Protocol for differential analysis of Trim71-associated protein complexes in mouse embryonic stem cells by mass spectrometry using Perseus.

Characterizing multi-protein complexes using mass spectrometry is crucial for understanding complex biochemical activities that govern cell fate. Investigating dynamic protein-protein interactions requires diverse qualitative and quantitative approaches. We present a protocol for differential analysis of Trim71-associated proteins in mouse embryonic stem cells under two conditions. We describe steps for cytoplasmic extraction, protein immunoprecipitation, sample preparation for mass spectrometry, and data analysis with Perseus. Our versatile tool enables in-depth exploration of protein complex compositional changes, contributing to a deeper understanding of cellular dynamics and making it suitable for various research domains. For complete details on the use and execution of this protocol, please refer to Rapone et al.1.

CTNND2 moderates the pace of synaptic maturation and links human evolution to synaptic neoteny.

Human-specific genes are potential drivers of brain evolution. Among them, SRGAP2C has contributed to the emergence of features characterizing human cortical synapses, including their extended period of maturation. SRGAP2C inhibits its ancestral copy, the postsynaptic protein SRGAP2A, but the synaptic molecular pathways differentially regulated in humans by SRGAP2 proteins remain largely unknown. Here, we identify CTNND2, a protein implicated in severe intellectual disability (ID) in Cri-du-Chat syndrome, as a major partner of SRGAP2. We demonstrate that CTNND2 slows synaptic maturation and promotes neuronal integrity. During postnatal development, CTNND2 moderates neuronal excitation and excitability. In adults, it supports synapse maintenance. While CTNND2 deficiency is deleterious and results in synaptic loss of SYNGAP1, another major ID-associated protein, the human-specific protein SRGAP2C, enhances CTNND2 synaptic accumulation in human neurons. Our findings suggest that CTNND2 regulation by SRGAP2C contributes to synaptic neoteny in humans and link human-specific and ID genes at the synapse.

SETDB1 modulates the TGFß response in Duchenne muscular dystrophy myotubes.

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.

The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells.

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.