Biology subjects

Motzik, A.

Publications and source records attributed to Motzik, A..

4 recordsLinked to original sources

bioRxiv2020

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. Author summaryCellular dynamics are underlined by numerous regulatory layers. The regulatory mechanism of interest in this work are enhancers. Enhancers are regulatory regions responsible, mainly, for increasing the possibility of transcription of a certain gene. Enhancers are marked by two distinct chemical groups-H3K4me1 and H3K27ac on the tail of histones. Histones are the proteins responsible for DNA packaging into condensed chromatin structure. In contrast, DNA methylation is a chemical modification often found on enhancers, and is traditionally associated with repression. A long debated question revolves around the functional relevance of DNA methylation in the context of enhancers. Here, we combined the two regulatory layers, histone marks and DNA methylation, to a single measurement that can highlight DNA methylation separately on each histone mark but at the same genomic region. When isolated with H3K4me1, enhancers showed higher levels of methylation compared to H3K27ac. As we measured the same genomic locations, we show that differences of DNA methylation between these marks can only be explained by cellular heterogeneity. We also demonstrated that these enhancers tend to play roles in stem cell differentiation and expression levels of the genes they control correlate with cell-to-cell variation.

molecular biology↗

bioRxiv2020

CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture

Single-cell assays have revealed the scope and importance of heterogeneity in many biological systems. However, in many cases, single cell limited sensitivity is a major hurdle for uncovering the full range of cellular variation. To overcome this limitation, we developed a complementary single cell technology, CloneSeq that combines clonal expansion under controlled culture conditions inside three-dimensional (3D) hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that unlike single cell transcriptomes, clonal cells maintain cell states and share similar transcriptional profiles. CloneSeq analysis of Non-small-cell lung carcinoma (NSCLC) cells revealed the presence of novel cancer-specific subpopulations, including cancer stem-like cells (CSLCs). Standard single cell RNA-seq assays as well as cell-to-clone tracing by genetic barcoding failed to identify these rare CSLCs. In addition to CSLCs, clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) that retained their pluripotent state in the absence of pluripotent media and improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. Our results demonstrate the capacity of CloneSeq, which can be effectively adapted to different biological systems, to discover rare and previously hidden subpopulations of cells, including CSLCs, by leveraging the broader expression space within clones.

molecular biology↗

bioRxiv2020

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

Mesenchymal stromal/stem cells (MSCs) are 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 conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of naive, matrix-conditioned, and early differentiation state cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We showed that soft matrices support adipogenesis of multipotent cells and endochondral ossification of non-adipogenic cells, whereas intramembranous ossification and pre-osteoblast proliferation are enhanced by stiff matrices. Using diffusion pseudotime mapping, we delineated hierarchical matrix-directed differentiation and identified mechanoresponsive genes. We found that tropomyosin-1 (TPM1) is highly sensitive to stiffness cues both at RNA and protein levels and that changes in expression of TPM1 determine adipogenic or osteogenic fates. Thus, cell-to-cell variation in tropomyosin-mediated matrix-sensing contributes to impaired differentiation with implications to the biomedical potential of MSCs.

cell biology↗

bioRxiv2019

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

Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein {beta}NAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

biochemistry↗