Robust co-immunoprecipitation with mass spectrometry for Caenorhabditis elegans using solid-phase enhanced sample preparation

Studying protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-immunoprecipitation with mass spectrometry detects protein–protein interactions with high throughput. The nematode Caenorhabditis elegans is a powerful genetic model organism for in vivo studies. Yet its rigid and complex tissues require optimization for biochemistry applications to ensure reproducibility. The authors optimized co-immunoprecipitation with mass spectrometry by combining a native co-immunoprecipitation procedure with single-pot, solid-phase enhanced sample preparation. The authors' results for the highly conserved chromatin regulator FACT subunits HMG-3 and HMG-4 demonstrated that single-pot, solid-phase enhanced sample preparation-integrated co-immunoprecipitation with mass spectrometry procedures for C. elegans samples are highly robust. Moreover, in an accompanying study about the chromodomain factor MRG-1 (MRG15 in humans), the authors demonstrated remarkably high reproducibility for ten replicate experiments

SUMOylation of the chromodomain factor MRG-1 in C. elegans affects chromatin-regulatory dynamics

Epigenetic mechanisms control chromatin accessibility and gene expression to ensure proper cell fate specification. Histone proteins are integral chromatin components, and their modification promotes gene expression regulation. Specific proteins recognize modified histones such as the chromodomain protein MRG-1. MRG-1 is the Caenorhabditis elegans ortholog of mammalian MRG15, which is involved in DNA repair. MRG-1 binds methylated histone H3 and is important for germline maturation and safeguarding. To elucidate interacting proteins that modulate MRG-1 activity, we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with mass spectrometry. We detected strong association with the Small ubiquitin-like modifier SUMO, and found that MRG-1 is post-translationally modified by SUMO. SUMOylation affects chromatin-binding dynamics of MRG-1, suggesting an epigenetic regulation pathway, which may be conserved

FACT sets a barrier for cell fate reprogramming in Caenorhabditis elegans and human cells

The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection

MRG-1/MRG15 is a barrier for germ cell to neuron reprogramming in Caenorhabditis elegans

Chromatin regulators play important roles in the safeguarding of cell identities by opposing the induction of ectopic cell fates and, thereby, preventing forced conversion of cell identities by reprogramming approaches. Our knowledge of chromatin regulators acting as reprogramming barriers in living organisms needs improvement as most studies use tissue culture. We used C. elegans as an in vivo gene discovery model and automated solid-phase RNAi screening, by which we identified 10 chromatin-regulating factors that protect cells against ectopic fate induction. Specifically, the chromodomain protein MRG-1 safeguards germ cells against conversion into neurons. MRG-1 is the ortholog of mammalian MRG15 (MORF-related gene on chromosome 15) and is required during germline development in C. elegans. However, MRG-1's function as a barrier for germ cell reprogramming has not been revealed previously. Here, we further provide protein-protein and genome interactions of MRG-1 to characterize its molecular functions. Conserved chromatin regulators may have similar functions in higher organisms and, therefore, understanding cell fate protection in C. elegans may also help to facilitate reprogramming of human cells