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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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Identification of Z.2.2. (A) qPCR with cDNA from different human cell lines and tissues using primers specific for Z.2.1 and Z.2.2. Data were normalized to HPRT1 and HMBS expression levels. Controls generated without reverse transcriptase (no RT) were used to assess amplification threshold. Shown are the levels of Z.2.2 mRNA as percentages of total Z.2 transcripts (Z.2.1 + Z.2.2). For an evaluation of absolute expression levels see Supplementary Figure S1C and D. (B) Amino acid alignment of human Z.1, Z.2.1 and Z.2.2 proteins using ClustalW Alignment (MacVector 10.0.2). Identical amino acids are highlighted in dark gray, similar amino acids in light gray and changes are set apart on white background. Known acetylation sites are depicted with stars and ubiquitination sites with circles. A schematic representation of the secondary structure of Z.1 and Z.2.1 is shown below the alignment, including depiction of the H3/H4 docking domain (50). M6 and M7 boxes indicate regions important for H2A.Z-specific biological functions in D. melanogaster (60). (C) IB analyses of RP–HPLC purified fractions from different human (HEK293, HeLa, HeLa Kyoto and U2OS) and mouse (NIH3T3) cell lines using a polyclonal rabbit αZ.2.2 and αH2A.Z (αZ, C-terminal) antibodies. Recombinant Z.2.2 protein (rZ.2.2) was loaded in the first lane as positive control for αZ.2.2 antibody. Similar results were obtained when using a monoclonal rat αZ.2.2 antibody (data not shown).
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gks267-F1: Identification of Z.2.2. (A) qPCR with cDNA from different human cell lines and tissues using primers specific for Z.2.1 and Z.2.2. Data were normalized to HPRT1 and HMBS expression levels. Controls generated without reverse transcriptase (no RT) were used to assess amplification threshold. Shown are the levels of Z.2.2 mRNA as percentages of total Z.2 transcripts (Z.2.1 + Z.2.2). For an evaluation of absolute expression levels see Supplementary Figure S1C and D. (B) Amino acid alignment of human Z.1, Z.2.1 and Z.2.2 proteins using ClustalW Alignment (MacVector 10.0.2). Identical amino acids are highlighted in dark gray, similar amino acids in light gray and changes are set apart on white background. Known acetylation sites are depicted with stars and ubiquitination sites with circles. A schematic representation of the secondary structure of Z.1 and Z.2.1 is shown below the alignment, including depiction of the H3/H4 docking domain (50). M6 and M7 boxes indicate regions important for H2A.Z-specific biological functions in D. melanogaster (60). (C) IB analyses of RP–HPLC purified fractions from different human (HEK293, HeLa, HeLa Kyoto and U2OS) and mouse (NIH3T3) cell lines using a polyclonal rabbit αZ.2.2 and αH2A.Z (αZ, C-terminal) antibodies. Recombinant Z.2.2 protein (rZ.2.2) was loaded in the first lane as positive control for αZ.2.2 antibody. Similar results were obtained when using a monoclonal rat αZ.2.2 antibody (data not shown).

Mentions: Next, we wanted to determine to what degree all three H2A.Z mRNAs are expressed in different human cell lines and tissues and performed quantitative PCR (qPCR). Z.2.2 mRNA was present to different degrees in all human cell lines and tissues tested, though less abundant than Z.1 and Z.2.1 mRNAs that are expressed in similar amounts (Supplementary Figure S1C and D). Z.2.2 constituted between 5% and 15% of total Z.2 transcripts in all cell lines and tissues, with the exception of brain, where it was statistically significant upregulated (p = 1.7 × 10−4; Figure 1A). In some regions of this particular organ Z.2.2 accounted for up to 50% of all Z.2 transcripts pointing toward an exciting brain-specific function of this novel variant.Figure 1.


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)

Identification of Z.2.2. (A) qPCR with cDNA from different human cell lines and tissues using primers specific for Z.2.1 and Z.2.2. Data were normalized to HPRT1 and HMBS expression levels. Controls generated without reverse transcriptase (no RT) were used to assess amplification threshold. Shown are the levels of Z.2.2 mRNA as percentages of total Z.2 transcripts (Z.2.1 + Z.2.2). For an evaluation of absolute expression levels see Supplementary Figure S1C and D. (B) Amino acid alignment of human Z.1, Z.2.1 and Z.2.2 proteins using ClustalW Alignment (MacVector 10.0.2). Identical amino acids are highlighted in dark gray, similar amino acids in light gray and changes are set apart on white background. Known acetylation sites are depicted with stars and ubiquitination sites with circles. A schematic representation of the secondary structure of Z.1 and Z.2.1 is shown below the alignment, including depiction of the H3/H4 docking domain (50). M6 and M7 boxes indicate regions important for H2A.Z-specific biological functions in D. melanogaster (60). (C) IB analyses of RP–HPLC purified fractions from different human (HEK293, HeLa, HeLa Kyoto and U2OS) and mouse (NIH3T3) cell lines using a polyclonal rabbit αZ.2.2 and αH2A.Z (αZ, C-terminal) antibodies. Recombinant Z.2.2 protein (rZ.2.2) was loaded in the first lane as positive control for αZ.2.2 antibody. Similar results were obtained when using a monoclonal rat αZ.2.2 antibody (data not shown).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks267-F1: Identification of Z.2.2. (A) qPCR with cDNA from different human cell lines and tissues using primers specific for Z.2.1 and Z.2.2. Data were normalized to HPRT1 and HMBS expression levels. Controls generated without reverse transcriptase (no RT) were used to assess amplification threshold. Shown are the levels of Z.2.2 mRNA as percentages of total Z.2 transcripts (Z.2.1 + Z.2.2). For an evaluation of absolute expression levels see Supplementary Figure S1C and D. (B) Amino acid alignment of human Z.1, Z.2.1 and Z.2.2 proteins using ClustalW Alignment (MacVector 10.0.2). Identical amino acids are highlighted in dark gray, similar amino acids in light gray and changes are set apart on white background. Known acetylation sites are depicted with stars and ubiquitination sites with circles. A schematic representation of the secondary structure of Z.1 and Z.2.1 is shown below the alignment, including depiction of the H3/H4 docking domain (50). M6 and M7 boxes indicate regions important for H2A.Z-specific biological functions in D. melanogaster (60). (C) IB analyses of RP–HPLC purified fractions from different human (HEK293, HeLa, HeLa Kyoto and U2OS) and mouse (NIH3T3) cell lines using a polyclonal rabbit αZ.2.2 and αH2A.Z (αZ, C-terminal) antibodies. Recombinant Z.2.2 protein (rZ.2.2) was loaded in the first lane as positive control for αZ.2.2 antibody. Similar results were obtained when using a monoclonal rat αZ.2.2 antibody (data not shown).
Mentions: Next, we wanted to determine to what degree all three H2A.Z mRNAs are expressed in different human cell lines and tissues and performed quantitative PCR (qPCR). Z.2.2 mRNA was present to different degrees in all human cell lines and tissues tested, though less abundant than Z.1 and Z.2.1 mRNAs that are expressed in similar amounts (Supplementary Figure S1C and D). Z.2.2 constituted between 5% and 15% of total Z.2 transcripts in all cell lines and tissues, with the exception of brain, where it was statistically significant upregulated (p = 1.7 × 10−4; Figure 1A). In some regions of this particular organ Z.2.2 accounted for up to 50% of all Z.2 transcripts pointing toward an exciting brain-specific function of this novel variant.Figure 1.

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