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In vitro Methylation Assay to Study Protein Arginine Methylation
10:01

In vitro Methylation Assay to Study Protein Arginine Methylation

Published on: October 5, 2014

Regulation of coactivator complex assembly and function by protein arginine methylation and demethylimination

Young-Ho Lee1, Scott A Coonrod, W Lee Kraus

  • 1Department of Pathology, University of Southern California, 2011 Zonal Avenue, HMR 301, Los Angeles, CA 90089, USA.

Proceedings of the National Academy of Sciences of the United States of America
|February 26, 2005
PubMed

Insights

Coactivator-associated arginine methyltransferase 1 (CARM1) methylates p300, impacting GRIP1 binding. Peptidyl deiminase 4 reverses this, regulating estrogen receptor transcription.

Area of Science:

  • Molecular Biology
  • Epigenetics
  • Gene Regulation

Background:

  • Nuclear receptors regulate gene transcription via coactivator recruitment.
  • p160 coactivators, histone acetyltransferases (CBP/p300), and CARM1 are crucial for this process.
  • CARM1's methyltransferase activity is essential for coactivator synergy.

Purpose of the Study:

  • To investigate the role of CARM1-mediated methylation in p300 and its impact on coactivator complex assembly.
  • To elucidate the specific methylation site on p300 and its functional consequences.
  • To understand the regulatory mechanism involving methylation and demethylation in coactivator function.

Main Methods:

  • In vitro and in vivo biochemical assays to study protein-protein interactions.
  • Site-directed mutagenesis to identify critical arginine residues.
  • Mass spectrometry or similar techniques to identify methylation sites.
  • Functional assays to assess transcriptional activity.

Main Results:

  • CARM1 methylates Arg-2142 in the GRIP1 binding domain (GBD) of p300.
  • Methylation of Arg-2142 inhibits the interaction between GRIP1 and p300.
  • Peptidyl deiminase 4 removes the methylation mark, enhancing the p300-GRIP1 interaction.
  • These modifications affect coactivator complex conformation and estrogen receptor-mediated transcription.

Conclusions:

  • CARM1-mediated methylation of p300 is a novel mechanism regulating coactivator complex assembly and function.
  • The interplay between methylation and demethylation by specific enzymes fine-tunes transcriptional regulation.
  • These findings reveal unique epigenetic mechanisms controlling nuclear receptor activity.

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