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The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9.

Thorstensen T, Fischer A, Sandvik SV, Johnsen SS, Grini PE, Reuter G, Aalen RB - Nucleic Acids Res. (2006)

Bottom Line: The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization.Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far.A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.

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SUVR4 interaction with histones. (A) Coomassie stained SDS–PAGE gel after GST pull-down of recombinant histone H3 using full-length GST-SUVR4. GST alone, and mock pull-down reactions without H3 input (-H3), were used as a negative controls. The undegraded GST-fusion proteins are indicated by asterisks. (B) Coomassie stained SDS–PAGE gel after GST pull-down with full-length GST-SUVR4 using calf thymus core histones as input. GST was used as a negative control. The undegraded GST-fusion proteins are indicated by asterisks. (C) Western analysis of the reactions in (B) using a diMeH3K9 antibody.
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fig5: SUVR4 interaction with histones. (A) Coomassie stained SDS–PAGE gel after GST pull-down of recombinant histone H3 using full-length GST-SUVR4. GST alone, and mock pull-down reactions without H3 input (-H3), were used as a negative controls. The undegraded GST-fusion proteins are indicated by asterisks. (B) Coomassie stained SDS–PAGE gel after GST pull-down with full-length GST-SUVR4 using calf thymus core histones as input. GST was used as a negative control. The undegraded GST-fusion proteins are indicated by asterisks. (C) Western analysis of the reactions in (B) using a diMeH3K9 antibody.

Mentions: Some proteins require certain histone tail modifications to bind histones (33,34). To investigate whether posttranslational modifications were required for targeting and binding of SUVR4, a pull-down experiment was performed. In this in vitro binding assay the GST-SUVR4 full-length protein was able to bind recombinant histone H3 which is devoid of posttranslational modifications (Figure 5A). In addition, we tested binding to calf thymus histones (Figure 5B), and by using antibodies against dimethylated histone H3K9 (Figure 5C) we could show that SUVR4 also bound histone H3 with this modification (Figure 5C), although dimethylated H3K9 is not a substrate for SUVR4 (Figure 4C). A 374 amino acid C-terminal SUVR4 fragment lacking the WIYLD domain pulled down calf thymus histone H3 equally well as the full-length GST-SUVR4 protein (data not shown). In conclusion, these pull-down experiments demonstrate that SUVR4 binds histone H3 irrespective of the methylation status of lysine 9.


The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9.

Thorstensen T, Fischer A, Sandvik SV, Johnsen SS, Grini PE, Reuter G, Aalen RB - Nucleic Acids Res. (2006)

SUVR4 interaction with histones. (A) Coomassie stained SDS–PAGE gel after GST pull-down of recombinant histone H3 using full-length GST-SUVR4. GST alone, and mock pull-down reactions without H3 input (-H3), were used as a negative controls. The undegraded GST-fusion proteins are indicated by asterisks. (B) Coomassie stained SDS–PAGE gel after GST pull-down with full-length GST-SUVR4 using calf thymus core histones as input. GST was used as a negative control. The undegraded GST-fusion proteins are indicated by asterisks. (C) Western analysis of the reactions in (B) using a diMeH3K9 antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636477&req=5

fig5: SUVR4 interaction with histones. (A) Coomassie stained SDS–PAGE gel after GST pull-down of recombinant histone H3 using full-length GST-SUVR4. GST alone, and mock pull-down reactions without H3 input (-H3), were used as a negative controls. The undegraded GST-fusion proteins are indicated by asterisks. (B) Coomassie stained SDS–PAGE gel after GST pull-down with full-length GST-SUVR4 using calf thymus core histones as input. GST was used as a negative control. The undegraded GST-fusion proteins are indicated by asterisks. (C) Western analysis of the reactions in (B) using a diMeH3K9 antibody.
Mentions: Some proteins require certain histone tail modifications to bind histones (33,34). To investigate whether posttranslational modifications were required for targeting and binding of SUVR4, a pull-down experiment was performed. In this in vitro binding assay the GST-SUVR4 full-length protein was able to bind recombinant histone H3 which is devoid of posttranslational modifications (Figure 5A). In addition, we tested binding to calf thymus histones (Figure 5B), and by using antibodies against dimethylated histone H3K9 (Figure 5C) we could show that SUVR4 also bound histone H3 with this modification (Figure 5C), although dimethylated H3K9 is not a substrate for SUVR4 (Figure 4C). A 374 amino acid C-terminal SUVR4 fragment lacking the WIYLD domain pulled down calf thymus histone H3 equally well as the full-length GST-SUVR4 protein (data not shown). In conclusion, these pull-down experiments demonstrate that SUVR4 binds histone H3 irrespective of the methylation status of lysine 9.

Bottom Line: The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization.Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far.A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

ABSTRACT
Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.

Show MeSH
Related in: MedlinePlus