SUMOylation of the chromodomain factor MRG-1 inC. elegansaffects chromatin-regulatory dynamics

Abstract

Epigenetic mechanisms to control chromatin accessibility and structure is important for gene expression in eukaryotic cells. Chromatin regulation ensures proper development and cell fate specification but is also essential later in life. Modifications of histone proteins as an integral component of chromatin can promote either gene expression or repression, respectively. Proteins containing specific domains such as the chromodomain recognize mono-, di- or tri-methylated lysine residues on histone H3. The chromodomain protein MRG-1 in Caenorhabditis elegans is the ortholog of mammalian MRG15, which belongs to the MORF4 Related Gene (MRG) family in humans. In C. elegans MRG-1 predominantly binds methylated histone H3 lysine residues at position 36 (H3K36me3). MRG-1 is important during germline maturation and for safeguarding the germ cell identity. However, it lacks enzymatic activity and depends on protein-protein interaction to cooperate with other factors to regulate chromatin. To elucidate the variety of MRG-1 interaction partners we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with mass-spectrometry. Besides previously described and novel interactions with other proteins, we also detected a strong association with the Small Ubiquitin-like Modifier (SUMO). Since SUMO is known to be attached to proteins in order to modulate the target proteins activity we assessed whether MRG-1 is post-translationally modified by SUMOylation. Notably, we provide evidence that MRG-1 is indeed SUMOylated and that this post-translational modification influences the chromatin-binding profile of MRG-1 in the C. elegans genome. Our presented study hints towards an important role of SUMOylation in the context of epigenetic regulation via the chromodomain protein MRG-1, which may be a conserved phenomenon also in mammalian species.

Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry forC. elegans

“…We confirmed predicted interactions such as with SPT-16 (Orphanides et al 1998(Orphanides et al , 1999Kolundzic et al 2018) in a highly robust manner. Moreover, all 11 CoIP-MS for MRG-1 showed a very strong correlation based on Pearson correlations also in our previous study (Baytek et al 2021). Overall, our protocol enables a efficient and robust examination of protein interactions in C. elegans based on CoIP-MS. For future applications, C. elegans also provides the potential to reveal tissue-specific protein interactions in particular when epitope-tagged proteins are expressed in confined tissue lineages.…”

“…Nevertheless, for interaction proteomics, the specific interactors of a given protein are detected if their enrichment levels are significant, which makes LFQ analysis the method of choice for interaction proteomics in C. elegans in our study. The LFQ analysis of HMG-3 and HMG-4 CoIP-MS, as well as of MRG-1 in a previous study (Baytek et al 2021), delivered highly reproducible results. We confirmed predicted interactions such as with SPT-16 (Orphanides et al 1998(Orphanides et al , 1999Kolundzic et al 2018) in a highly robust manner.…”

“…We introduced the 3xHA tag, which can be targeted using commercially available anti-HA antibodies in a highly efficient manner, by CRISPR to two proteins of interest, HMG-3, and HMG-4. In a parallel study, which is reported in an accompanying manuscript, we also tagged the chromatin regulator MRG-1 with 3xHA using CRISPR editing (Baytek et al 2021). This strategy allows that the wild-type animals without the knock-in can be used to identify the background binders of the antibody and purification resin.…”

“…We demonstrate based on the subunits of the heteromeric chromatin remodeler FACT (FAcilitates Chromatin Transcription) (Orphanides et al 1998(Orphanides et al , 1999Kolundzic et al 2018) that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. In a previous study (Baytek et al 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), where we found that MRG-1 is SUMOylated.…”

“…Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings.…”