1 Department of Cell Biology, University of Virginia Health Science Center, PO Box 800732, Charlottesville, VA 22908, USA
2 Laboratory of Chromatin Biology, The Rockefeller University, 230 York Avenue, New York, NY 10021, USA
3 Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
* Author for correspondence (e-mail: scc2003{at}med.cornell.edu)
Accepted 25 May 2004
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Summary |
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Key words: Epigenetics, Histone modifications, Oocyte, Early embryo
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Introduction |
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Although not rigorously tested, it has long been predicted that, during embryonic reprogramming, specific histone modifications are removed from the chromatin template and new marks are placed onto different regions of chromatin that facilitate expression of the embryonic genome (Li, 2002). Here, we investigate this problem at a global level using a wide range of modification-selective antibodies. By documenting a collection of remarkable and highly reproducible changes during oocyte maturation and early embryonic development, our findings lend support to the histone modification `resetting' prediction. Of particular relevance to animal cloning endeavors, our results suggest that the egg cytoplasm may contain enzymatic activities that are capable of removing both acetyl and arginine methyl modifications from specific residues within histone proteins.
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Materials and Methods |
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Indirect immunofluorescence
Oocytes and embryos were fixed for 20 minutes in 4% paraformaldehyde in PBS and then washed three times with 1% BSA-supplemented PBS. Fixed oocytes and embryos were permeabilized in 0.5% Triton-X100 in 1% BSA-supplemented PBS for 20 minutes, washed, and incubated overnight at 4°C in the appropriate antibody diluted in 1% BSA-supplemented PBS. The anti-histone modification antibodies were generated in rabbits and the dilutions used in this study were as follows: Me(Lys9)H3, 1:100; Me(Lys4)H3, 1:500; Ph(Ser1)H4/H2A, 1:2000; hyperacetylated H4, 1:500; Me(Arg17)H3, 1:100; Me(Arg3)H4, 1:200. The embryos and oocytes were then washed extensively and incubated in 2.5 µg/ml Texas red-conjugated donkey anti-rabbit IgG secondary antibody for 1 hour at room temperature. The embryos and oocytes were then placed in 0.4 mg/ml RNase in PBS with 1% BSA for 10 minutes and stained with 20 nM Sytox in PBS with 1% BSA. The embryos and oocytes were again extensively washed and placed in slow fade equilibration medium for approximately 1 minute, mounted on slides in glycerol-slow fade mounting media.
Scanning confocal microscopy
Images were obtained on a Zeiss 410 Axiovert 100 micro systems LSM confocal microscope. For each developmental panel, attenuation, contrast, brightness and pinhole aperture remained constant. For all panels, four-second scans were averaged four times per line using a x40 oil lens equipped with a zoom capacity of approximately two. Z-steps were carried out every 5 microns and false color was added as appropriate. Approximately 15-25 sections were analyzed per oocyte/embryo with only the relevant optical sections being shown. The z-steps were specifically not combined because this reduces the amount of information available from each optical section. For each of the 8 stages, 15-25 embryos were analyzed over at least three independent trials. All reported observations were highly repeatable.
Indirect immunofluorescence on capacitated mouse sperm
Sperm were collected from the cauda epididymis of an ICR retired breeder male mouse and capacitated as described previously (Coonrod et al., 1999). Following capacitation, sperm were centrifugally washed three times in PBS for 5 minutes at 1000 g and air dried onto wells of Teflon coated slides (Polysciences). Sperm were fixed and permeablized as described above. Sperm were incubated with the same dilutions of primary antibody in PBS containing 1% BSA as the eggs/embryos overnight at 4°C. Sperm were then washed four times with PBST and then incubated with 2.5 µg/ml of Texas red-conjugated donkey anti-rabbit IgG for 1 hour at room temperature. Sperm were then washed four times with PBST, once with PBS and mounted in slow-fade medium. A mouse monoclonal antibody (1:100 primary antibody dilution; 1:200 secondary FITC-conjugated donkey anti-mouse IgG dilution) generated against protamine was included as a control to ensure that the sperm were sufficiently permeabilized.
Antisera absorption with peptides
Germinal vesicle (GV) oocytes were collected, fixed and permeabilized as above. The working dilution of each antibody (in PBS/1% BSA) was mixed with the corresponding peptide to give a final concentration of 5 µg/ml peptide and incubated at 4°C for 1 hour with rocking. Prepared GV oocytes were added to the absorbed antisera or non-absorbed sera and processed as above for immunofluorescence.
Peptidylarginine deiminase (PAD) assay and western blot analysis
Total histones were isolated from 293T cells by acid extraction. Histones H4 and H2A were further purified using HPLC. Biochemically purified skeletal muscle peptidylarginine deiminase (smPAD) was purchased from Sigma-Aldrich. For the PAD assay, 0.5 µg of smPAD or heat-inactivated smPAD (95°C, 10 minutes) was incubated with 2 µg purified histones for 30 minutes at 37°C in buffer containing 50 mM Tris HCl (pH 7.6), 5 mM Ca2+ and 5 mM dithiothreitol. The reaction was stopped by adding 2x Laemmli buffer and then heating to 100°C for 3 minutes. The proteins were loaded on a 15% SDS PAGE gel, resolved and electroblotted onto a nitrocellulose membrane. The blot was stained with Ponceau S and imaged. Citrullinated proteins were detected using a modified citrulline antibody (-Mod-Cit) that was generously provided by Dr Tatsuo Senshu (Department of Protein Chemistry, Tokyo, Japan). For the
-Mod-Cit antibody, blots were treated as described previously (Hagiwara et al., 2002
) to expose the Mod-Cit epitope. Blots were then blocked with 5% non-fat milk and incubated overnight at 4°C with the appropriate antibody. Primary antibody dilutions were as follows: Me(Arg3)H4, 1:2000; hyperacetylated H4, 1:5000; Mod-Cit, 1:5000. The blots were then washed, incubated with a 1:5000 dilution of HRP-conjugated anti-rabbit secondary antibody and the labeled proteins were detected using enhanced chemiluminescence.
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Results |
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Histone H3 lysine 9 methylation, histone H3 lysine 4 methylation and histone H4/H2A phosphoserine 1 modifications are stable epigenetic marks
Multiple lysine residues on the histone H3 tail can be methylated. Two of the better studied modifications occur at lysines 4 [Me(Lys4)H3)] and 9 [Me(Lys9)H3] (Lachner et al., 2003). Interestingly, although Me(Lys4)H3 is largely associated with activation of gene expression, Me(Lys9)H3 methylation is associated with repression of gene expression (Rice and Allis, 2001
). Results from this study indicate that the majority of Me(Lys9)H3 staining in immature oocytes (Fig. 1E, arrow) appears to closely co-localize with the DNA (Fig. 1I, arrow) that is surrounding the nucleolus (SN). At fertilization (Fig. 1F), strong Me(Lys9)H3 staining is associated with maternal metaphase chromatin (MII Plate) whereas no staining is seen in the decondensing sperm nucleus (SpN). Similarly, at the pronuclear stage of development, Me(Lys9)H3 staining was limited to the female pronucleus (FPN) whereas the male pronucleus shows an almost complete lack of staining (Fig. 1G,K, arrow). The arrow in Fig. 1C indicates the polar body (PB) for reference. At the morula stage (Fig. 1H), chromatin from both metaphase (Met) and interphase (Int) stage blastomeres stain positive for this modification. Interestingly, when we probed oocytes, eggs and early embryos with antibodies to Me(Lys4)H3 (Fig. 2), we found the localization and staining intensity patterns to be similar to Me(Lys9)H3 staining at earlier developmental stages. As with the Me(Lys9)H3 modification, the metaphase plate of the fertilized egg (Fig. 2F, asterisk) stains strongly for this modification whereas the decondensing sperm nucleus (Fig. 2F, arrow) does not stain. Similarly, the Me(Lys4)H3 mark strongly stains the maternal pronucleus adjacent to the polar body but the paternal pronucleus shows little or no staining. Subtle differences in staining patterns between the two modifications become apparent by the morula/blastocyst stage of development, however, with Me(Lys4)H3 staining being somewhat uniform, the Me(Lys9)H3 staining forms many speckles, which could reflect the appearance of condensed heterochromatic foci.
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The N-terminal residues of the mammalian H4 and H2A histone tails contain the identical SGRGK sequence and investigators have shown that the terminal serine on both tails can be phosphorylated (Zhang and Reinberg, 2001). Athough little is known about the function of this mark, recent studies have demonstrated that the modification is increased during both the S phase and M phase of the cell cycle in HeLa cells (Barber et al., 2004
). In this study, the majority of histone H4/H2A phosphoserine 1 [Ph(Ser1)H4/H2A] staining appears to co-localize with DNA (Fig. 3I) surrounding the nucleolus in the immature oocyte (Fig. 3E). As with the above methylation modifications, strong staining is associated with maternal metaphase chromatin at fertilization (Fig. 3F, asterisk). However, in contrast to the Me(Lys9)H3 and Me(Lys4)H3 modifications, decondensing sperm nuclei (Fig. 3F, arrow) also stain positively for Ph(Ser1)H4/H2A. In the pronuclear stage zygote, Ph(Ser1)H4/H2A staining was of equal intensity in both male and female pronuclei and appears to be more abundant in the nuclear cortical regions (Fig. 3G). Chromatin at all subsequent developmental stages stained positive for Ph(Ser1)H4/H2A. At the blastocyst stage of development note that the staining remains cortical in the nuclei and is more intense in the trophectodermal blastomeres when compared to blastomeres of the inner cell mass (the arrow in Fig. 3D indicates the inner cell mass).
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Hyperacetylated histone H4, histone H3 arginine 17 methylation and histone H4 arginine 3 methylation are dynamic marks
Four highly conserved lysine residues (Lys5, Lys8, Lys12, and Lys16) on the histone H4 tail, can be acetylated singularly or in combination (Rice and Allis, 2001). Hyperacetylation of H4 is thought to unfold the chromatin template for gene expression (Urnov and Wolffe, 2001
) and DNA repair (Kurdistani and Grunstein, 2003
). In this study, hyperacetylated H4 co-localized with DNA surrounding the immature oocyte nucleolus (Fig. 4F). As with the Ph(Ser1)H4/H2A modification, staining is also observed in decondensing sperm nuclei in fertilized eggs (Fig. 4G, arrow). Interestingly, as opposed to all previous modifications, hyperacetylated H4 appears to be only weakly associated with the metaphase plate of maternal chromatin at fertilization (Fig. 4G, asterisk). Also, at the four-cell stage almost no staining is observed in metaphase stage blastomeres (Fig. 4I, arrow) whereas strong staining is seen in interphase stage blastomeres. However, after the four-cell stage of development, hyperacetylated H4 staining again becomes weakly associated with the chromatin of metaphase stage blastomeres (Fig. 4J, arrow).
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Recent studies have suggested that histone arginine methylation may play a role in the nuclear receptor signaling pathway (Stallcup, 2001). For example, the histone H3 Arg17 and Arg26 methyltransferase CARM1 and the histone H4 Arg3 methyltransferase PRMT1 form complexes with other coactivators to regulate the expression of steroid hormone responsive genes (Bauer et al., 2002
; Wang et al., 2001
). In order to determine if arginine-based methyl marks appear to be stable like the Me(Lys9)H3, Me(Lys4)H3 and Ph(Ser1)H4/H2A modifications or are more dynamic like the hyperacetylated H4 modification, we next investigated the amount and distribution of the Me(Arg17)H3 and Me(Arg3)H4 modifications in the egg and early embryo. Immature oocyte nuclei displayed high levels of Me(Arg17)H3 in a punctate manner throughout the entire nuclear region excluding the nucleolus (Fig. 5F). Strikingly, at fertilization, diffuse punctate cytoplasmic Me(Arg17)H3 staining is detected with no apparent staining observed on the condensed metaphase chromosomes (Fig. 5G, asterisk). At the pronuclear stage, Me(Arg17)H3 staining is associated with chromatin at equal levels in both the male and female pronuclei (Fig. 5H). In all of the subsequent developmental stages tested, punctate cytoplasmic staining with little or no chromatin staining is observed in metaphase stage blastomeres (Fig. 5J,O, arrows). Of interest, however, note that there is little or no cytoplasmic staining in the anaphase stage blastomere shown in Fig. 5I,J,N (arrows). This modification also stains cumulus cell nuclei and polar bodies.
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Staining for the Me(Arg3)H4 modification exhibits yet another distinct staining pattern. Weak diffuse nuclear staining for the Me(Arg3)H4 modification is seen within the germinal vesicle of immature oocytes (Fig. 6B). At fertilization this modification appears to be particularly affected by egg cytoplasmic factor(s), as it is undetectable on metaphase chromosomes of fertilized oocytes (indicated by asterisk in Fig. 6E). At the pronuclear stage, nuclear Me(Arg3)H4 staining becomes weakly apparent in some zygotes (data not shown) whereas other zygotes do not appear to stain positive for this modification (Fig. 6I). Staining becomes evident with increasing levels in interphase stage blastomeres at all subsequent developmental stages. Weak staining is seen associated with chromatin in cleavage stage blastomeres at metaphase (Fig. 6L) but by the blastocyst stage of development the Me(Arg3)H4 modification does appear to partially re-associate with the metaphase stage chromatin (Fig. 6R, arrow).
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Global levels of the acetylated lysine and methylated arginine modifications are dramatically reduced on egg chromatin during metaphase
In order to evaluate more rigorously the effect of the egg cytoplasm on global abundance of specific histone modifications, we next directly compared staining levels in immature oocyte nuclei and on metaphase II-arrested egg chromatin (Fig. 7). Results showed that global levels of the Me(Lys4)H3 (Fig. 7, MII C), Me(Lys9)H3 (Fig. 7, MII G) and Ph(Ser1)H4/H2A (data not shown) modifications did not appear to be reduced following dissolution of the nuclear envelope during oocyte maturation. However, staining for the hyperacetylated H4 (Fig. 7, MII K), Me(Arg17)H3 (Fig. 7, MII O) and Me(Arg3)H4 (Fig. 7, MII S) modifications was almost completely absent from the egg chromatin in metaphase II-arrested eggs when compared to staining levels in the immature oocyte nucleus. These results support the hypothesis that the egg cytoplasm contains histone deacetylase activity and possibly also arginine demethylase activities. Also of interest is the observation that in the immature oocyte nuclei, the Me(Lys4)H3 (Fig. 7, GV C), Me(Lys9)H3 (Fig. 7, GV G) and hyperacetylated H4 (Fig. 7, GV K) modifications all appeared to intimately co-localize with the Sytox DNA stain whereas the Me(Arg17)H3 (Fig. 7, GV O) and Me(Arg3)H4 (Fig. 7, GV S) modifications were found throughout the nucleus in a punctate staining pattern and only weakly co-localized with the DNA stain (arrows in GV P and GV T indicate several spots where the arginine methyl modifications do co-localize with DNA).
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Tentative identification of an enzymatic activity capable of altering methylated arginine residues on histones
To test the hypothesis that peptidylarginine deiminase (PAD) activity may alter arginine methyl modifications on histones, we investigated whether a commercially available PAD preparation could affect levels of the Me(Arg3)H4 modification (Fig. 8). In this assay, HPLC-purified 293T cell histone H4 was treated with either heat-inactivated or active skeletal muscle PAD (smPAD), and the samples were processed for western blot analysis. Results show that there was a dramatic loss of staining for the Me(Arg3)H4 modification following PAD treatment whereas staining levels for hyperacetylated histone H4 appeared unaffected. Strong staining for citrullinated H4 was observed following PAD treatment but citrullinated H4 was undetectable in the control sample. Other proteins contained within the treatment sample were also citrullinated including smPAD and possibly H2A. The mass of the citrullinated band observed in the control sample corresponded to that of smPAD and therefore probably indicates that smPAD can deiminate itself in vitro. As with histone acetylation (Georgieva and Sendra, 1999), the altered mobility of histone H4 following PAD treatment probably results from the neutralization of multiple arginine residues following conversion to citrulline. These results provide indirect evidence that the observed loss of staining for methylated arginine residues on egg and embryonic histones might be a result of PAD activity.
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Discussion |
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Stable modifications
The first histone modification tested in our model was H3 Lys9 dimethylation. As with previous reports, we found that the Me(Lys9)H3 mark is abundant in the maternal pronucleus, absent from the male pronucleus (Arney et al., 2002; Cowell et al., 2002
) and then continues to stain the nuclei and metaphase plates of blastomeres (Erhardt et al., 2003
) at similar levels until at least the blastocyst stage of development. Our result is somewhat different than that of Santos et al., who found that staining for both the histone H3 Lys9 methyl and acetyl modifications appeared to be globally reprogrammed between the two-cell and morula stage of development and that these modifications (along with DNA methylation) were incorrectly reprogrammed in cloned bovine embryos (Santos et al., 2003
). In their report, there is a significant decrease in levels of H3 Lys9 acetyl staining during the 8-16 cell stage of development (presumably owing to nuclear HDAC activity), however, the decrease in staining for the Me(Lys9)H3 modification during the 4-8 cell stage appears to be modest. Differences between our results and those of Santos et al. (Santos et al., 2003
) might be explained by the fact that the antibody they used to detect Me(Lys9)H3 levels (anti-4X-methyl K9) was generated against a `branched' peptide containing four K9-dimethylated H3 N-termini (Peters et al., 2001
). This branched peptide was made to more closely simulate a compact heterochromatic state and the cognate antibody has a staining pattern that is strikingly different from the Me(Lys9)H3 antibody (Peters et al., 2001
) used in our study. Alternatively, this discrepancy could reside in inherent developmental differences that exist between mice and cattle. In this study we expand on previous findings by demonstrating that that the Me(Lys9)H3 mark co-localizes with DNA in the immature oocyte nucleus, remains closely associated with DNA on the metaphase II plate of the fertilized egg and is not associated with decondensing sperm chromatin.
Unexpectedly, when we probed oocytes, eggs and early embryos with antibodies to the Me(Lys4)H3 modification, we found the localization and staining intensity patterns was similar to the Me(Lys9)H3 modification until at least the pronuclear stage of development. This finding was somewhat surprising given that, although Me(Lys9)H3 is mainly correlated with gene silencing, Me(Lys4)H3 is largely associated with permissive chromatin regions and gene activation (Lachner et al., 2001; Nishioka et al., 2002
). It is known that the male pronucleus is transcriptionally active whereas the female pronucleus is not (Bouniol et al., 1995
; Ram and Schultz, 1993
) and therefore one might predict that the male pronucleus would contain the activating Me(Lys4)H3 mark and the female pronucleus would contain the repressing Me(Lys9)H3 mark. Given the complex nature of the histone code however, this prediction is probably somewhat simplistic. For example, it is known that dimethylation of histone H3 Lys4 occurs at both inactive and active euchromatic genes whereas trimethylation is present exclusively on active euchromatic genes (Santos-Rosa et al., 2002
). Importantly, global levels of the lysine methyl and phosphoserine modifications do not appear to dramatically fluctuate during oocyte to embryo transition and thus do not represent strong candidates for embryonic reprogramming.
Dynamic modifications
Analysis of the global levels of hyperacetylated H4 in the egg and early embryo revealed that, at all stages tested, hyperacetylated H4 was found to be intimately associated with the DNA in interphase nuclei (see Figs 4, 7). However, levels of this modification appear to be dramatically reduced on maternal chromatin in metaphase II-arrested eggs, fertilized eggs and in early embryonic metaphase blastomeres. Interestingly, by the blastocyst stage of development this modification appears, at least partially, to re-associate with chromatin on metaphase stage blastomeres (see Fig. 4). It is also worth noting that at fertilization, although staining for hyperacetylated H4 on the maternal metaphase chromatin is negative, decondensing sperm chromatin stains positive for this modification. The lack of acetylated H4 staining on metaphase chromatin in eggs and fertilized zygotes and the presence of acetylated H4 staining on decondensing sperm chromatin correlates well with previous literature (Adenot et al., 1997; Kim et al., 2003
).
Methylation of histone arginine residues represents a more recently characterized post-translational modification and, as with histone acetylation, this mark is also associated with activation of gene expression (Davie and Dent, 2002). However, as opposed to acetylation, arginine methylation is thought to be a relatively stable biochemical event (Byvoet, 1972
) and therefore probably represents more of a `long term' activating mark. This prediction has been supported by the fact that, despite exhaustive efforts, an enzyme possessing demethylase activity remains to be conclusively identified (Bannister et al., 2002
). However, there are instances when rapid removal of arginine methyl marks may be necessary. For example, previous reports have found that nuclear hormone-regulated gene expression can be rapidly turned on and off (Bauer et al., 2002
; Wang et al., 2001
). Therefore, if as believed, histone arginine methylation is required for mediating an active state for nuclear hormone activated gene expression, then an enzymatic activity that could remove the arginine methylation mark will probably be required to mediate the inactive state of the target gene. Increasingly, PRMT1 (Berthet et al., 2002
), CARM1 (Chen et al., 2002
), and protein arginine methylation (Aletta et al., 1998
) are becoming associated with cellular differentiation. Therefore, the arginine methyl marks investigated in this study represent particularly attractive modifications to target for `resetting' during early embryonic reprogramming.
Our results show that in immature oocytes, pronuclear stage zygotes, and interphase stage early embryonic blastomeres, both the Me(Arg3)H4 and Me(Arg17)H3 modifications localize to the nucleus. However, as opposed to the Me(Lys9)H3, Me(Lys4)H3 and hyperacetylated H4, the arginine methyl marks are found throughout the nucleus and are not necessarily correlated with regions of the nucleus that stain strongly for DNA. This especially appears to be the case for Me(Arg3)H4 (see Fig. 6). Interestingly, we also found that in early stage Drosophila embryos, the Me(Arg3)H4 modification appears to associate with transcriptionally active developmental puffs of DNA that stain poorly for DNA using Sytox (data not shown). Importantly, our results also show that as opposed to the lysine-methyl and phosphoserine modifications, levels of both arginine-methyl and lysine-acetyl modifications are dramatically reduced on chromatin in metaphase II-arrested eggs, fertilized zygotes and in metaphase-staged early embryonic blastomeres. Also, with respect to Me(Arg3)H4, we find that this modification, as opposed to all other modifications, is either significantly lower or absent from the majority of both male and female pronuclei. We and others (Kim et al., 2003) predict that the loss of staining for histone H4 acetyl modifications is due to the enzymatic activity of a histone deacetylase that localizes to the egg cytoplasm. We also predict that the loss of staining for the histone arginine methyl modifications is caused by histone demethylase activity that probably localizes to the egg cytoplasm. At present, however, we cannot rule out the possibility that the observed lack of staining for the acetyl and arginine methyl modifications at metaphase is due to epitope masking during chromatin condensation. Furthermore, it is also possible that the observed loss of staining for the H4 acetyl and arginine methyl modification results from removal of the modified histones followed by replication-independent deposition of histone variants into the nucleosome. Experiments are currently ongoing in an effort to resolve these questions.
The loss of staining for the arginine-methyl and lysine-acetyl modifications upon exposure to the egg cytoplasm is particularly interesting with respect to nuclear cloning. Nuclear transfer experiments have shown that when somatic cell nuclei are injected into the Xenopus egg cytoplasm, many somatic cell nuclear proteins rapidly relocate to the egg cytoplasm and numerous cytoplasmic proteins (including histone acetyltransferases and histone deacetylases) relocate to the somatic cell nucleus (Wade and Kikyo, 2002). Previous reports also found that, although linker histone proteins were released from somatic cell nuclei into the egg cytoplasm, core histone proteins were not (Dimitrov and Wolffe, 1996
). One might then predict that reprogramming of somatic cell nuclei is facilitated by the activities of cytoplasmic histone modification enzymes following translocation into the somatic cell nucleus. In support of this prediction we have performed preliminary experiments showing that staining for arginine-methyl modifications on somatic cell nuclei is significantly diminished following microinjection into the mouse egg cytoplasm (data not shown).
Our results show that there appears to be little or no staining for the Me(Arg17)H3 or Me(Arg3)H4 modification in metaphase II egg chromatin and on the metaphase chromatin of early cleavage stage blastomeres. However, there did appear to be some Me(Arg3)H4 staining on the chromatin of metaphase stage blastomeres by the blastocyst stage of development. As stated earlier, prior reports have implicated histone arginine methylation in cellular differentiation. Therefore, one could speculate that the histone arginine methyl mark is being removed from the chromatin in early embryonic cell divisions by cytoplasmic factors (possibly of maternal origin) to inhibit differentiation and stimulate cellular proliferation. Following a diminution of this factor at the blastocyst stage of development, the arginine-methyl mark would then be maintained on the chromatin throughout the cell cycle leading to increased cellular differentiation. If, in fact, histone arginine `demethylation' does occur during early embryonic development it could, perhaps, be related to the wave DNA demethylation that also occurs during this developmental period (Jaenisch and Bird, 2003).
Might peptidylarginine deiminases (PADs) alter histone methylarginine residues?
Histone acetyltransferases, deacetylases (Wade and Kikyo, 2002) and arginine methyltransferases (Pawlak et al., 2000
) are present in many cell types including eggs. The identity, however, of an enzymatic activity capable of removing the arginine methylation modification has proved to be elusive. PAD enzymes are known to catalyze the conversion of arginine residues to citrulline in proteins (reviewed by Vossenaar et al., 2003
). Previous studies have found that PADs can target histones H2A, H3 and H4 for deimination (Hagiwara et al., 2002
). We recently cloned and characterized ePAD, a novel highly abundant egg and embryo specific PAD-like protein (Wright et al., 2003
). Based on the above observations, we predicted that PAD activity in the egg might directly affect histone methylarginine modifications and thus give rise to the observed loss of staining for the Me(Arg3)H4 and Me(Arg17)H3 modifications in the egg and early embryo. To test this prediction, we treated 293T cell histone H4 with a commercially available skeletal muscle PAD. Western blot analysis showed that staining for the histone Me(Arg3)H4 modification was dramatically reduced following PAD treatment with a concurrent increase in staining for citrullinated H4. Staining for hyperacetylated histone H4 was virtually unaffected. These results suggest that PADs can alter the Me(Arg3)H4 modification in such a way as to prevent it from being recognized by the Me(Arg3)H4 antibody. The specific nature of this reaction is currently being investigated.
In summary, this report finds that, as opposed to the Me(Lys4)H3, Me(Lys9)H3 or Ph(Ser1)H4/H2A modifications, there appears to be a dramatic reduction in levels of the histone H4 hyperacetylated lysine, Me(Arg17)H3 and Me(Arg3)H4 modifications in metaphase II eggs, fertilized eggs and early embryonic metaphase stage blastomeres. These results suggest that the cytoplasm of the egg and early embryo contains histone deacetylase and probably histone arginine demethylase activity and that removal of the acetyl and methyl modifications from the chromatin template by these as yet unidentified enzymes may be an important component of genomic reprogramming during the egg to embryo transition.
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Acknowledgments |
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Footnotes |
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References |
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Adenot, P. G., Mercier, Y., Renard, J. P. and Thompson, E. M. (1997). Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development 124, 4615-4625.
Aletta, J. M., Cimato, T. R. and Ettinger, M. J. (1998). Protein methylation: a signal event in post-translational modification. Trends Biochem. Sci. 23, 89-91.[CrossRef][Medline]
Arney, K. L., Bao, S., Bannister, A. J., Kouzarides, T. and Surani, M. A. (2002). Histone methylation defines epigenetic asymmetry in the mouse zygote. Int. J. Dev. Biol. 46, 317-320.[Medline]
Bannister, A. J., Schneider, R. and Kouzarides, T. (2002). Histone methylation: dynamic or static? Cell 109, 801-806.[Medline]
Barber, C. M., Turner, F. B., Wang, Y., Hagstrom, K., Taverna, S. D., Mollah, S., Ueberheide, B., Meyer, B. J., Hunt, D. F., Cheung, P. et al. (2004). The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved. Chromosoma 112, 360-371.[CrossRef][Medline]
Bauer, U. M., Daujat, S., Nielsen, S. J., Nightingale, K. and Kouzarides, T. (2002). Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep. 3, 39-44.
Berthet, C., Guehenneux, F., Revol, V., Samarut, C., Lukaszewicz, A., Dehay, C., Dumontet, C., Magaud, J. P. and Rouault, J. P. (2002). Interaction of PRMT1 with BTG/TOB proteins in cell signalling: molecular analysis and functional aspects. Genes Cells 7, 29-39.
Bouniol, C., Nguyen, E. and Debey, P. (1995). Endogenous transcription occurs at the 1-cell stage in the mouse embryo. Exp. Cell Res. 218, 57-62.[CrossRef][Medline]
Byvoet, P. (1972). In vivo turnover and distribution of radio-N-methyl in arginine-rich histones from rat tissues. Arch. Biochem. Biophys. 152, 887-888.[Medline]
Chen, S. L., Loffler, K. A., Chen, D., Stallcup, M. R. and Muscat, G. E. (2002). The coactivator-associated arginine methyltransferase is necessary for muscle differentiation: CARM1 coactivates myocyte enhancer factor-2. J. Biol. Chem. 277, 4324-4333.
Coonrod, S. A., Naaby-Hansen, S., Shetty, J., Shibahara, H., Chen, M., White, J. M. and Herr, J. C. (1999). Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion. Dev. Biol. 207, 334-349.[CrossRef][Medline]
Cowell, I. G., Aucott, R., Mahadevaiah, S. K., Burgoyne, P. S., Huskisson, N., Bongiorni, S., Prantera, G., Fanti, L., Pimpinelli, S., Wu, R. et al. (2002). Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals. Chromosoma 111, 22-36.[CrossRef][Medline]
Cullen, B. R., Emigholz, K. and Monahan, J. J. (1980). Protein patterns of early mouse embryos during development. Differentiation 17, 151-160.[Medline]
Davie, J. K. and Dent, S. Y. (2002). Transcriptional control: an activating role for arginine methylation. Curr. Biol. 12, R59-R61.[CrossRef][Medline]
Dean, W., Santos, F. and Reik, W. (2003). Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin. Cell Dev. Biol. 14, 93-100.[CrossRef][Medline]
Dimitrov, S. and Wolffe, A. P. (1996). Remodeling somatic nuclei in Xenopus laevis egg extracts: molecular mechanisms for the selective release of histones H1 and H1(0) from chromatin and the acquisition of transcriptional competence. EMBO J. 15, 5897-5906.[Abstract]
Erhardt, S., Su, I. H., Schneider, R., Barton, S., Bannister, A. J., Perez-Burgos, L., Jenuwein, T., Kouzarides, T., Tarakhovsky, A. and Surani, M. A. (2003). Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development. Development 130, 4235-4248.
Fischle, W., Wang, Y. and Allis, C. D. (2003). Histone and chromatin crosstalk. Curr. Opin. Cell Biol. 15, 172-183.[CrossRef][Medline]
Georgieva, E. I. and Sendra, R. (1999). Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 269, 399-402.[CrossRef][Medline]
Hagiwara, T., Nakashima, K., Hirano, H., Senshu, T. and Yamada, M. (2002). Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes. Biochem. Biophys. Res. Commun. 290, 979-983.[CrossRef][Medline]
Hansen, J. C., Tse, C. and Wolffe, A. P. (1998). Structure and function of the core histone N-termini: more than meets the eye. Biochemistry 37, 17637-17641.[CrossRef][Medline]
Jaenisch, R. and Bird, A. (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33, 245-254.[CrossRef][Medline]
Jenuwein, T. and Allis, C. D. (2001). Translating the histone code. Science 293, 1074-1080.
Kim, J. M., Liu, H., Tazaki, M., Nagata, M. and Aoki, F. (2003). Changes in histone acetylation during mouse oocyte meiosis. J. Cell Biol. 162, 37-46.
Kurdistani, S. K. and Grunstein, M. (2003). Histone acetylation and deacetylation in yeast. Nat. Rev. Mol. Cell Biol. 4, 276-284.[CrossRef][Medline]
Lachner, M., O'Carroll, D., Rea, S., Mechtler, K. and Jenuwein, T. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120.[CrossRef][Medline]
Lachner, M., O'Sullivan, R. J. and Jenuwein, T. (2003). An epigenetic road map for histone lysine methylation. J. Cell Sci. 116, 2117-2124.
Levinson, J., Goodfellow, P., Vadeboncoeur, M. and McDevitt, H. (1978). Identification of stage-specific polypeptides synthesized during murine preimplantation development. Proc. Natl. Acad. Sci. USA 75, 3332-3336.[Abstract]
Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662-673.[CrossRef][Medline]
Nakao, M. (2001). Epigenetics: interaction of DNA methylation and chromatin. Gene 278, 25-31.[CrossRef][Medline]
Nishioka, K., Chuikov, S., Sarma, K., Erdjument-Bromage, H., Allis, C. D., Tempst, P. and Reinberg, D. (2002). Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 16, 479-489.
Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J. and Ruley, H. E. (2000). Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol. Cell. Biol. 20, 4859-4869.
Peters, A. H., O'Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A. et al. (2001). Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323-337.[Medline]
Pickard, B., Dean, W., Engemann, S., Bergmann, K., Fuermann, M., Jung, M., Reis, A., Allen, N., Reik, W. and Walter, J. (2001). Epigenetic targeting in the mouse zygote marks DNA for later methylation: a mechanism for maternal effects in development. Mech. Dev. 103, 35-47.[CrossRef][Medline]
Ram, P. T. and Schultz, R. M. (1993). Reporter gene expression in G2 of the 1-cell mouse embryo. Dev. Biol. 156, 552-556.[CrossRef][Medline]
Rice, J. C. and Allis, C. D. (2001). Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr. Opin. Cell Biol. 13, 263-273.[CrossRef][Medline]
Santos, F., Zakhartchenko, V., Stojkovic, M., Peters, A., Jenuwein, T., Wolf, E., Rreik, W. and Dean, W. (2003). Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr. Biol. 13, 1116-1121.[CrossRef][Medline]
Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B. E., Emre, N. C., Schreiber, S. L., Mellor, J. and Kouzarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature 419, 407-411.[CrossRef][Medline]
Stallcup, M. R. (2001). Role of protein methylation in chromatin remodeling and transcriptional regulation. Oncogene 20, 3014-3020.[CrossRef][Medline]
Strahl, B. D. and Allis, C. D. (2000). The language of covalent histone modifications. Nature 403, 41-45.[CrossRef][Medline]
Urnov, F. D. and Wolffe, A. P. (2001). Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20, 2991-3006.[CrossRef][Medline]
Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J. and Pruijn, G. J. (2003). PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 25, 1106-1118.[CrossRef][Medline]
Wade, P. A. and Kikyo, N. (2002). Chromatin remodeling in nuclear cloning. Eur. J. Biochem. 269, 2284-2287.
Wang, H., Huang, Z. Q., Xia, L., Feng, Q., Erdjument-Bromage, H., Strahl, B. D., Briggs, S. D., Allis, C. D., Wong, J., Tempst, P. et al. (2001). Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science 293, 853-857.
Wright, P. W., Bolling, L. C., Calvert, M. E., Sarmento, O. F., Berkeley, E. V., Shea, M. C., Hao, Z., Jayes, F. C., Bush, L. A., Shetty, J. et al. (2003). ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets. Dev. Biol. 256, 73-88.[Medline]
Zhang, Y. and Reinberg, D. (2001). Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 15, 2343-2360.
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