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1 Institute of Molecular and Cellular Biosciences, University of Tokyo,1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
2 ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
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Abstract |
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Introduction |
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Among the nucleosomal core histones, H2A has the largest number of known non-allelic variants, among which H2A.Z is the most evolutionarily conserved from primitive eukaryotes to mammals. In fact, less than 10% of the amino acid sequences in the H2A.Z homologues vary across the species, whereas H2A.Z shares only 60% homology with its major counterpart, H2A (Redon et al. 2002; Kamakaka & Biggins 2005). The biological significance of the histone H2A.Z has been demonstrated in several distant vertebrate and non-vertebrate species by generation of H2A.Z null mutants, homozygotes of which were found to die at early stages of embryonic development (Redon et al. 2002; Kamakaka & Biggins 2005; Guillemette & Gaudreau 2006).
H2A.Z appears to play an important regulatory role in diverse biological processes including gene activation, heterochromatin silencing and cell cycle progression (Guillemette & Gaudreau 2006; Raisner & Madhani 2006; Zlatanova & Thakar 2008). Much of our current knowledge of H2A.Z functions came, however, from studies of its Saccharomyces cerevisiae homologue Htz1. Loss of Htz1 has been shown to associate with defects in transcriptional activation of various yeast genes and disruption in the transcriptional silencing of normally heterochromatic loci (Santisteban et al. 2000; Meneghini et al. 2003; Babiarz et al. 2006). Whole genome expression profiling of Htz1 and its deletion mutants suggests that this histone variant functions to counteract the spreading of Sir2-mediated heterochromatin-type silencing (Guillemette et al. 2005; Raisner et al. 2005; Venkatasubrahmanyam et al. 2007). In contrast to the yeast studies, analyses of H2A.Z function in higher eukaryotes produced controversial and confusing results, apparently reflecting a more complex and diverse functions of the histone variant in complex organisms. Structural and biophysical studies on reconstituted chromatin reported both stabilizing and destabilizing effects of mammalian H2A.Z on the nucleosomal structure and on the stability of oligonucleosomal arrays (Guillemette & Gaudreau 2006; Raisner & Madhani 2006; Jin & Felsenfeld 2007; Zlatanova & Thakar 2008). Drosophila melanogaster H2A.Z homologue, H2AvD displays nonrandom distribution on both euchromatic and heterochromatic regions of polytene chromosomes, whereas chromatin immunoprecipitation studies showed that H2AvD is localized to transcribing and non-transcribing genes (van Daal & Elgin 1992; Clarkson et al. 1999; Leach et al. 2000; Swaminathan et al. 2005). Fractionation of H2A.Z-containing nucleosomes showed that specific covalent modifications, such as monoubiquitylation and acetylation, may distinguish H2A.Z association between euchromatin and facultative heterochromatin (Ren & Gorovsky 2001; Bruce et al. 2005; Babiartz et al. 2006; Keogh et al. 2006; Osley et al. 2006; Sarcinella et al. 2007).
Progress in the identification of biological functions of histone variants in vivo is confounded by the lack of appropriate experimental model systems. In this study we have generated transgenic Drosophila lines expressing H2AvD fused at the C- or N-terminus with the green fluorescent protein (GFP) as an experimental system to study physiological activities of H2AvD in vivo. Using this model Drosophila, we have shown that conditional activation of facultative heterochromatic loci is associated with increased H2AvD acetylation.
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Results |
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H2AvD consists of 140 amino acid (a.a) residues, and its N-terminal tail harbors five lysine residues that are known to be substrates for histone acetyltransferases (HATs) (Keogh et al. 2006) (Fig. 1A). In the H2AvDl(3)810 allele, the first 26 amino acids including the five lysine residues are deleted and the resulted protein has been designates as 
H2AvD mutant. H2AvDl(3)810 homozygous flies (further referred as H2AvD/H2AvD genotype) die during transition from the third instar larva into pupa stages of development (van Daal & Elgin 1992; Clarkson et al. 1999; Swaminathan et al. 2005). This indicates that the N-terminal segment plays an indispensable role in the vital functions of Drosophila H2AvD and its mammalian homologue H2A.Z. The functional significance of the C-terminal tails of H2AvD and its mammalian counterpart is less clear. To further elucidate potential roles of the terminal domains in H2AvD, we generated several independent transgenic Drosophila lines that express histone H2AvD protein with either N- or C-terminally fused GFP, that is, GFP-H2AvD or H2AvD-GFP, respectively (Fig. 1A) under the control of constitutive Drosophila Actin 5C (dActin5C) promoter (Fig. 1B).
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First, we investigated whether N-terminal and C-terminal modification would affect polytene chromosome chromatin incorporation of GFP-H2AvD and H2AvD-GFP mutants. Both chimeric H2AvD proteins were visualized by immunostaining with anti-GFP antibody. H2AvD-GFP was readily detectable and distributed throughout polytene chromosomes, including pericentromeric regions (Fig. 2A). The H2AvD-GFP distribution appears to replicate previously demonstrated pattern of the wild-type H2AvD distribution determined by immunostaining with anti-C-terminal H2AvD antibody (van Daal & Elgin 1992; Clarkson et al. 1999; Leach et al. 2000; Swaminathan et al. 2005). In contrast, the chromosomal incorporation of GFP-H2AvD mutant was low and its visibility under the same microscope sensitivity setting was limited to a very few foci (Fig. 2A). This data suggest that the N-terminal tail plays an important regulatory role in the mechanisms of H2AvD chromatin deposition and modifications at the N-terminus are less tolerated than modifications at the C-terminus of H2AvD. In fact, acetylation of H2A.Z (Htz1) in its N-terminus was shown to control gene expression and heterochromatin boundaries in yeast (Millar et al. 2006; Babiarz et al. 2006). To address the impact of the N-terminal tail in deposition of H2AvD, we generated transgenic Drosophila line in which five lysine (K) sites on N-terminal tail of H2AvD; K4, K7, K11, K13 and K15 were mutated individually to arginine (R) and GFP was fused to C-terminal of H2AvD, that is, N-terminal tail mutated KR-H2AvD-GFP under the control of constitutive dActin5C promoter (Fig. 1A,B). The expression levels of GFP-H2AvD, H2AvD-GFP and KR-H2AvD-GFP in these three transgenic Drosophila lines were comparable (Fig. 2B), without overt influence on the expression of the control white gene located downstream of the integrated transgenes (Fig. 2C). Like GFP-H2AvD, KR-H2AvD-GFP was much less deposited on polytene chromosome than H2AvD-GFP (Fig. 2A).
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In yeast, H2A.Z (Htz1) localizes at promoter region and regulates gene expressions (Bruce et al. 2005; Guillemette et al. 2005; Raisner et al. 2005; Zhang et al. 2005; Millar et al. 2006). In Drosophila, actively transcribed loci can be visible as puffs on polytene chromosomes from salivary glands of Drosophila third instar larvae. Using heat shock-induced polytene chromosome puff formation as a model system, we analyzed in vivo possible effects of the H2AvD termini modifications on transcription. The heat shock-induced puff formation appeared to be also normal in the H2AvD-GFP Drosophila line. At the same time, however, heat shock puffs in the GFP-H2AvD larvae as well as KR-H2AvD-GFP larvae seemed to be smaller and incomplete (Fig. 3A). This suggests that gene activation in response to heat shock was impaired in the GFP-H2AvD expressing line.
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Interestingly, the induction of hsp70 gene was also lower in homozygous H2AvD larvae, which are viable until the pupa stage. The impaired activation of the hsp70 gene expression in response to heat shock in GFP-H2AvD and KR-H2AvD-GFP larvae is consistent with observed abnormal heat shock puff formation in these lines (Fig. 3A).
Modification at H2AvD N-terminus affects its acetylation
Next we investigated possible molecular basis of observed functional difference between GFP-H2AvD, H2AvD-GFP and KR-H2AvD-GFP mutants.
Transcriptional activation is known to be closely linked to histone acetylation (Rice & Allis 2001; Clayton et al. 2006; Shahbazian & Grunstein 2007). Therefore, we investigated whether impaired puff formation in GFP-H2AvD line was associated with abnormal acetylation at the mutant N-terminal tail lysine residues. Using antibodies that specifically recognize N-terminally acetylated H2A.Z and H2AvD for Western blot visualization (Bruce et al. 2005), we found that H2AvD-GFP mutant was acetylated. However, acetylation of GFP-H2AvD was expectedly undetectable (Fig. 4A). This suggests that GFP which fused at the N-terminal tail inhibits acetylation of the H2AvD N-terminal region.
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Using a ChIP assay of the transgenic lines, we analyzed change in the content of endogenous H2AvD and GFP-fused mutants in chromatin at a heat shock-induced hsp70 gene promoter (Fig. 4B). In the GFP-H2AvD expressing line, in response to exposure at high temperature, GFP-H2AvD was depleted at the hsp70 gene promoter (Fig. 4C), but acetylation of endogenous H2AvD was induced. Similar heat shock response was seen in the KR-H2AvD-GFP line. As GFP-H2AvD and KR-H2AvD-GFP are deficient in ability to be acetylated (Fig. 4A), this result suggests that GFP-H2AvD and KR-H2AvD-GFP might be dismissed from the promoter, whereas acetylated endogenous H2AvD might be deposited to support the physiological response to heat shock.
Having shown deficiency of the N-terminally modified H2AvD in transcriptional function, we next examined whether GFP-H2AvD mutant is compatible with normal Drosophila development and would be able to compensate for functional deficiency of the lethal H2AvD mutant. We designed a crossing scheme to generate H2AvDl(3)810 homozygous flies with GFP-H2AvD gene. Expectedly, transgenic flies with genotype of homozygous H2AvD/H2AvD died at early stages (adult stage; 0%), however, rescued lines of genotypes of GFP-H2AvD/CyO; H2AvD/H2AvD as well as GFP-H2AvD/GFP-H2AvD; H2AvD/H2AvD survived to adult stage in 12% and 27%, respectively (Table 1). This suggests that the GFP-H2AvD protein is partially competent and able to a limited degree compensate for the N-terminally truncated H2AvD mutant. This idea could be further pronounced by no viability of those of KR-H2AvD-GFP/KR-H2AvD-GFP; H2AvD/H2AvD. However, unexpectedly, the lines of H2AvD-GFP/CyO; H2AvD/H2AvD and H2AvD-GFP/H2AvD-GFP; H2AvD/H2AvD exhibited shorter lives than those of GFP-H2AvD/CyO; H2AvD/H2AvD as well as GFP-H2AvD/GFP-H2AvD; H2AvD/H2AvD, revealing that the C-terminal tail of H2AvD is also significant for development.
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Discussion |
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Histones are modified at their N- or C-terminal tails exposed outwards from the nucleosomal core octamer. It has been reported that H2A.Z can be acetylated at the N-terminus and ubiquitinylated at the C-terminus, and suggested that differential H2A.Z modifications might distinguish actively transcribed and silenced chromatin loci (Ren & Gorovsky 2001; Bruce et al. 2005; Boyne et al. 2006; Keogh et al. 2006; Osley et al. 2006; Sarcinella et al. 2007). In general, histone acetylation has been established as a definitive mark of gene activation (Rice & Allis 2001; Clayton et al. 2006; Shahbazian & Grunstein 2007). To investigate biological activities of the fly H2A.Z homologue, H2AvD in vivo, we have generated transgenic Drosophila lines expressing H2AvD with either N-terminally or C-terminally fused GFP. In this study we focused on regulation of facultatively repressed genomic loci using heat-shock-induced puff formation and activation of hsp70 gene expression as an in vivo experimental model system.
We found that chromosomal incorporation of the GFP-H2AvD chimeric mutant was significantly reduced in comparison with the C-terminally fusion H2AvD-GFP chimera. In addition, we have demonstrated that GFP-H2AvD mutant is deficient in the ability to be acetylated, suggesting that the intact N-terminus is required for functional interaction of H2A.Z with HATs. These findings implicate structural elements of the N-terminus in the mechanisms of H2A.Z chromatin deposition. It has been established in yeast that SWR1 chromatin remodeling complex is involved in exchange of H2A for H2A.Z (Kobor et al. 2004; Mizuguchi et al. 2004). As HATs are common subunits shared with multiple complexes like Ino80 chromatin remodeling complex, GFP in the N-terminal of H2AvD might disrupt functional associations of such HAT complexes and chromatin. Alternative possibility could be raised that the GFP-H2AvD is defective to recruit histone chaperon or histone deposition machinery. In this respect, it would be interesting to define a factor/complex interacting with GFP-H2AvD and KR-H2AvD-GFP mutants on polytene chromosomes.
In the tested these transgenic lines, the ratio of the expression levels of exogenous H2AvD mutants to that of endogenous H2AvD was comparable. By a Western blotting with the antibody against acetylated — K4, K7 and K11 — H2A.Z, acetylation of GFP-H2AvD was not observed. However, it still remains to be clarified if the K13 and K15 of GFP-H2AvD are acetylated, because the K14 of Htz1 is preferentially acetylated than K3, K8 and K11 in yeast (Millar et al. 2006). As in the KR-H2AvD-GFP line, all five lysine residues are mutated, it is also conceivable that KR-H2AvD-GFP acts as a dominant negative mutant against endogenous H2AvD.
A detailed analysis of heat shock-induced puff formation on the polytene chromosomes and expression of the heat-activated hsp70 gene in our transgenic Drosophila lines showed that GFP-H2AvD and KR-H2AvD-GFP mutant also impaired both puff formation and hsp70 gene activation. These results suggest that activation of transiently silenced Drosophila loci requires acetylation of histone H2AvD.
Overall, based on the presented findings we can conclude that the N-terminal region of histone H2A.Z plays a pivotal role in the mechanisms of transcriptional activation of facultative heterochromatin and that acetylation of N-terminal H2A.Z is required to support induction of transiently silenced gene expression.
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Experimental procedures |
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DNA constructs encoding GFP-H2AvD, H2AvD-GFP and KR-H2AvD-GFP proteins were cloned into the pCaSpeR vector (Drosophila Genomics Resource Center). For germ-line transformation, the transgene plasmids were microinjected into Drosophila embryo of yw strain (i.e. with deficient white gene). The microinjection was a customer service of the BestGene Inc. The yw strain was used as a control in all experiments. Histone H2AvD mutant Drosophila H2AvDl(3)810 line was obtained from the Bloomington Drosophila Stock Center. All flies were raised at 25 °C on standard agar/cornmeal/yeast medium. In the rescue experiment, H2AvD heterozygous flies were crossed with GFP-H2AvD expressing flies, and generated transgenic flies in genotype with GFP-H2AvD/CyO; H2AvD/TM3. "CyO" and "TM3" are representing balancer chromosome on second and third chromosome, respectively. Each balancer enables chromosomes escaping homologous recombination; as a result, transgenic flies can be kept stable genetically. Second, male and female of GFP-H2AvD/CyO; H2AvD/TM3 were crossed, and H2AvD heterozygous flies with GFP-H2AvD (GFP-H2AvD/CyO; H2AvD/TM3, GFP-H2AvD/GFP-H2AvD; H2AvD/TM3) and H2AvD homozygous flies with GFP-H2AvD (GFP-H2AvD/CyO; H2AvD/H2AvD, GFP-H2AvD/GFP-H2AvD; H2AvD/H2AvD) were obtained. The same crosses were carried out in H2AvD-GFP line as well as KR-H2AvD-GFP line.
Immunostaining of polytene chromosomes
Salivary glands were dissected from wandering third instar larvae. In heat shock experiments, larvae were incubated at 37 °C for 30 min before dissection. Polytene chromosome samples were prepared and immunostaining carried out following published protocols (Furuhashi et al. 2006), with the following modifications. Salivary glands were dissected and placed in 0.7% NaCl with 0.5% NP-40 for 5 min, and fixed in 3.7% formaldehyde, 45% acetic acids and 0.1% Triton X-100 mix for 2 min before squashing. Immunostaining was carried out with primary antibodies: rabbit polyclonal anti-GFP antibody (Molecular Probes) and mouse monoclonal anti-Drosophila RNA polymerase II antibody (Covance) in combination with secondary antibodies: donkey anti-rabbit IgG antibody-FITC conjugate and goat anti-mouse IgM antibody–TexasRed conjugate (Jackson ImmunoResearch). DNA was visualized with DAPI (Roche). Images were captured using Zeiss Confocal Laser Scanning System 510. At least six pairs of salivary glands samples in each of at least three independent experiments were analyzed to verify the staining pattern reproducibility.
RNA isolation and Northern blotting
Total RNA was extracted from 10 third instar larvae using Trizol (Invitrogen). In heat shock experiments, larvae were incubated at 37 °C for 30 min before homogenization. Precisely measured 5 µg RNA in each line were fractionated in 1% formaldehyde-agarose gel. RNA was transferred onto nitrocellurose membrane using 20 x SSC and cross-linked in the UV-stratalinker (Stratagene). The membranes were prehybridized with DIG Easy Hyb Buffer (Roche) for 30 min at 50 °C and then hybridized with DIG-labeled hsp70 and rp49 RNA antisense cDNA probes overnight at 50 °C. The membranes were washed under constant agitation two times for 15 min in 2 x SSC, 0.1% SDS at room temperature followed by two times for 15 min in 0.1 x SSC 0.1% SDS at 50 °C. The membranes were incubated for 30 min in blocking solution (1% blocking reagent (Roche), 0.1 M maleic acid, pH7.4. 0.15 M NaCl), followed by 30 min of incubation with sheep anti-DIG-AP antibodies (Roche) in blocking solution. The membranes were washed extensively, soaked with CDP-Star reagent (Roche) and exposed to High performance chemiluminescence film (GE Healthecare). Probe sequences were, at the hsp70Ab gene coding region: 5'-GGCTAAGAACCAGGTGGCCATGAA-3' and 5'-GCACCTCGAACAGTGATCCCTCGT-3'; and at the rp49 gene coding region: 5'-CCACCAGTCGGATCGATATGCTAAC-3' and 5'-CAGAAATGACAATTGAACTCGGCA-3'.
Western blotting
Salivary glands from 10 larvae were dissected, homogenized and sonicated in RIPA lysis buffer (140 mM NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.3% SDS, 0.1% sodium deoxycholate). Lysate proteins were resolved in 10% and 15% SDS-PAGE and transferred onto Immobilon-P transfer membranes (Millipore). Western blot staining were carried out with rabbit anti-GFP polyclonal antibody (Molecular Probes), sheep polyclonal H2A.Z antibody (ab18263, Abcam), and sheep polyclonal anti-K4, K7, K11 acetylated H2A.Z antibody (ab18262, Abcam) and corresponding HRP-conjugated secondary antibodies (Dacocytomation or Abcam).
White gene expression measurement
Expression levels of the white gene were evaluated by colorimetric spectrometry measurement of the pigment in adult fly eye as previously described (Pal-Bhadra et al. 2004).
Chromatin immunoprecipitation
Chromatin immunoprecipitation was carried out essentially as described previously (Kouzmenko et al. 2004), with the following modifications. In heat shock experiments, larvae were incubated at 37 °C for 30 min before dissection. Forty pairs of salivary glands from each of the investigated lines and/or treatment conditions were dissected and fixed with 1% formaldehyde at 37 °C for 20 min. After washing three times with cold 1 x PBS containing protease inhibitor cocktail (Roche), glands were homogenized in SDS lysis buffer and resulted homogenates were sonicated to obtain DNA fragments with length in the range between 400 and 1000 base pairs. Immunoprecipitation was carried out with rabbit polyclonal anti-GFP antibody (Molecular Probes), anti-K4, K7, K11-acetyl H2A.Z antibodies (ab18262, Abcam), or rabbit pre-immune serum IgG (Santa Cruz Biotechnology). DNA fragments were purified with QIAquick DNA purification kit (Qiagen). Primer sequences for PCR at the hsp70Ab gene promoter region: 5'-CTCTGCGATTATCTCTAACATAATTAACTT-3' and 5'-CTTTGCTTGTTTGAATAGAATTGACTCTCC-3'; and at the rp49 gene coding region: 5'-TCCGGCAAGGTATGT GCGTGATTT-3' and 5'-ATATCGATCCGACTGGTGGCG GAT-3'.
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Acknowledgements |
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Footnotes |
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* Correspondence:uskato{at}mail.ecc.u-tokyo.ac.jp
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References |
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Received: 21 August 2008
Accepted: 15 September 2008
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