DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation.

In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.

Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling.

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor.

BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

CloneSeq - Single-cell clonal 3D culture and analysis protocol.

This CloneSeq protocol combines clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing to resolve the limited sensitivity of single-cell approaches. CloneSeq can reveal rare subpopulations and support cellular stemness. CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging clonal enhanced sensitivity. Important considerations include the hydrogel composition, adaptation of 3D cultured clones to the inDrops system, and inherent adhesive properties of the cells. CloneSeq is only validated for cell lines so far. For complete details on the use and execution of this protocol, please refer to (Bavli et al., 2021).

CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones.

Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins.

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein-protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.

Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells.

Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.

Correction: Serine 207 phosphorylated lysyl-tRNA synthetase predicts disease-free survival of non-small-cell lung carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.18053.].

Serine 207 phosphorylated lysyl-tRNA synthetase predicts disease-free survival of non-small-cell lung carcinoma.

It has been shown that various tRNA synthetases exhibit non-canonical activities unrelated to their original role in translation. We have previously described a signal transduction pathway in which serine 207 phosphorylated lysyl-tRNA synthetase (P-s207 LysRS) is released from the cytoplasmic multi-tRNA synthetase complex (MSC) into the nucleus, where it activates the transcription factor MITF in stimulated cultured mast cells and cardiomyocytes. Here we describe a similar transformation of LysRS due to EGFR signaling activation in human lung cancer. Our data shows that activation of the EGFR results in phosphorylation of LysRS at position serine 207, its release from the MSC and translocation to the nucleus. We then generated a P-s207 LysRS rabbit polyclonalantibody and tested 242 tissue micro-array samples derived from non-small-cell lung cancer patients. Highly positive nuclear staining for P-s207 LysRS was noted in patients with EGFR mutations as compared to WT EGFR patients and was associated with improved mean disease-free survival (DFS). In addition, patients with mutated EGFR and negative lymph node metastases had better DFS when P-s207 LysRS was present in the nucleus. The data presented strongly suggests functional and prognostic significance of P-s207 LysRS in non-small-cell lung cancer.

Non-canonical roles of lysyl-tRNA synthetase in health and disease.

Lysyl-tRNA synthetase (LysRS) is a highly conserved enzyme that is part of the translational machinery in all living cells. Besides its canonical role in translation, LysRS gained additional domains and functions throughout evolution. These include its essential role in HIV replication and its roles in transcriptional regulation, cytokine-like signaling, and transport of proteins to the cell membrane. These diverse processes are tightly regulated through post-transcriptional modifications, interactions with other proteins, and targeting to the various cell compartments. The emerging variety of tasks performed by LysRS may therefore be utilized by various processes and pathological conditions that are described in this review, and their ongoing investigation is of extreme importance for our understanding of basic cellular regulatory mechanisms.

Importin beta plays an essential role in the regulation of the LysRS-Ap(4)A pathway in immunologically activated mast cells.

We recently reported that diadenosine tetraphosphate hydrolase (Ap(4)A hydrolase) plays a critical role in gene expression via regulation of intracellular Ap(4)A levels. This enzyme serves as a component of our newly described lysyl tRNA synthetase (LysRS)-Ap(4)A biochemical pathway that is triggered upon immunological challenge. Here we explored the mechanism of this enzyme's translocation into the nucleus and found its immunologically dependent association with importin beta. Silencing of importin beta prevented Ap(4)A hydrolase nuclear translocation and affected the local concentration of Ap(4)A, which led to an increase in microphthalmia transcription factor (MITF) transcriptional activity. Furthermore, immunological activation of mast cells resulted in dephosphorylation of Ap(4)A hydrolase, which changed the hydrolytic activity of the enzyme.