Ridgway P et al. (2004), Unique residues on the H2A.Z containing nucleos...

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J Biol Chem 2004 Oct 15;27942:43815-20. doi: 10.1074/jbc.M408409200.

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Unique residues on the H2A.Z containing nucleosome surface are important for Xenopus laevis development.

Ridgway P , Brown KD , Rangasamy D , Svensson U , Tremethick DJ .

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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.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 h2az1 hvcn1 slc15a4 tbx2

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	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.
	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).
	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.
	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 -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).
	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).