Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M408409200 on August 6, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43815-43820, October 15, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/42/43815    most recent
M408409200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ridgway, P.
Right arrow Articles by Tremethick, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ridgway, P.
Right arrow Articles by Tremethick, D. J.
Related Collections
Right arrow Papers Of The Week
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Unique Residues on the H2A.Z Containing Nucleosome Surface Are Important for Xenopus laevis Development*{diamondsuit}

Patricia Ridgway{ddagger}, Karl D. Brown, Danny Rangasamy, Ulrica Svensson, and David J. Tremethick{ddagger}

From the John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 0200, Australia

Received for publication, July 26, 2004 , and in revised form, August 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Critical to vertebrate development is a complex program of events that establishes specialized tissues and organs from a single fertilized cell. Transitions in chromatin architecture, through alterations in its composition and modification markings, characterize early development. A variant of the H2A core histone, H2A.Z, is essential for development of both Drosophila and mice. We recently showed that H2A.Z is required for proper chromosome segregation. Whether H2A.Z has additional specific functions during early development remains unknown. Here we demonstrate that depletion of H2A.Z by RNA interference perturbs Xenopus laevis development at gastrulation leading to embryos with malformed, shortened trunks. Consistent with this result, whole embryo in situ hybridization indicates that endogenous expression of H2A.Z is highly enriched in the notochord. H2A.Z modifies the surface of a canonical nucleosome by creating an extended acidic patch and a metal ion-binding site stabilized by two histidine residues. To examine the significance of these specific surface regions in vivo, we investigated the consequences of overexpressing H2A.Z and mutant proteins during X. laevis development. Overexpression of H2A.Z slowed development following gastrulation. Altering the extended acidic patch of H2A.Z reversed this effect. Remarkably, modification of a single stabilizing histidine residue located on the exposed surface of an H2A.Z containing nucleosome was sufficient to disrupt normal trunk formation mimicking the effect observed by RNA interference. Taken together, these results argue that key determinants located on the surface of an H2A.Z nucleosome play an important specific role during embryonic patterning and provide a link between a chromatin structural modification and normal vertebrate development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Variant histones have been identified in all eukaryotes and evidence exists that they define specialized functional domains within the genome. At the centromere, for example, the core histone H3 is replaced by the centromere protein A (CENP-A) variant to impart a unique structure to centromeric chromatin (1). A number of H2A variants have been described at defined locations in the genome: H2A.X at sites of DNA double-strand breaks (2), H2A-Bbd deficient from the Barr body (3), and macroH2A on the mammalian inactive X chromosome (4). H2A.Z histone variant is highly conserved suggesting an important role; however, to date, defining the function and localization of H2A.Z has remained elusive. The first hypothesis, derived from its preferential localization in Tetrahymena to the transcriptionally active macronucleus and the micronucleus during a brief transcriptional period prior to conjugation, proposed that H2A.Z has a role in the activation of transcription (5, 6). In budding yeast, H2A.Z was suggested to be necessary for gene activation at the PHO5 and GAL1 promoters in cooperation with a chromatin remodelling factor (7). On the other hand, others suggested that H2A.Z was involved in gene silencing at telomeres and the HMR locus (8). More recently it was shown that H2A.Z containing chromatin may act as a buffer between active and inactive regions by structurally altering chromatin to prevent the spreading of silencing factors (9).

Although these investigations have defined a role for H2A.Z in the transcription of some genes in yeast, it is important to note that in budding yeast H2A.Z is not essential. We propose that H2A.Z has additional roles in higher eukaryotes. A role at constitutive heterochromatin is indicated by the enrichment of H2A.Z at pericentric heterochromatin, with heterochromatin protein 1{alpha} (HP1{alpha}), in early mouse extra-embryonic cells (10). Importantly, a reduction in H2A.Z expression in mammalian tissue culture cells by RNAi1 leads to a loss in heterochromatin stability and abnormal chromosome segregation (11). Consistent with these findings, deletion of the H2A.Z gene in fisson yeast Saccharomyces pombe results in genome instability (12).

H2A.Z may have additional specific functions during early development. Loss of H2A.Z in Drosophila and mice is lethal with the defect occurring early in embryonic development (13–15). Subsequent investigation during mouse blastocyst outgrowth revealed that the localization and expression pattern of H2A.Z differed in early cell lineages (10). Furthermore, analysis of the H2A.Z gene promoter in human embryonal carcinoma cells identified an element that regulates its expression during differentiation suggesting that H2A.Z may be acting at defined stages of cell specialization (16). Therefore, H2A.Z may also have a specific role in the development of certain tissues.

Xenopus laevis is an ideal system to investigate the role of chromatin in regulating early developmental events. On fertilization, the activated X. laevis egg undergoes a global transcriptional silencing for 12 cleavage divisions until the midblastula transition (MBT) when embryonic transcription commences (17, 18). It appears that this early silencing is maintained by vast pools of maternal histones, which inhibit the formation of the basal transcriptional machinery and translation (19, 20). Histone variants of the H1 linker histone have been reported to specifically control embryonic gene expression patterns in X. laevis. The accumulation of somatic H1 linker histone regulates the loss of mesodermal competence during gastrulation (21, 22) and overexpression of somatic H1, but not the maternal H1 linker variant B4, reduces mesoderm-specific myoD induction (21). Therefore, evidence suggests that general chromatin components are involved in pattern formation processes during early X. laevis development. Interestingly, and in contrast to early mouse development (15), Xenopus H2A.Z is expressed at a low level during early development,2 but its expression increases and peaks at gastrulation (23).

Previously we used H2A to H2A.Z domain swap experiments in Drosophila to identify a region in the H2A.Z C terminus, the docking domain, as being essential for development in flies (14). The docking domain stabilizes the interaction between the H2A/H2B dimer and the H3/H4 tetramer and makes a significant contribution to the nucleosomal surface. The crystal structure of an H2A.Z containing nucleosome showed that the major difference from a canonical nucleosome is an altered surface due to amino acid changes in the docking domain. Interestingly, one major difference was two histidine residues that bound a metal ion and are indicative of a protein/protein interaction interface (24). Our objective in this investigation was to determine whether H2A.Z has a specific developmental role and to define the essential amino acid residues in H2A.Z required for such a function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs—To express wild-type murine H2A, wild-type murine H2A.Z, or H2A.Z with alterations to specific amino acid residues, H2A.Z cDNA fragments were generated by PCR and subcloned into the RNA expression vector p{beta}GFP/RN3 (25). Alterations to the wild-type H2A.Z sequence were made to produce the following H2A.Z derivatives: H2A.ZNQ (the two evolutionarily conserved histidine residues at positions 112 and 114 changed to asparagine and glutamine residues, which are the identically positioned amino acid residues in H2A), H2A.ZNH (only one histidine at position 112 altered to asparagine), H2A.ZCS (the C-terminal {alpha}-helix of H2A.Z (amino acid residues swapped with the equivalent region of H2A), H2A.ZNK (two amino acid residues within the C-terminal {alpha}-helix of H2A.Z at amino acid positions 97 and 98 in H2A.Z changed from aspartic acid to asparagine and serine to lysine, respectively, to correspond to H2A residues at the same position), and H2A.Z/EGFP (wild-type H2A.Z fused at its C terminus to the EGFP cDNA as described previously (26)).

Embryo Culture and Microinjection—X. laevis embryos were obtained from in vitro fertilized eggs according to standard protocols (26). Embryos were microinjected into the animal pole of one or both blastomeres at the two-cell stage of development with 1–10 ng of in vitro transcribed mRNA in a total volume of 9.2–27.6 nl using a nanoject injector (Drummond) depending on experimental demands and as described previously (19). Synthetic annealed 21-bp H2A.Z RNAi (Ambion) (100 µM) was injected into each blastomere of a two-cell stage embryo in a volume of 27.6 nl. Staging of embryos was according to Nieuwkoop and Faber (27).

In Vitro RNA Preparation and RNA Interference—RNA for microinjection was transcribed in vitro from cDNA using T3 RNA polymerase. The RNA was 5'-capped and contains stabilizing {beta}-globin 5'- and 3'-untranslated regions specifically designed to maximize exogenous protein expression in X. laevis embryos (25). RNA was purified and quantified by both electrophoresis and UV analysis and diluted to required concentrations in injection buffer as described previously (26, 28). For details of H2A.Z RNAi sequences from Xenopus, mouse, and scrambled control, see supplementary Fig. 1 in Ref. 11.

In Situ hybridization—Whole mount in situ hybridization was performed as described previously (29) except without the use of xylene for removal of lipids or without the hydrolysis of probes given the short length of the RNA (28). Probes were hybridized at 60 °C. The H2A.Z probe was an antisense RNA incorporating the whole translated region produced by the same in vitro technique used to produce mRNA for microinjection but with a digoxigenin-substituted ribonucleotide. Control embryos were treated in the same manner as the test embryos with the omission of probe. 5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Sigma) was used as the color substrate. Embryos were dehydrated with a series of glycerol solutions of increasing concentration and mounted in 70% glycerol. A Leica MZFL III microscope with the IM50 software was used for image capture.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Depletion of H2A.Z Disrupts Embryo Development following Gastrulation—To determine whether H2A.Z is important for Xenopus development we chose two parallel complementary approaches to perturb H2A.Z activity. Endogenous H2A.Z function was disrupted using double-stranded RNA interference (RNAi) and by a dominant-negative approach (see below). When RNAi complementary to Xenopus H2A.Z was microinjected into two blastomeres of a two-cell stage embryo over 35% of embryos displayed a specific defective phenotype (Fig. 1). The level of total H2A.Z mRNA was reduced by 20–30% in these embryos (determined by RT-PCR normalized to H4 mRNA levels) consistent with other reports of RNAi in Xenopus (30, 31) (data not shown). The abnormal phenotype was first evident at gastrulation as a failure of the blastopore to close leading to embryos with shortened trunks and a malformed neural plate at the tailbud stage. This effect was specific, since neither mouse H2A.Z RNAi nor a scrambled RNAi produced a marked increase in defective embryos relative to uninjected controls (Fig. 1A). To confirm that the malformation was indeed attributed specifically to H2A.Z, we co-injected H2A.Z RNA in a series of rescue experiments (Fig. 1, B–F). Compared with control embryos that developed normally (Fig. 1B), over half of Xenopus H2A.Z RNAi-injected embryos displayed a gastrulation defect as illustrated in Fig. 1C. It is important to point out that embryo trunks were severely shortened and malformed, whereas the anterior head region appeared normal suggesting that specific tissues were affected. The percentage of malformed embryos was reduced following H2A.Z RNA co-injection, in a dose-dependant manner, to similar levels as those observed with injection of mouse or scrambled RNAi (~10%; Fig. 1F). Not only were there fewer malformed embryos in the H2A.Z rescued embryos, but the malformations were generally less severe (Fig. 1, compare C with D and E). These experiments indicate that H2A.Z has a role in progression of Xenopus early development beyond gastrulation in specific tissues.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1.
Depletion of H2A.Z disrupts embryonic development following gastrulation. A, Xenopus embryos were injected into two blastomeres at the two-cell stage with either Xenopus, mouse, or a scrambled H2A.Z RNAi as described under "Experimental Procedures." At stage 13 the number of embryos with gastrulation defects were counted. The number of embryos scored is given above each histogram. B–E show embryos at approximately stage 31 following: no injection (B), Xenopus H2A.Z RNAi (C), Xenopus H2A.Z RNAi + 0.5 ng of H2A.Z RNA (D), and Xenopus H2A.Z RNAi + 1 ng of H2A.Z RNA (E). F, percentage of above embryos that display gastrulation defects scored at stage 13.

 
Microinjected H2A.Z mRNA Is Expressed and in the Nucleus—We propose that macromolecular determinants located on the nucleosome surface play a fundamental role in controlling chromatin function. To identify specific molecular determinants located on the surface of an H2A.Z containing nucleosome that are required for normal development, we chose an overexpression strategy. First, to confirm that injected H2A.Z mRNA is expressed and nuclear an H2A.Z/EGFP fusion protein was used to visually monitor the expression of exogenous H2A.Z because levels of protein were too low for standard immunodetection. Fig. 2 shows that the injected H2A.Z/EGFP fusion protein was first detected at approximately stage 7 of development before the initiation of embryonic transcription at the MBT. Significantly, before the MBT the H2A.Z/EGFP fusion protein is found in a diffuse pattern, not associated with chromatin, within cells of the early cleavage stages (Fig. 2B). During these early divisions of rapid genome replication, the interphase nucleus is not reconstructed, and DNA remains highly methylated suggesting that chromatin has a unique structure until the MBT (32). Following MBT, in contrast to early cleavage stages, we detected green fluorescence clearly localized specifically in the cell nucleus (Fig. 2, C–F) in a pattern consistent with the localization of DNA (see Fig. 2, E and F). We conclude that injected H2A.Z mRNA is translated into protein early in development but is not targeted to chromatin until gastrulation, after zygotic transcription and asynchronous cell divisions commence. Consistent with this result, H2A.F/Z zebrafish embryos displayed similar developmental localization patterns (33). These results therefore support our hypothesis that H2A.Z is required at gastrulation perhaps to initiate or maintain a particular developmental process. In agreement with this conclusion, endogenous H2A.Z expression peaks at gastrulation (23).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 2.
H2A.Z/EGFP fusion protein expression commences before the MBT. Xenopus embryos were injected with 1 ng of H2A.Z/EGFP RNA. A, at stage 5–6 green EGFP cannot be detected. B, by stage 7–8 green fusion protein shows diffuse, cytoplasmic localization pattern visible using fluorescent light and a Leica GFPII filter set. C–F, after the MBT H2A.Z/EGFP is localized to the nucleus. C and D, shows transmitted and fluorescent light with GFP II filter set for cell orientation (C) and fluorescent light alone (D). E and F, another view of a post-MBT embryo illustrating the localization of H2A.Z/EGFP to chromatin during cell division. E and F show transmitted and fluorescent light with GFP II filter set for cell orientation (E) and fluorescent light alone (F).

 
H2A.Z Overexpression Slows Developmental Progression after Gastrulation—Interestingly, overexpression of wild-type H2A.Z in Xenopus embryos, by RNA injection into the two-cell stage embryo, delayed development compared with uninjected or H2A-injected control embryos (Fig. 3). Following in vitro fertilization, development proceeded normally in the H2A.Z-injected embryos until the gastrula stage (Fig. 3F) after which a delay of approximately two developmental stages was observed (Fig. 3, I and L). Despite the delay, embryos were morphologically indistinguishable from control embryos to the swimming tadpole stage (data not shown) suggesting that they are competent to develop normally. Next we investigated whether changing the extended acidic patch of H2A.Z back to H2A would generate a dominant-negative phenotype (H2A.ZNK; Fig. 4, B and D, panels E and F). Although no dominant-negative phenotype was observed, this mutant did not show the pronounced developmental delay observed with the overexpression of wild-type H2A.Z (Fig. 4D, panels C and D). This raises the possibility that H2A.ZNK mimics the core H2A histone (Fig. 3) and that the acidic patch functionally distinguishes H2A.Z from H2A. In vitro structural analyses led us to suggest that the extended acidic patch of H2A.Z is responsible for the ability of H2A.Z to produce a more compact secondary chromatin structure (34).3



View larger version (141K):
[in this window]
[in a new window]
  		FIG. 3.
H2A.Z overexpression delays Xenopus development at gastrulation. Xenopus embryos were injected as described in the legend to Fig. 2. A, D, G, and J show normal development in uninjected control embryos. B, E, H, and K show embryos injected with H2A RNA, and C, F, I, and L show embryos injected with H2A.Z RNA. Stages of development are given at the left.

 



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 4.
H2A.Z dominant-negative proteins perturb development after gastrulation. A, amino acid sequence and secondary structure of the H2A.Z C terminus. Amino acid residues differing from H2A are given in red. The black bar shows the docking domain including the essential M6 and M7 regions. The diagram was modified from Rangasamy et al. (10). B, schematic diagram of proteins overexpressed in Xenopus embryos. The upper gray bar illustrates H2A protein. C-terminal {alpha}-helix is indicated above bar. White bars depict H2A.Z and H2A.Z derivative proteins. Relevant amino acid residues are shown in bold black letters. H2A.Z derivatives are listed to the left and described under "Experimental Procedures." C, graph of embryo survival and developmental defects. Embryos were injected as described in the legend to Fig. 2. RNA injected is indicated at the left and corresponds to the proteins illustrated in B. The gray histogram shows the percentage of embryos surviving to the tailbud stage, and the black histogram shows the percentage of embryos with developmental defect evident at gastrulation. The total number of embryos counted for each variable is indicated at the right. Results are pooled from a number of experiments. D, morphology of embryos at gastrulation and tailbud stages of development. RNA injected is given at the left. Embryo morphology was monitored at gastrulation (panels A, C, E, G, and I) and tailbud (panels B, D, F, H, and J). Dorsal views of defective tailbud embryos are shown in panels H and J. E, sequence comparison of H2A.Z protein from different species. The two histidine residues required for normal development are highlighted in bold, the M6 and M7 essential domains for Drosophila development are boxed, and the docking domain is indicated with a dashed line. Sequences were from the following: X. laevis H2A and mouse H2A.Z (24), X. laevis H2A.Z1 (23), Drosophila H2A.vD (14), Tetrahymena hv1 (42), sea urchin H2A.F/Z (43), S. pombe pht1 (12), and Saccharomyces cerevisiae HTZ1 (44).

 

The Unique H2A.Z Containing Nucleosomal Surface Is Essential for Correct Mesoderm Development—Based on our predicted functionally significant regions of the protein, we made other mutations in H2A.Z cDNA that may be important to its activity in vivo during X. laevis early development (Fig. 4, A and B). Injection of RNA expressing some H2A.Z mutant proteins produced a striking defect beginning at gastrulation and resulting in aberrant development and failure of the blastopore to close (Fig. 4, C and D). It is worth noting that the RNA interference and dominant-negative approaches yielded a defect in only a fraction of injected embryos (as observed in other studies using a similar strategy (28, 30, 35)), since injected RNA levels were intentionally kept to a minimum to eliminate nonspecific effects. Significantly, this defect was uniform for all mutant embryos and was similar to the phenotype observed using RNA interference. Embryos ultimately displayed a shortened trunk and opening of varying size in their dorsal surface (Fig. 4D, panels H and J). The "H2A.Z dominant-negative" phenotype was only observed with a subset of mutations suggesting that they have lost functionally important regions of H2A.Z.

The most significant mutations were in the two histidine residues that lie in the proposed region for protein/protein interactions (24). When one (H2A.ZNH) or both (H2A.ZNQ) of these residues are altered, it gives rise to the dominant-negative phenotype in over 50% of embryos (Fig. 4C). Interestingly, these histidine residues in H2A.Z are not found in yeast (Fig. 4E). Since these sites are functionally relevant in X. laevis, they may define a distinct activity for development of higher eukaryotes.

The second mutation, in which a region encompassing the {alpha}-helix of H2A.Z was replaced with the equivalent region of H2A (H2A.ZCS), produced a dominant-negative phenotype in ~40% of embryos. In Drosophila this mutant protein could not rescue the H2A.Z null phenotype, and flies died early in development (14). We conclude that two regions that include the C-terminal {alpha}-helix and the two histidine residues, located on the surface of an H2A.Z nucleosome, are vital for H2A.Z function, and overexpression of these mutant proteins interferes with X. laevis development in a tissue-specific and developmentally specific manner.

H2A.Z Is Enriched in Tissues of Chordamesodermal Origin—The ability of some H2A.Z mutants to generate a uniform dominant-negative phenotype affecting mesoderm formation raises the possibility that it plays a specific role in the development of these tissues. If this is the case H2A.Z expression may be regulated differently in mesoderm specific cells. Previous reports of developmental H2A.Z expression patterns in X. laevis embryos showed that H2A.Z RNA is present in oocytes, but on fertilization H2A.Z is poorly expressed until gastrulation when it has a ubiquitous high expression profile (23). However, when we investigated the localization of endogenous H2A.Z RNA in more detail, we found that it is clearly enriched in specialized tissues (Fig. 5 compare A–E to F, G, I, and J). X. laevis H2A.Z RNA is preferentially expressed in the notochord and defined regions of the primitive ear. This pattern corresponds to tissues of mesoderm origin that arise from convergent extension during gastrulation.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 5.
Endogenous H2A.Z is enriched in specific tissues. A–E, embryos hybridized with digoxigenin-labeled H2A.Z antisense RNA as described under "Experimental Procedures" with the time of color development given in parentheses. A, whole embryo (17 min). B, head (39 min) showing localization to notochord and otic vesicle. C, transverse section through otic vesicles (39 min). D, transverse section through mid-trunk (39 min). E, exposed notochord (37 min). F–J, control embryos exposed to anti-digoxygenin-AP fab fragments only. Control embryos demonstrate nonspecific staining that develops with longer periods of color development. F, whole embryo (2 h). G, head (2 h). H, diagram illustrating where transverse sections were cut. I, transverse section through mid-trunk (3 h). J, exposed notochord (1.25 h).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Results presented here show enrichment of an H2A histone variant within a specific tissue and its necessity for normal development. Perturbation of H2A.Z by an RNA interference and an overexpression and dominant-negative approach demonstrate that this variant is required at gastrulation for correct mesoderm formation. Notably, a region on the surface of the H2A.Z containing nucleosome is critical, providing a vital link between a specific structural chromatin remodelling event and a developmental pathway. The nature of the unique H2A.Z containing nucleosomal surface suggests that H2A.Z acts by recruiting nuclear proteins or by altering nucleosome/nucleosome interactions. In support of this hypothesis, mutation of a single histidine residue within the surface domain gives rise to a uniform developmental defect in mesoderm formation. The essential histidine residues are not present in yeast H2A.Z suggesting that H2A.Z may have evolved additional roles necessary for development in higher eukaryotes. One role requires the NK residues on the acidic patch that we have recently shown can modify the structure of nucleosomal arrays.3 We believe that this acidic patch region is important for heterochromatin formation and thus necessary for proper chromosome segregation. Here we identify a unique surface domain that is required for a second role being vital for early development.

Gastrulation is a critical stage in mesoderm development when two pathways of mesoderm progression are defined. The regions that involute first form the prechordal mesoderm pathway and the later involuting regions form the chordamesoderm, which gives rise to the notocord. The prechordal mesoderm cells develop by active cell migration leading to the formation of the head region, whereas chordamesoderm progresses by convergent extension resulting in the movement of cells to define the anterioposterior axis and the notocord (36–38). Recent evidence implicates the transcription factor Xenopus brachyury as an essential factor for convergent extension that actively inhibits cell migration and thereby distinguishes these two processes during gastrulation (39). Our results demonstrate that H2A.Z is enriched in chordamesoderm (Fig. 5). One attractive possibility is that H2A.Z plays a regulatory role in the expression of factors, such as Xenopus brachyury, that are vital for correct convergent extension. Alternatively, since overexpression of H2A.Z slows development (Fig. 3), regulating the level of H2A.Z expression in a particular cell type may control the rate of cellular proliferation that is intimately linked to cellular differentiation perhaps by increasing heterochromatin formation. Overexpression of the H2A.Z dominant-negative proteins may thereby interfere with normal development primarily in the cell lineage that expresses endogenous H2A.Z at a high level. Importantly, our experimental evidence clearly demonstrates that general chromatin components can be specifically involved in pattern-formation processes during early X. laevis development.

Our work in mouse embryos and mammalian tissue culture cells demonstrates that H2A.Z has an essential role in chromosome segregation (10, 11). In contrast to early mouse development, where H2A.Z is transcribed from the 2-cell stage,4 Xenopus H2A.Z expression peaks at gastrulation indicating that H2A.Z may have a specialized role in Xenopus. When we deplete or overexpress dominant-negative H2A.Z protein in Xenopus embryos, developmental perturbations are not evident until after gastrulation once asynchronous cell divisions are established. Possibly H2A.Z is not required for early cleavage divisions and only becomes necessary at the onset of asynchronous divisions when the cell cycle lengthens. The diffuse localization of the H2A.Z/EGFP fusion protein during the early cleavage divisions is consistent with this notion. Also consistent are reports that H2A.Z protein levels are elevated in terminally differentiated cells when the cell cycle slows (40) and reduced during rapid proliferation in cells of the mouse embryo inner cell mass (10). It is important to point out that cells of the axial mesoderm (notocord) also display characteristics of terminally differentiated cells (41). In conclusion, this study demonstrates that a chromatin protein has a tissue-specific role in complex patterning during early development.


   FOOTNOTES
 
* This work was supported by Australian National Health and Medical Research Project Grants 179823 (to P. R. and D. J. T.) and 224226 (to D. R. and D. J. T.) and Australian Academy of Science visits to Europe Grant RI 146.1 (to P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{diamondsuit} This article was selected as a Paper of the Week. Back

{ddagger} To whom correspondence may be addressed. Tel.: 61-2-61252326; Fax: 61-2-6125-0415; E-mail: David.Tremethick{at}anu.edu.au or Pat. Ridgway{at}anu.edu.au.

1 The abbreviations used are: RNAi, RNA interference; MBT, midblastula transition; GFP, green fluorescent protein; EGFP, enhanced GFP. Back

2 K. Brown, personal communication. Back

3 J. Y. Fan, D. Rangasamy, K. Luger, and D. J. Tremethick, submitted for publication. Back

4 D. Tremethick, unpublished data. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge J-P. Quivy and G. Almouzni for their ongoing intellectual input and exchange of ideas. We also thank E. Ball for advice with in situ hybridization.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sullivan, K. F. (2001) Curr. Opin. Genet. Dev. 11, 182–188[Medline] [Order article via Infotrieve]
  2. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000) Curr. Biol. 10, 886–895[CrossRef][Medline] [Order article via Infotrieve]
  3. Chadwick, B. P., and Willard, H. F. (2001) J. Cell Biol. 152, 375–384[Abstract/Free Full Text]
  4. Costanzi, C., Stein, P., Worrad, D. M., Schultz, R. M., and Pehrson, J. R. (2000) Development (Camb.) 127, 2283–2289[Abstract]
  5. Allis, C. D., Glover, C. V., Bowen, J. K., and Gorovsky, M. A. (1980) Cell 20, 609–617[CrossRef][Medline] [Order article via Infotrieve]
  6. Allis, C. D., Ziegler, Y. S., Gorovsky, M. A., and Olmsted, J. B. (1982) Cell 31, 131–136[CrossRef][Medline] [Order article via Infotrieve]
  7. Santisteban, M. S., Kalashnikova, T., and Smith, M. M. (2000) Cell 103, 411–422[CrossRef][Medline] [Order article via Infotrieve]
  8. Dhillon, N., and Kamakaka, R. T. (2000) Mol. Cell 6, 769–780[CrossRef][Medline] [Order article via Infotrieve]
  9. Meneghini, M. D., Wu, M., and Madhani, H. D. (2003) Cell 112, 725–736[CrossRef][Medline] [Order article via Infotrieve]
  10. Rangasamy, D., Berven, L., Ridgway, P., and Tremethick, D. J. (2003) EMBO J. 22, 1599–1607[CrossRef][Medline] [Order article via Infotrieve]
  11. Rangasamy, D., Greaves, I., and Tremethick, D. J. (2004) Nat. Struct. Mol. Biol. 11, 650–655[CrossRef][Medline] [Order article via Infotrieve]
  12. Carr, A. M., Dorrington, S. M., Hindley, J., Phear, G. A., Aves, S. J., and Nurse, P. (1994) Mol. Gen. Genet 245, 628–635[Medline] [Order article via Infotrieve]
  13. van Daal, A., and Elgin, S. C. (1992) Mol. Biol. Cell 3, 593–602[Abstract]
  14. Clarkson, M. J., Wells, J. R., Gibson, F., Saint, R., and Tremethick, D. J. (1999) Nature 399, 694–697[CrossRef][Medline] [Order article via Infotrieve]
  15. Faast, R., Thonglairoam, V., Schulz, T. C., Beall, J., Wells, J. R., Taylor, H., Matthaei, K., Rathjen, P. D., Tremethick, D. J., and Lyons, I. (2001) Curr. Biol. 11, 1183–1187[CrossRef][Medline] [Order article via Infotrieve]
  16. Hatch, C. L., and Bonner, W. M. (1996) Biochim. Biophys. Acta 1305, 59–62[Medline] [Order article via Infotrieve]
  17. Newport, J., and Kirschner, M. (1982) Cell 30, 675–686[CrossRef][Medline] [Order article via Infotrieve]
  18. Newport, J., and Kirschner, M. (1982) Cell 30, 687–696[CrossRef][Medline] [Order article via Infotrieve]
  19. Almouzni, G., and Wolffe, A. P. (1995) EMBO J. 14, 1752–1765[Medline] [Order article via Infotrieve]
  20. Prioleau, M. N., Huet, J., Sentenac, A., and Mechali, M. (1994) Cell 77, 439–449[CrossRef][Medline] [Order article via Infotrieve]
  21. Steinbach, O. C., Wolffe, A. P., and Rupp, R. A. (1997) Nature 389, 395–399[CrossRef][Medline] [Order article via Infotrieve]
  22. Vermaak, D., Steinbach, O. C., Dimitrov, S., Rupp, R. A., and Wolffe, A. P. (1998) Curr. Biol. 8, 533–536[CrossRef][Medline] [Order article via Infotrieve]
  23. Iouzalen, N., Moreau, J., and Mechali, M. (1996) Nucleic Acids Res. 24, 3947–3952[Abstract/Free Full Text]
  24. Suto, R. K., Clarkson, M. J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121–1124[CrossRef][Medline] [Order article via Infotrieve]
  25. Zernicka-Goetz, M., Pines, J., Ryan, K., Siemering, K. R., Haseloff, J., Evans, M. J., and Gurdon, J. B. (1996) Development (Camb.) 122, 3719–3724[Abstract]
  26. Ridgway, P., Rangasamy, D., Berven, L., Svensson, U., and Tremethick, D. J. (2004) Methods Enzymol. 375, 239–252[CrossRef][Medline] [Order article via Infotrieve]
  27. Nieuwkoop, P. D., and Faber, J. (eds) (1994) Normal Table of Xenopus laevis (Daudin), 3rd Ed., Garland Publishing, Inc., New York
  28. Sive, H. L., Grainger, R. M., and Harland, R. M. (2000) Early Development of Xenopus laevis: A Laboratory Mannual, pp. 39–79; 249–274, Cold Spring Harbour Laboratory Press, New York
  29. Hayward, D. C., Catmull, J., Reece-Hoyes, J. S., Berghammer, H., Dodd, H., Hann, S. J., Miller, D. J., and Ball, E. E. (2001) Dev. Genes Evol. 211, 10–19[CrossRef][Medline] [Order article via Infotrieve]
  30. Nakano, H., Amemiya, S., Shiokawa, K., and Taira, M. (2000) Biochem. Biophys. Res. Commun. 274, 434–439[CrossRef][Medline] [Order article via Infotrieve]
  31. Zhou, Y., Ching, Y. P., Kok, K. H., Kung, H. F., and Jin, D. Y. (2002) Nucleic Acids Res. 30, 1664–1669[Abstract/Free Full Text]
  32. Lemaitre, J. M., Geraud, G., and Mechali, M. (1998) J. Cell Biol. 142, 1159–1166[Abstract/Free Full Text]
  33. Pauls, S., Geldmacher-Voss, B., and Campos-Ortega, J. A. (2001) Dev. Genes Evol. 211, 603–610[CrossRef][Medline] [Order article via Infotrieve]
  34. Fan, J. Y., Gordon, F., Luger, K., Hansen, J. C., and Tremethick, D. J. (2002) Nat. Struct. Biol. 9, 172–176[Medline] [Order article via Infotrieve]
  35. Glavic, A., Maris Honore, S., Gloria Feijoo, C., Bastidas, F., Allende, M. L., and Mayor, R. (2004) Dev. Biol. 272, 89–103[CrossRef][Medline] [Order article via Infotrieve]
  36. Gerhart, J., and Keller, R. (1986) Annu. Rev. Cell Biol. 2, 201–229[CrossRef][Medline] [Order article via Infotrieve]
  37. Winklbauer, R. (1990) Dev. Biol. 142, 155–168[CrossRef][Medline] [Order article via Infotrieve]
  38. Keller, R., and Winklbauer, R. (1992) Curr. Top. Dev. Biol. 27, 39–89[Medline] [Order article via Infotrieve]
  39. Kwan, K. M., and Kirschner, M. W. (2003) Development (Camb.) 130, 1961–1972[Abstract/Free Full Text]
  40. Wunsch, A. M., Reinhardt, K., and Lough, J. (1991) Mech. Ageing Dev. 59, 299–305[CrossRef][Medline] [Order article via Infotrieve]
  41. Saka, Y., and Smith, J. C. (2001) Dev. Biol. 229, 307–318[CrossRef][Medline] [Order article via Infotrieve]
  42. White, E. M., Shapiro, D. L., Allis, C. D., and Gorovsky, M. A. (1988) Nucleic Acids Res. 16, 179–198[Abstract/Free Full Text]
  43. Ernst, S. G., Miller, H., Brenner, C. A., Nocente-McGrath, C., Francis, S., and McIsaac, R. (1987) Nucleic Acids Res. 15, 4629–4644[Abstract/Free Full Text]
  44. Jackson, J. D., Falciano, V. T., and Gorovsky, M. A. (1996) Trends Biochem. Sci. 21, 466–467[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
I. K. Greaves, D. Rangasamy, M. Devoy, J. A. Marshall Graves, and D. J. Tremethick
The X and Y Chromosomes Assemble into H2A.Z, Containing Facultative Heterochromatin, following Meiosis.
Mol. Cell. Biol., July 1, 2006; 26(14): 5394 - 5405.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Gordon, K. Luger, and J. C. Hansen
The Core Histone N-terminal Tail Domains Function Independently and Additively during Salt-dependent Oligomerization of Nucleosomal Arrays
J. Biol. Chem., October 7, 2005; 280(40): 33701 - 33706.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
R. T. Kamakaka and S. Biggins
Histone variants: deviants?
Genes & Dev., February 1, 2005; 19(3): 295 - 316.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/42/43815    most recent
M408409200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ridgway, P.
Right arrow Articles by Tremethick, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ridgway, P.
Right arrow Articles by Tremethick, D. J.
Related Collections
Right arrow Papers Of The Week
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement