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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
ORTH/VIM proteins that regulate DNA methylation are functional ubiquitin E3 ligases
Edward Kraft1, Magnolia Bostick, Steven E Jacobsen
1Department of Molecular and Cellular Biology, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA.
The Plant Journal : for Cell and Molecular Biology
|July 23, 2008
Summary
The ORTHRUS (ORTH) gene family in Arabidopsis thaliana acts as E3 ligases, crucial for regulating DNA methylation. Overexpression of ORTH genes alters flowering time and leads to hypomethylation, demonstrating their role in epigenetic control.
Area of Science:
- Plant Molecular Biology
- Epigenetics
- Gene Regulation
Background:
- Genomic methylation is vital for regulating gene expression.
- The ORTHRUS (ORTH) gene family in Arabidopsis thaliana is implicated in DNA methylation.
- ORTH proteins possess distinct domain structures, including Plant Homeodomain (PHD), Really Interesting New Gene (RING), and Set Ring Associated (SRA) domains.
Purpose of the Study:
- To investigate the function of the ORTHRUS (ORTH) gene family in DNA methylation.
- To determine the enzymatic activity and domain requirements of ORTH proteins as E3 ligases.
- To analyze the in vivo effects of ORTH gene overexpression on plant development and epigenetics.
Main Methods:
- Isolation and expression of ORTH gene cDNAs as glutathione-S-transferase (GST) fusion proteins.
- In vitro ubiquitylation assays using E2 conjugating enzymes (AtUBC11, AtUBC8).
- Site-directed mutagenesis and domain deletion analysis of ORTH1 RING domains.
- Nuclear localization studies using GFP-tagged ORTH proteins in transgenic Arabidopsis.
- Phenotypic analysis of ORTH overexpression lines, including flowering time and DNA methylation analysis at FWA and Cen180 repeats.
Main Results:
- ORTH1, ORTH2, ORTH5, and ORTHlike-1 proteins promoted ubiquitylation in vitro.
- Both RING domains of ORTH1 are capable of catalyzing ubiquitylation; the PHD domain is not required.
- GFP-ORTH1 and GFP-ORTH2 proteins are localized to the nucleus in Arabidopsis.
- Overexpression of ORTH1 or ORTH2 resulted in altered flowering times and hypomethylation of FWA and Cen180 repeats.
- A late-flowering phenotype persisted after transgene removal, suggesting stable epigenetic changes.
Conclusions:
- ORTH proteins function as E3 ligases involved in mediating DNA methylation status in vivo.
- The RING domains are essential for the E3 ligase activity of ORTH proteins.
- ORTH gene family members play a significant role in epigenetic regulation affecting plant development.
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These groups modify specific amino acids in a protein.
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01:37Epigenetic Regulation
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