MRG Proteins Are Shared by Multiple Protein Complexes With Distinct Functions
Maëva Devoucoux1, Céline Roques1, Catherine Lachance1
1St. Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Oncology Division of CHU de Québec-Université Laval Research Center, Quebec City, Quebec, Canada.
Abstract:
MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2-BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.
Insights
MRG15 and MRGX proteins associate with key chromatin regulators and DNA repair complexes. A novel human TINTIN complex involving MRG15/X, MRGBP, BRD8, and EP400NL plays a role in gene transcription.
Area of Science:
- Molecular Biology
- Epigenetics
- Chromatin Biology
Background:
- MRG15/MORF4L1 is a conserved eukaryotic protein with a chromodomain (CHD) that binds histone H3 lysine 36 trimethylation (H3K36me3).
- MRG15 is known to interact with factors involved in chromatin modification, gene regulation, mRNA splicing, and DNA repair.
- A comprehensive understanding of MRG15's native interactome under physiological conditions is lacking.
Purpose of the Study:
- To identify and characterize the stable native protein complexes associated with human MRG15, MRGX/MORF4L2, and MRGBP.
- To investigate the functional significance of these protein associations in chromatin-based nuclear processes.
- To elucidate the composition and function of a newly identified MRG15/X-MRGBP-BRD8-EP400NL complex.
Main Methods:
- Genome editing and tandem affinity purification were used to analyze the native interactome of MRG15, MRGX, and MRGBP in K562 cells.
- CRISPR tagging of endogenous proteins and isoform comparison were employed for confirmation.
- Point mutations were introduced based on structural information to disrupt specific complex associations.
Main Results:
- MRG15 and MRGX showed stable, interchangeable associations with NuA4/TIP60, Sin3B, ASH1L, and PALB2-BRCA2 complexes.
- A novel, abundant native complex, MRG15/X-MRGBP-BRD8-EP400NL, was identified, functionally resembling the yeast TINTIN complex.
- EP400NL competes for BRD8 binding, forming TINTIN, which is implicated in the transcription of specific genes via BRD8's H4ac-binding and MRG15's H3K36me3-binding domains.
Conclusions:
- The study provides a detailed map of human MRG protein-associated complexes, revealing their dynamic interactions.
- The newly discovered human TINTIN complex plays a role in gene transcription, integrating epigenetic marks.
- These findings enhance the understanding of MRG proteins' diverse functions in chromatin regulation and nuclear processes.
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