Biology subjects

Alajem, A.

Publications and source records attributed to Alajem, A..

3 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

Esrrb is a cell cycle dependent priming factor balancing between pluripotency and differentiation

Cell cycle and differentiation decisions are tightly linked; however, the underlying principles that drive these decisions are not fully understood. Here, we combined cell-cycle reporter system and single-cell RNA-seq profiling to study the transcriptomes of mouse embryonic stem cells (ESCs) in the context of cell cycle states and differentiation. By applying retinoic acid, a multi-linage differentiation assay, on G1 and G2/M pre-sorted ESCs, we show that only G2/M ESCs were capable of differentiating into extraembryonic endoderm cells (XENs), whereas cells in the G1 phase predominantly produce Epiblast Stem Cells. We identified ESRRB, a key pluripotency factor that is upregulated during G2/M phase, as a central driver of XEN differentiation. Furthermore, enhancer chromatin states based on WT and Esrrb-KO ESCs show association of ESRRB with XEN poised enhancers. Cells engineered to overexpress Esrrb during G1 allow ESCs to produce XENs, while ESRRB-KO ESCs lost their potential to differentiate into XEN. In addition, Embryonic bodies (EBs) are not affected by deletion of ESRRB but trigger apoptosis upon attempts to apply direct XEN differentiation. Taken together, this study reveals an important functional link between Esrrb and cell-cycle states during the exit from pluripotency. Finally, the experimental scheme of single cell RNA-seq in the context of cell cycle can be further expanded into other cellular systems to better understand differentiation decisions and cancer models.

genomics↗