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Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2.

Horikoshi N, Sato K, Shimada K, Arimura Y, Osakabe A, Tachiwana H, Hayashi-Takanaka Y, Iwasaki W, Kagawa W, Harata M, Kimura H, Kurumizaka H - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical.It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2.These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

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Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.

ABSTRACT
The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

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The mobilities of H2A.Z.1 and H2A.Z.2 are different in HeLa cells. (a) GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S and GFP-H2A were stably expressed in HeLa cells. Fluorescence images of HeLa cells stably expressing GFP-H2A (clone 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S are presented in the upper panels. The scale bar indicates 10 µm. The lower panel shows the distribution of the fluorescence intensities of GFP-H2A (clones 4 and 6), GFP-H2A.Z.1 (clones 2 and 5), GFP-H2A.Z.2 (clones 3 and 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S represented in arbitrary units. (b) HeLa cells expressing GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A, GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S were subjected to FRAP analysis. The mobility of GFP-histones in living cells was analyzed by bleaching one-half of the nucleus in the presence of 100 µg ml−1 cycloheximide. Representative images before bleaching (left column), upon bleaching (0 min, centre column) and 180 min after bleaching (right column) are shown. The images for GFP-H2A, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in the top, middle and bottom rows, respectively. The scale bar indicates 10 µm. (c) The average relative fluorescence intensities of the bleached areas were plotted with their standard deviations (n = 11–36). The FRAP curves of GFP-H2A.Z.1, GFP-H2A.Z.2 and GFP-H2A are presented in blue, magenta and green, respectively. (d) Salt-resistance assay. The H2A nucleosomes (lanes 1–4), H2A.Z.1 nucleosomes (lanes 5–8) or H2A.Z.2 nucleosomes (lanes 9–12) were incubated in the presence of 0.4 M (lanes 1, 5 and 9), 0.6 M (lanes 2, 6 and 10), 0.7 M (lanes 3, 7 and 11) and 0.8 M NaCl (lanes 4, 8 and 12) at 328 K for 1 h. The samples were then analyzed by nondenaturing 6% PAGE with ethidium bromide staining. Bands corresponding to nucleosome monomers and nucleosome–nucleosome aggregates are indicated. Asterisks represent bands corresponding to non-nucleosomal DNA–histone complexes. (e) FRAP analysis of the H2A.Z.1 S38T and H2A.Z.2 T38S mutants. The average relative fluorescence intensities of the bleached areas were plotted with the standard deviations (n = 10–15). The FRAP curves of GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in dark blue, red, blue and magenta, respectively.
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fig4: The mobilities of H2A.Z.1 and H2A.Z.2 are different in HeLa cells. (a) GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S and GFP-H2A were stably expressed in HeLa cells. Fluorescence images of HeLa cells stably expressing GFP-H2A (clone 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S are presented in the upper panels. The scale bar indicates 10 µm. The lower panel shows the distribution of the fluorescence intensities of GFP-H2A (clones 4 and 6), GFP-H2A.Z.1 (clones 2 and 5), GFP-H2A.Z.2 (clones 3 and 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S represented in arbitrary units. (b) HeLa cells expressing GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A, GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S were subjected to FRAP analysis. The mobility of GFP-histones in living cells was analyzed by bleaching one-half of the nucleus in the presence of 100 µg ml−1 cycloheximide. Representative images before bleaching (left column), upon bleaching (0 min, centre column) and 180 min after bleaching (right column) are shown. The images for GFP-H2A, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in the top, middle and bottom rows, respectively. The scale bar indicates 10 µm. (c) The average relative fluorescence intensities of the bleached areas were plotted with their standard deviations (n = 11–36). The FRAP curves of GFP-H2A.Z.1, GFP-H2A.Z.2 and GFP-H2A are presented in blue, magenta and green, respectively. (d) Salt-resistance assay. The H2A nucleosomes (lanes 1–4), H2A.Z.1 nucleosomes (lanes 5–8) or H2A.Z.2 nucleosomes (lanes 9–12) were incubated in the presence of 0.4 M (lanes 1, 5 and 9), 0.6 M (lanes 2, 6 and 10), 0.7 M (lanes 3, 7 and 11) and 0.8 M NaCl (lanes 4, 8 and 12) at 328 K for 1 h. The samples were then analyzed by nondenaturing 6% PAGE with ethidium bromide staining. Bands corresponding to nucleosome monomers and nucleosome–nucleosome aggregates are indicated. Asterisks represent bands corresponding to non-nucleosomal DNA–histone complexes. (e) FRAP analysis of the H2A.Z.1 S38T and H2A.Z.2 T38S mutants. The average relative fluorescence intensities of the bleached areas were plotted with the standard deviations (n = 10–15). The FRAP curves of GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in dark blue, red, blue and magenta, respectively.

Mentions: We next examined the mobilities of H2A.Z.1 and H2A.Z.2 as GFP-fusion proteins in living cells by fluorescence recovery after photobleaching (FRAP; Kimura, 2005 ▶). The canonical H2A tagged with GFP was used as a control. HeLa cells stably expressing GFP-fused H2A (clones 4 and 6), H2A.Z.1 (clones 2 and 5) or H2A.Z.2 (clones 3 and 4) were generated. The fluorescence intensity measurements indicated that the expression levels among the different GFP-histones were similar (Fig. 4 ▶a), and cells showing similar fluorescence intensities were used for the FRAP experiments. As previously reported, the fluorescence of GFP-H2A in the bleached area recovered slowly, consistent with its incorporation into nucleosomes in living cells (Figs. 4 ▶b and 4 ▶c; Kimura & Cook, 2001 ▶; Gautier et al., 2004 ▶; Bönisch et al., 2012 ▶). Interestingly, the fluorescence recovery of GFP-H2A.Z.1 was substantially faster than that of GFP-H2A (Figs. 4 ▶b and 4 ▶c). This suggested that in living cells the nucleosomal H2A.Z.1 is more rapidly exchanged than the canonical H2A. Consistently, a salt-resistance assay revealed that the reconstituted H2A.Z.1 nucleosome was unstable compared with the canonical H2A nucleosome (Fig. 4 ▶d). However, the fluorescence recovery of GFP-H2A.Z.2 was almost the same as that of GFP-H2A (Figs. 4 ▶b and 4 ▶c), although the stability of the reconstituted H2A.Z.2 nucleosome was clearly different from that of the canonical H2A nucleosome (Fig. 4 ▶d). Therefore, the mobilities of H2A.Z.1 and H2A.Z.2 may be independently regulated in living cells, probably by histone chaperones and/or nucleosome re­modellers that are specific for H2A.Z.1 and H2A.Z.2.


Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2.

Horikoshi N, Sato K, Shimada K, Arimura Y, Osakabe A, Tachiwana H, Hayashi-Takanaka Y, Iwasaki W, Kagawa W, Harata M, Kimura H, Kurumizaka H - Acta Crystallogr. D Biol. Crystallogr. (2013)

The mobilities of H2A.Z.1 and H2A.Z.2 are different in HeLa cells. (a) GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S and GFP-H2A were stably expressed in HeLa cells. Fluorescence images of HeLa cells stably expressing GFP-H2A (clone 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S are presented in the upper panels. The scale bar indicates 10 µm. The lower panel shows the distribution of the fluorescence intensities of GFP-H2A (clones 4 and 6), GFP-H2A.Z.1 (clones 2 and 5), GFP-H2A.Z.2 (clones 3 and 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S represented in arbitrary units. (b) HeLa cells expressing GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A, GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S were subjected to FRAP analysis. The mobility of GFP-histones in living cells was analyzed by bleaching one-half of the nucleus in the presence of 100 µg ml−1 cycloheximide. Representative images before bleaching (left column), upon bleaching (0 min, centre column) and 180 min after bleaching (right column) are shown. The images for GFP-H2A, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in the top, middle and bottom rows, respectively. The scale bar indicates 10 µm. (c) The average relative fluorescence intensities of the bleached areas were plotted with their standard deviations (n = 11–36). The FRAP curves of GFP-H2A.Z.1, GFP-H2A.Z.2 and GFP-H2A are presented in blue, magenta and green, respectively. (d) Salt-resistance assay. The H2A nucleosomes (lanes 1–4), H2A.Z.1 nucleosomes (lanes 5–8) or H2A.Z.2 nucleosomes (lanes 9–12) were incubated in the presence of 0.4 M (lanes 1, 5 and 9), 0.6 M (lanes 2, 6 and 10), 0.7 M (lanes 3, 7 and 11) and 0.8 M NaCl (lanes 4, 8 and 12) at 328 K for 1 h. The samples were then analyzed by nondenaturing 6% PAGE with ethidium bromide staining. Bands corresponding to nucleosome monomers and nucleosome–nucleosome aggregates are indicated. Asterisks represent bands corresponding to non-nucleosomal DNA–histone complexes. (e) FRAP analysis of the H2A.Z.1 S38T and H2A.Z.2 T38S mutants. The average relative fluorescence intensities of the bleached areas were plotted with the standard deviations (n = 10–15). The FRAP curves of GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in dark blue, red, blue and magenta, respectively.
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fig4: The mobilities of H2A.Z.1 and H2A.Z.2 are different in HeLa cells. (a) GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S and GFP-H2A were stably expressed in HeLa cells. Fluorescence images of HeLa cells stably expressing GFP-H2A (clone 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S are presented in the upper panels. The scale bar indicates 10 µm. The lower panel shows the distribution of the fluorescence intensities of GFP-H2A (clones 4 and 6), GFP-H2A.Z.1 (clones 2 and 5), GFP-H2A.Z.2 (clones 3 and 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S represented in arbitrary units. (b) HeLa cells expressing GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A, GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S were subjected to FRAP analysis. The mobility of GFP-histones in living cells was analyzed by bleaching one-half of the nucleus in the presence of 100 µg ml−1 cycloheximide. Representative images before bleaching (left column), upon bleaching (0 min, centre column) and 180 min after bleaching (right column) are shown. The images for GFP-H2A, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in the top, middle and bottom rows, respectively. The scale bar indicates 10 µm. (c) The average relative fluorescence intensities of the bleached areas were plotted with their standard deviations (n = 11–36). The FRAP curves of GFP-H2A.Z.1, GFP-H2A.Z.2 and GFP-H2A are presented in blue, magenta and green, respectively. (d) Salt-resistance assay. The H2A nucleosomes (lanes 1–4), H2A.Z.1 nucleosomes (lanes 5–8) or H2A.Z.2 nucleosomes (lanes 9–12) were incubated in the presence of 0.4 M (lanes 1, 5 and 9), 0.6 M (lanes 2, 6 and 10), 0.7 M (lanes 3, 7 and 11) and 0.8 M NaCl (lanes 4, 8 and 12) at 328 K for 1 h. The samples were then analyzed by nondenaturing 6% PAGE with ethidium bromide staining. Bands corresponding to nucleosome monomers and nucleosome–nucleosome aggregates are indicated. Asterisks represent bands corresponding to non-nucleosomal DNA–histone complexes. (e) FRAP analysis of the H2A.Z.1 S38T and H2A.Z.2 T38S mutants. The average relative fluorescence intensities of the bleached areas were plotted with the standard deviations (n = 10–15). The FRAP curves of GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in dark blue, red, blue and magenta, respectively.
Mentions: We next examined the mobilities of H2A.Z.1 and H2A.Z.2 as GFP-fusion proteins in living cells by fluorescence recovery after photobleaching (FRAP; Kimura, 2005 ▶). The canonical H2A tagged with GFP was used as a control. HeLa cells stably expressing GFP-fused H2A (clones 4 and 6), H2A.Z.1 (clones 2 and 5) or H2A.Z.2 (clones 3 and 4) were generated. The fluorescence intensity measurements indicated that the expression levels among the different GFP-histones were similar (Fig. 4 ▶a), and cells showing similar fluorescence intensities were used for the FRAP experiments. As previously reported, the fluorescence of GFP-H2A in the bleached area recovered slowly, consistent with its incorporation into nucleosomes in living cells (Figs. 4 ▶b and 4 ▶c; Kimura & Cook, 2001 ▶; Gautier et al., 2004 ▶; Bönisch et al., 2012 ▶). Interestingly, the fluorescence recovery of GFP-H2A.Z.1 was substantially faster than that of GFP-H2A (Figs. 4 ▶b and 4 ▶c). This suggested that in living cells the nucleosomal H2A.Z.1 is more rapidly exchanged than the canonical H2A. Consistently, a salt-resistance assay revealed that the reconstituted H2A.Z.1 nucleosome was unstable compared with the canonical H2A nucleosome (Fig. 4 ▶d). However, the fluorescence recovery of GFP-H2A.Z.2 was almost the same as that of GFP-H2A (Figs. 4 ▶b and 4 ▶c), although the stability of the reconstituted H2A.Z.2 nucleosome was clearly different from that of the canonical H2A nucleosome (Fig. 4 ▶d). Therefore, the mobilities of H2A.Z.1 and H2A.Z.2 may be independently regulated in living cells, probably by histone chaperones and/or nucleosome re­modellers that are specific for H2A.Z.1 and H2A.Z.2.

Bottom Line: The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical.It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2.These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.

ABSTRACT
The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

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