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Extended Recognition of the Histone H3 Tail by Histone Demethylase KDM5A
Nektaria Petronikolou1, James E Longbotham1, Danica Galonić Fujimori1,2
1Department of Cellular and Molecular Pharmacology , University of California , 600 16th Street, Genentech Hall , San Francisco , California 94158 , United States.
Abstract:
Human lysine demethylase KDM5A is a chromatin-modifying enzyme associated with transcriptional regulation, because of its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in many cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer, and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp, compared to wild-type 18mer peptide, suggesting that this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A, as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.
Insights
Human lysine demethylase KDM5A, crucial in cancer, recognizes histone H3 at residue Q5. Distal H3 tail regions and modifications also impact its demethylation activity, revealing complex chromatin regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Epigenetics
Background:
- Human lysine demethylase KDM5A regulates transcription by removing methyl groups from histone H3 lysine 4 (H3K4me3).
- KDM5A amplification is linked to various cancers, including breast, prostate, and lung cancer.
- Understanding KDM5A's substrate recognition is vital for cancer research.
Purpose of the Study:
- To investigate substrate recognition mechanisms of KDM5A using alanine scanning mutagenesis.
- To identify critical histone H3 tail residues involved in KDM5A-catalyzed demethylation.
- To explore how distal histone modifications influence KDM5A activity.
Main Methods:
- Alanine scanning mutagenesis was used to probe KDM5A-histone H3 interactions.
- Demethylation activity assays were performed on modified H3 peptides.
- Kinetic analysis (KMapp) was used to quantify the effect of H3 tail mutations.
Main Results:
- Histone H3 residue Q5 was identified as critical for KDM5A substrate recognition.
- Mutations or deletions in H3 tail residues 14-18 significantly increased KMapp (8-fold), indicating their importance in histone engagement.
- Post-translational modifications on the distal H3 tail epitope were shown to modulate KDM5A activity.
Conclusions:
- KDM5A exhibits specific recognition of the H3 tail, involving both proximal (Q5) and distal (14-18) regions.
- Chromatin context, including distal modifications, plays a significant role in regulating KDM5A activity and H3K4 methylation.
- These findings enhance understanding of KDM5A function in transcriptional regulation and cancer.
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