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H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization.

Bönisch C, Schneider K, Pünzeler S, Wiedemann SM, Bielmeier C, Bocola M, Eberl HC, Kuegel W, Neumann J, Kremmer E, Leonhardt H, Mann M, Michaelis J, Schermelleh L, Hake SB - Nucleic Acids Res. (2012)

Bottom Line: Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain.Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus.Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, 80336 Munich, Germany.

ABSTRACT
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

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Z.2.2 localizes to the nucleus and is partially incorporated into chromatin. (A) Fluorescence imaging of stably transfected HeLa Kyoto cells shows nuclear localization of all GFP-H2A variants (middle). DNA was counterstained with DAPI (top). Overlay of both channels in color is shown at the bottom (Merge; GFP: green, DAPI: blue). Scale bar = 5 µm. (B) Deconvolved images of metaphase spreads of HeLa Kyoto cells stably expressing GFP-H2A variants (middle). Merged images in color are shown below (GFP: green; DAPI: blue). Scale bar = 10 µm. (C) Chromatin from HeLa Kyoto cells stably expressing GFP-Z.2.2 was digested with MNase followed by a purification of mononucleosomes using sucrose gradient centrifugation. Isolated DNA from subsequent sucrose gradient fractions was analyzed by agarose gel electrophoresis (left). Fractions containing pure mononucleosomes (marked with asterisk) were combined and analyzed by IB (right) using αGFP antibody for the presence of GFP-Z.2.2 (top), and αH3 (bottom).
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gks267-F2: Z.2.2 localizes to the nucleus and is partially incorporated into chromatin. (A) Fluorescence imaging of stably transfected HeLa Kyoto cells shows nuclear localization of all GFP-H2A variants (middle). DNA was counterstained with DAPI (top). Overlay of both channels in color is shown at the bottom (Merge; GFP: green, DAPI: blue). Scale bar = 5 µm. (B) Deconvolved images of metaphase spreads of HeLa Kyoto cells stably expressing GFP-H2A variants (middle). Merged images in color are shown below (GFP: green; DAPI: blue). Scale bar = 10 µm. (C) Chromatin from HeLa Kyoto cells stably expressing GFP-Z.2.2 was digested with MNase followed by a purification of mononucleosomes using sucrose gradient centrifugation. Isolated DNA from subsequent sucrose gradient fractions was analyzed by agarose gel electrophoresis (left). Fractions containing pure mononucleosomes (marked with asterisk) were combined and analyzed by IB (right) using αGFP antibody for the presence of GFP-Z.2.2 (top), and αH3 (bottom).

Mentions: In fluorescence microscopy, GFP-Z.2.2 exhibited a sole but rather diffuse nuclear distribution similar to GFP-Bbd, suggesting that both variants might have similar properties (Figure 2A). Additionally, GFP-Z.2.2 was detected in condensed mitotic chromosomes, with a faint residual staining in the surrounding area (Figure 2B), suggesting that it is incorporated into chromatin, although to a lesser extent than other GFP-H2A variants. To discriminate between a potential non-specific DNA binding and nucleosomal incorporation of Z.2.2 we purified mononucleosomes by sucrose gradient centrifugation. GFP-Z.2.2 was detected by IB in fractions containing mononucleosomes (Figure 2C), suggesting that Z.2.2 is indeed a nucleosomal constituent.Figure 2.


H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization.

Bönisch C, Schneider K, Pünzeler S, Wiedemann SM, Bielmeier C, Bocola M, Eberl HC, Kuegel W, Neumann J, Kremmer E, Leonhardt H, Mann M, Michaelis J, Schermelleh L, Hake SB - Nucleic Acids Res. (2012)

Z.2.2 localizes to the nucleus and is partially incorporated into chromatin. (A) Fluorescence imaging of stably transfected HeLa Kyoto cells shows nuclear localization of all GFP-H2A variants (middle). DNA was counterstained with DAPI (top). Overlay of both channels in color is shown at the bottom (Merge; GFP: green, DAPI: blue). Scale bar = 5 µm. (B) Deconvolved images of metaphase spreads of HeLa Kyoto cells stably expressing GFP-H2A variants (middle). Merged images in color are shown below (GFP: green; DAPI: blue). Scale bar = 10 µm. (C) Chromatin from HeLa Kyoto cells stably expressing GFP-Z.2.2 was digested with MNase followed by a purification of mononucleosomes using sucrose gradient centrifugation. Isolated DNA from subsequent sucrose gradient fractions was analyzed by agarose gel electrophoresis (left). Fractions containing pure mononucleosomes (marked with asterisk) were combined and analyzed by IB (right) using αGFP antibody for the presence of GFP-Z.2.2 (top), and αH3 (bottom).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3401452&req=5

gks267-F2: Z.2.2 localizes to the nucleus and is partially incorporated into chromatin. (A) Fluorescence imaging of stably transfected HeLa Kyoto cells shows nuclear localization of all GFP-H2A variants (middle). DNA was counterstained with DAPI (top). Overlay of both channels in color is shown at the bottom (Merge; GFP: green, DAPI: blue). Scale bar = 5 µm. (B) Deconvolved images of metaphase spreads of HeLa Kyoto cells stably expressing GFP-H2A variants (middle). Merged images in color are shown below (GFP: green; DAPI: blue). Scale bar = 10 µm. (C) Chromatin from HeLa Kyoto cells stably expressing GFP-Z.2.2 was digested with MNase followed by a purification of mononucleosomes using sucrose gradient centrifugation. Isolated DNA from subsequent sucrose gradient fractions was analyzed by agarose gel electrophoresis (left). Fractions containing pure mononucleosomes (marked with asterisk) were combined and analyzed by IB (right) using αGFP antibody for the presence of GFP-Z.2.2 (top), and αH3 (bottom).
Mentions: In fluorescence microscopy, GFP-Z.2.2 exhibited a sole but rather diffuse nuclear distribution similar to GFP-Bbd, suggesting that both variants might have similar properties (Figure 2A). Additionally, GFP-Z.2.2 was detected in condensed mitotic chromosomes, with a faint residual staining in the surrounding area (Figure 2B), suggesting that it is incorporated into chromatin, although to a lesser extent than other GFP-H2A variants. To discriminate between a potential non-specific DNA binding and nucleosomal incorporation of Z.2.2 we purified mononucleosomes by sucrose gradient centrifugation. GFP-Z.2.2 was detected by IB in fractions containing mononucleosomes (Figure 2C), suggesting that Z.2.2 is indeed a nucleosomal constituent.Figure 2.

Bottom Line: Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain.Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus.Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, 80336 Munich, Germany.

ABSTRACT
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

Show MeSH