Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development

Abstract

Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.

Fig. 1. H2A.Z expression is switched on when the ICM begins to differentiate. (A) Early mouse embryos were stained with phallodium (red) and immunostained with H2A.Z antibodies (green) or co-immunostained with diacetylated histone H3 (red) and H2A.Z (green) antibodies. Both antibodies were used at a 1:1000 dilution. The centre and right panels display two different confocal views highlighting trophoblast nuclei, and the ICM plus endoderm nuclei, respectively. The absence of H2A.Z at the ICM (red) and the co-localization of H2A.Z and diacetylated histone H3 at trophoblast and endoderm cells (yellow) can be seen clearly. (B) Immunostaining of ICM and endoderm nuclei at the periphery of the ICM with H2A.Z antibodies. An ICM nucleus (cell-1), an endoderm nucleus (cell-3) and an apparent differentiation intermediate between these two cell types (cell-2) are shown. Other cells in this image, which are also located at the edge of the ICM mound, are not in the same confocal plane. The right panels show cell-2, and a similar cell type, co-immunostained with H2A.Z (green) and diacetylated histone H3 (red) antibodies.

Fig. 2. Analysis of H2A.Z and H2A transcript levels in the ICM. Transcript levels of β-actin, endo-β-N-acetylglucosaminidase, fibroblast growth factor, H2A and H2A.Z were determined by semi-quantitative RT–PCR as described in Materials and methods. Approximately 32 whole embryos, 90 ICMs and 130 trophoblast samples were used to yield approximately the same total number of cells. Rabbit α-globin mRNA was used as an internal control to correct for differences between samples in RNA recovery and efficiency of RT–PCR.

Fig. 3. H2A.Z and HP1-α co-localize at pericentric heterochromatin. Early mouse embryos were immunostained with H2A.Z (1:1000), HP1-α (1:250), and CREST (1:500) antibodies or stained with propidium iodide (PI). The images show extraembryonic endoderm cells (A, D and F) and trophoblast cells (B, C, E and G). For the merged panel, the first and second panels are artificially coloured red and green, respectively. Undifferentiated cells of the ICM are also seen next to endoderm cells in (D, upper right).

Fig. 4. H2A is not enriched at pericentric heterochromatin. Mouse trophoblast cells were immunostained with H2A (1:1000), HP1-α (1:250) and CREST (1:500) antibodies, and the DNA was stained with PI. For the merged panel, the first and second panels are artificially coloured red and green, respectively.

Fig. 5. H2A.Z and macroH2A1.2 do not co-localize. Mouse trophoblast cells were co-immunostained with H2A.Z (1:1000) and macroH2A1.2 (1:100) (Costanzi et al., 2000) antibodies. For the merged panel, the first and second panels are artificially coloured red and green, respectively.

Fig. 6. H2A.Z interacts with the pericentric heterochromatin binding protein INCENP. (A) Schematic showing the regions of INCENP shared by vertebrates (Uren et al., 2000; Adams et al., 2001b,c). (B) The interaction between H2AZ and INCENP, and various mutations of these two proteins, were analysed using a yeast two-hybrid approach and visualized using an agarose overlay assay. Undiluted cultures and cultures diluted 1 in 10 are shown. (C) The quantification of protein–protein interactions using a β-galactosidase liquid culture assay. The relative fold stimulation was calculated from the β-galactosidase activities of individual co-transformants using the equation [(pGBT7X + pACT2Y) – (pGBT7 + pACT2Y)] / (pGBT7X+pACT2), where X is H2AZ and Y is INCENP protein. The average of three independent experiments are shown. ‘Bait’ constructs included the GAL4 DNA binding domain alone (B.D.) or fused with H2A, H2A.Z, H2A.Z_ΔN (the N-terminus removed), H2A.Z_CS (the C-terminal α-helix of H2A.Z swapped with the equivalent region of H2A), or with H2A.Z_NQ (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). ‘Prey’ constructs included the activation domain by itself (A.D.) or fused with full-length INCENP, or with different deletion mutants. A ‘prey’ construct was also generated that contained the activation domain fused with HP1-α. (D) The amino acid sequence and secondary structure of mouse H2A.Z are shown. Amino acid residues that differ from H2A are shown in red. The docking domain, encompassing the essential M6 and M7 regions, is also shown. (E) Either GST, GST–H2A.Z, GST–H2A.Z_ΔN, GST–H2A.Z_CS or GST–H2A were expressed in E.coli, bound to glutathione–agarose and, following purification, incubated with in vitro-labelled translation products of INCENP and various deletion mutants. Histone H2B was included in all binding reactions.