IRB, Leica Microsystems) equipped with a laser confocal imaging system (TCS SP2, Leica Microsystems). Images were acquired and analyzed using the FRAP application of the Leica Microsystems confocal software version 2.61. Salivary glands were dissected from third instar larvae and incubated in Schneider's insect medium (Sigma) containing 50 μg/ml aphidicolin (Sigma), or the equivalent volume of DMSO, for 4 hr. Glands were then transferred to a coverslip and covered in mineral oil for FRAP analysis. For each experiment, 10 single imaging scans were acquired followed by 15 bleach pulses of 600 ms within a square region of interest (ROI) measuring 6 × 6 μm. Images were then collected every 0.6 sec (10 images), every 3 sec (10 images), and every 5 sec (30 images). For imaging, the laser power was attenuated to 16% of the bleach intensity. A second ROI measuring 6 × 6 μm within the same polytene nucleus was used to normalize fluorescence values against background. FRAP recovery curves were generated and analyzed using Microsoft Excel. The recovery curves described represent the average values of eight or more experiments.
To investigate ATP dependence of H1 exchange, salivary glands were treated with agents that block oxidative phosphorylation. Salivary glands were dissected from third instar larvae expressing H2AvD-GFP or H1-CFP, and incubated for 1 hr in 1× PBS containing 100 mm sodium azide (Sigma), 100 μm antimycin A (Sigma), or 2 hr in 1× PBS containing 250 mm rotenone (MP Biomedicals, LLC). Control glands were incubated for 1 hr in 1× PBS. Glands were then transferred to a coverslip and covered in mineral oil for live analysis. Sections of 1 μm whole salivary gland nuclei were acquired. Seventeen azide-treated H1-CFP and 10 azide-treated H2AvD-GFP nuclei were analyzed. For untreated control experiments, 11 H1-CFP and 6 H2AvD-GFP nuclei were analyzed. Similar treatments have been shown to reduce ATP levels in Drosophila salivary glands by two- to fourfold within 2 hr (Leenders et al. 1974).
H1 is essential for Drosophila development:
Unlike other higher eukaryotes, Drosophila contains only one H1 subtype (His1) that is highly related to mammalian H1. Classical genetic approaches cannot be used to study His1 since >100 copies of this gene are present in the Drosophila genome. We therefore used RNA interference (RNAi) to study His1 function. Strains bearing a transgene encoding a His1 hairpin-loop RNA under the control of a GAL4-inducible promoter (UAS-His1-dsRNA) were generated by P-element-mediated transformation. To induce the expression of this transgene, transformants were crossed to strains bearing a daughterless-GAL4 (da-GAL4) transgene that is ubiquitously expressed at high levels. The expression of the His1 hairpin RNA under the control of the da-GAL4 driver resulted in death during late larval or early pupal stages, indicating that H1 is essential for development (Table 1). We were unable to completely eliminate His1 expression in imaginal discs or larval neuroblasts, but occasionally observed interphase nuclei with highly disorganized chromatin in these tissues, accompanied by a severe reduction in the number of metaphase chromosomes (data not shown). These data suggest that H1 is essential for progression through mitosis.
Histone H1 is a major determinant of chromosome structure in vivo:
We next analyzed phenotypes resulting from H1 knockdown in the larval salivary gland. Repeated rounds of DNA replication in the absence of cytokinesis in this tissue leads to the formation of polytene chromosomes that serve as a useful model for interphase chromosomes. The expression of His1 dsRNA in the salivary gland led to a significant reduction in H1 levels (Figure 1A) accompanied by highly penetrant changes in chromosome structure (Figure 1, C, D, and F). Similar phenotypes resulted from the expression of three independent insertions of the UAS-His1-dsRNA transgene, but were never observed in larvae bearing only the da-GAL4 driver or UAS-His1-dsRNA transgene (Figure 1B and data not shown). The most common phenotype resulting from His1 knockdown was the broadening of chromosome arms without an obvious disruption of their banding pattern (Figure 1, C and D). The increase in chromosome size was not due to extra rounds of replication, since the DNA content of chromosomes of control larvae and larvae expressing His1 dsRNA were similar (Figure 1E). Decondensed regions of chromatin and ectopic contacts between chromosome arms were occasionally observed (data not shown). In extreme cases, the banding pattern was completely disrupted and individual arms were no longer distinguishable (Figure 1F). The chromosome defects resulting from His1 knockdown were not limited to euchromatin, as evidenced by the dispersion of the heterochromatic chromocenter (supporting information, Figure S1). H1 is thus a major determinant of chromosome structure in vivo.
Similar chromosome defects result from the loss of H1 and ISWI function:
To clarify the functional relationship between ISWI and histone H1, we compared phenotypes resulting from their loss of function. We previously demonstrated that ISWI plays a global role in chromatin compaction that is antagonized by the acetylation of lysine 16 of the histone H4 tail (H4K16) (Deuring et al. 2000; Corona et al. 2002). The male X chromosome— which is acetylated on H4K16 by the dosage compensation complex—is therefore particularly sensitive to the loss of ISWI function. The partial loss of ISWI function leads to the decondensation of the male X chromosome (Figure 1, B, D, and G) accompanied by the loss of H1 (Corona et al. 2007). A further reduction in ISWI function (due to the expression of the dominant-negative ISWIK159R protein) leads to the decondensation of all chromosomes (Figure 1, B and H) accompanied by the loss of H1 (Corona et al. 2007).
The spectrum of chromosome defects resulting from the loss of ISWI function and H1 are similar, but not identical. The X chromosome of ISWI mutant males appears much broader than normal, but usually retains its banding pattern (Figure 1, D and G). H1 knockdown caused similar defects (Figure 1, C, D, and G) but did not have as pronounced an effect on chromosome length (Figure 1, B and C). In general, the expression of His1 dsRNA led to a much greater increase in the size of polytene chromosomes than the expression of ISWIK159R (compare Figure 1, F to H). This may be due to a reduction in DNA replication in larvae expressing ISWIK159R (Figure 1I). Overall, the similarities between the phenotypes resulting from the loss of His1 and ISWI function support our proposal that ISWI regulates chromatin structure by promoting the incorporation of H1 into chromatin.
To verify that H1 acts downstream of ISWI to regulate chromatin structure, we examined whether the loss of H1 altered either the expression of ISWI or its association with chromatin. The expression of His1 dsRNA dramatically reduced the expression of H1 in salivary gland nuclei without decreasing the overall level of ISWI protein (Figure 2A). We also failed to observe obvious differences in the level of ISWI associated with the polytene chromosomes of larvae expressing His1 dsRNA and control larvae (Figure 2, B and C). Thus, H1 does not appear to modulate chromosome structure by altering the expression of ISWI or its association with chromatin.
Histone H1 undergoes rapid, replication-independent exchange in vivo:
How does ISWI promote the association of H1 with chromatin? ISWI can promote the assembly of nucleosome arrays containing H1 in vitro (Lusser et al. 2005; Maier et al. 2008), suggesting that it may be required for replication-coupled chromatin assembly in vivo. ISWI could also promote H1 incorporation via a replication-independent mechanism, since H1 undergoes rapid, ATP-dependent exchange throughout the cell cycle in other organisms (Catez et al. 2006). As a first step toward distinguishing between these possibilities, we used fluorescence recovery after photobleaching (FRAP) to analyze interactions between H1 and chromosomes in a strain expressing CFP-tagged histone H1. We found that the majority of H1 associated with chromosomes undergoes rapid exchange in vivo (Figure 3A). As observed in mammalian cells (Misteli et al. 2000), approximately half the H1 underwent exchange within 50 sec. This exchange must be replication independent due to the short duration of our experiment and the fact that only two to three rounds of DNA replication occur over 48 hr in the salivary glands of third instar larvae (Rodman 1967). Furthermore, treatment of salivary glands with aphidicolin, an inhibitor of DNA replication, did not affect the rate of H1 exchange (Figure 3A). H1 exchange therefore occurs independently of replication-coupled chromatin assembly in this tissue.
H1 knockdown decreases nucleosome repeat length:
The above findings indicated that ISWI might promote the association of H1 with chromatin during either replication-coupled chromatin assembly or replication-independent H1 exchange. To help distinguish between these possibilities, we compared changes in nucleosome repeat length (NRL) resulting from the loss of H1 and ISWI function. Incorporation of H1 during de novo chromatin assembly increases the average distance between nucleosomes and there is a strong correlation between NRL and the amount of H1 incorporated during chromatin assembly (Blank and Becker 1995; Woodcock et al. 2006; Routh et al. 2008). Thus, the reduced expression of H1—or factors that promote replication-coupled H1 assembly—should cause a significant decrease in NRL. By contrast, the loss of factors required for replication-independent H1 exchange should have little or no effect on NRL, since this process occurs after genomewide nucleosome density has been established. We previously demonstrated that the loss of ISWI function has no apparent effect on NRL in the larval salivary gland, even though it leads to the loss of H1 from chromosomes (Corona et al. 2007). By contrast, reducing the level of H1 in the salivary gland via expression of His1 dsRNA leads to a reproducible 14-bp decrease in NRL from 172 to 158 bp (Figure 3B). These data suggest that ISWI is not required for replication-dependent H1 assembly in salivary gland nuclei.
ATP is required for the association of histone H1 with interphase chromosomes:
If ISWI is required for replication-independent H1 assembly, a reduction in cellular ATP levels should lead to the loss of H1 from chromosomes. To test this prediction, we monitored the association of histone H1-CFP with polytene chromosomes by live analysis following exposure to inhibitors of oxidative phosphorylation. Within 1 hr of azide treatment, H1-CFP was detected in the nucleoplasm in >90% of nuclei (Figure 3C, second panel); this was never observed in untreated salivary glands. In ∼15% of nuclei, all the H1-CFP had dissociated from chromosomes and was found in the nucleoplasm (Figure 3C, third panel). Similar results were obtained with other inhibitors of oxidative phosphorylation, including antimycin A and rotenone (data not shown). By contrast, azide treatment had no effect on the association of a tagged core histone, H2AvD-GFP (Clarkson and Saint 1999), with chromosomes (Figure 3C, fourth panel). Since ATP-depletion affects many cellular processes, it is possible that azide treatment triggers the dissociation of H1 from polytene chromosomes via an ISWI-independent mechanism. However, our data suggest that replication-independent H1 assembly is an energy-dependent process that is subject to regulation by ISWI or other factors.
Characterization of chromosome defects resulting from the loss of H1 and ISWI in living cells:
Live analysis revealed that the dissociation of H1 from chromosomes following treatment with inhibitors of oxidative phosphorylation was not accompanied by obvious changes in nuclear diameter or chromosome volume (Figure 3C). This was surprising, since traditional methods for fixing and squashing polytene chromosomes showed that the loss of H1 significantly increased the size of polytene chromosomes (see above). To gain a more accurate impression of the relative roles of H1 and ISWI in chromosome organization, we visualized chromosomes in living cells expressing H2AvD-GFP. The expression of His1 dsRNA caused a two- to fivefold increase in their volume (Figure 4, A, B, E, and G–I). This increase was not due to extra rounds of replication, since the DNA content of chromosomes of control larvae and larvae expressing His1 dsRNA were similar (Figure 1E). Interestingly, we never observed obvious changes in the banding pattern of chromosomes following H1 knockdown in living cells, even when the chromosome volume increased dramatically (Figure 4, H and I). Thus, His1 RNAi caused much greater changes in salivary gland polytene chromosome structure than ATP depletion, even though both conditions led to a significant reduction in the level of H1 associated with chromatin.
Live analysis of salivary gland nuclei expressing H2AvD-GFP also revealed differences between the chromosome defects resulting from the expression of His1 dsRNA and the dominant-negative ISWIK159R protein. The expression of ISWIK159R did not cause obvious changes in chromosome size (Figure 4, C, D, and F), even though these chromosomes contain reduced levels of H1 (Corona et al. 2007). Indeed, when normalized for DNA content, the volume of chromosomes of control larvae and larvae expressing ISWIK159R were indistinguishable (Figure 4G). The banding pattern of polytene chromosomes was often disrupted, however, and we frequently observed “holes,” which may represent regions of decondensed chromatin (Figure 4, C, D, H, and J). These defects are similar to those observed following the treatment of salivary glands with inhibitors of oxidative phosphorylation (compare Figures 3C and 4D).
Our findings provide direct evidence that H1 is a major determinant of interphase chromosome structure and support our proposal that ISWI regulates higher-order chromatin structure by promoting the association of H1 with chromatin. The incorporation of H1 during replication-coupled chromatin assembly has a particularly dramatic effect on chromatin compaction. After chromatin has been assembled, the continued association of H1 with chromosomes, while important, appears to have more subtle effects on chromosome structure.
An independent analysis of phenotypes resulting from the knockdown of Drosophila His1 by RNAi was recently reported (Lu et al. 2009). Consistent with our data, the authors of this study found that histone H1 is essential for Drosophila development. However, they observed relatively mild defects in salivary gland polytene chromosome structure following H1 knockdown. These defects appear similar to the weakest phenotypes we observed following H1 knockdown (Figure 1C), which may reflect differences in the extent of H1 knockdown achieved in our studies. On the basis of the analysis of fixed polytene chromosome squashes following H1 depletion, Lu et al. (2009) concluded that H1 is required for the alignment of sister chromatids in polytene chromosomes. Although we observed an even stronger disruption of the banding pattern of polytene chromosome squashes following H1 knockdown, we rarely observed such defects via live analysis. Our data therefore argue against a major role for H1 in sister chromatid alignment and illustrate the importance of using live analysis to study factors involved in the regulation of higher-order chromatin structure.
The incorporation of H1 during replication-coupled chromatin assembly increases the average distance between nucleosomes, thus leading to a decrease in genomewide nucleosome density (Woodcock et al. 2006). Accordingly, we observed a significant decrease in NRL following H1 knockdown (Figure 3B). By contrast, the loss of ISWI function leads to a dramatic reduction in the level of H1 associated with chromosomes without causing obvious changes in NRL (Corona et al. 2007). These data strongly suggest that ISWI promotes the association of H1 with salivary gland polytene chromosomes via a replication-independent mechanism. It remains possible that an additional role for ISWI in replication-coupled H1 assembly escaped detection in our genetic studies due to the failure to completely eliminate ISWI function during the stages of salivary gland development when the bulk of DNA replication occurs. Further experiments, including the analysis of fast-acting conditional ISWI alleles, will be required to address this issue.
How does ISWI promote the association of H1 with chromatin? By altering the structure, accessibility or fluidity of chromatin, ISWI may facilitate the binding of H1 to chromatin during dynamic exchange. Consistent with this possibility, we found that inhibitors of oxidative phosphorylation lead to the dissociation of H1 from polytene chromosomes accompanied by its accumulation in the nucleoplasm. Alternatively, ISWI may stabilize the binding of H1 to chromatin by influencing its phosphorylation. H1 is phosphorylated in most organisms, including Drosophila (Villar-Garea and Imhof 2008). In both Tetrahymena and mammals, the phosphorylation of H1 weakens its association with chromatin, leading to an increased frequency of H1 exchange (Dou et al. 2002; Contreras et al. 2003). Thus, ISWI may indirectly promote the association of H1 with chromatin by altering the level or activity of an H1 kinase or phosphatase.
The chromatin of stem cells is hyperdynamic, with both histone H1 and other chromatin-associated proteins undergoing highly elevated rates of exchange (Meshorer and Misteli 2006). This property of pluripotent cell types appears to be functionally important, since a mutant form of H1 that tightly binds chromatin blocks stem cell differentiation (Meshorer et al. 2006). These findings suggest that ISWI and other factors that regulate the association of H1 with chromatin may play important roles in the regulation of cellular pluripotency and differentiation. This possibility is intriguing in light of recent studies implicating ISWI in both nuclear reprogramming and stem cell self-renewal (Kikyo et al. 2000; Xi and Xie 2005).
Previous studies have shown that the dosage compensation machinery antagonizes ISWI function via the acetylation of its nucleosome substrate on H4K16 (Corona et al. 2002; Shogren-Knaak et al. 2006). Furthermore, increased linker histone exchange has been observed in active chromatin enriched in core histone acetylation (Misteli et al. 2000). It is therefore tempting to speculate that the dynamic association of H1 with chromatin is modulated by the interplay of chromatin-remodeling and -modifying enzymes, thus providing a straightforward mechanism for creating rapid, readily reversible changes in higher-order chromatin structure and gene expression. Further work will be required to test this hypothesis and clarify the molecular mechanisms that regulate the association of H1 with chromatin in vivo.
We thank the Bloomington Stock Center for the strains and Grant Hartzog, Susan Strome, Rohinton Kamakaka, and the members of our laboratories for numerous helpful discussions. This work was supported by National Institutes of Health grant GM49883 (to J.W.T.).
Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.102053/DC1.
Communicating editor: F. Winston
- Received February 25, 2009.
- Accepted April 8, 2009.
- Copyright © 2009 by the Genetics Society of America