Periodic Reporting for period 4 - SC-EpiCode (Decoding the Epigenomic Regulatory Code by the Use of Single Cell Technologies)
Reporting period: 2022-04-01 to 2023-03-31
Throughout the course of the project, we have pioneered innovative technologies that have been applied in various biological studies related to embryonic stem cells. These advancements are summarized as follows:
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 to provide insights into enhancer-based functional variation in complex biological systems.
1. Alajem A*LM, Roth H*S, Ratgauzer S*S, Bavli DPD, Motzik APD, Lahav SS, Peled IS, Ram OPI. DNA Methylation Patterns Expose Variations in Enhancer-Chromatin Modifications during Embryonic Stem Cell Differentiation. BioRxiv: doi: https://doi.org/10.1101/2020.11.25.39728. PLoS Genet. 2021. Impact factor: 5.917.
Limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq that combines clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq).
2.Bavli D*PD, Sun X*S, Kozulin C*S, Ennis DS, Motzik APD, Biran APD, Brielle SS, Alajem ALB, Meshorer ECoPI, Buxboim A*PI, Ram O*PI. CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture. BioRxiv: https://doi.org/10.1101/2020.11.24.395541. Dev Cell. 2021. Impact factor: 12.270. #Cit=NA. Our cover illustration was selected.
3.Sun X*S, Bavli D*PD, Kozulin C*S, Motzik APD, Buxboim ACoPI, Ram O*PI. CloneSeq - single-cell clonal 3D culture and analysis protocol. STAR protocols. 2021. Impact factor: NA.
Cell cycle and differentiation decisions are tightly linked; however, the underlying principles that drive these decisions are not fully understood. We show that while both G1 and G2/M cells have the capacity to differentiate towards Epiblast Stem Cells (EpiSCs), only G2/M ESCs could differentiate into extraembryonic endoderm cells (XENs).
We identified ESRRB, a key pluripotency factor that is upregulated during G2/M phase, as a central driver of XEN differentiation. 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.
4.Herchcovici LS*S, Feldman S*S, Arnon L*S, Alajem ALM, Bavli DPD, Sun XS, Buganim YCoPI, Ram OPI. Esrrb is a cell cycle dependent associated factor balancing between pluripotency and XEN differentiation. Stem Cell Rep. 2021. Impact factor: 7.765. #Cit=NA.
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 to provide insights into enhancer-based functional variation in complex biological systems.
1. Alajem A*LM, Roth H*S, Ratgauzer S*S, Bavli DPD, Motzik APD, Lahav SS, Peled IS, Ram OPI. DNA Methylation Patterns Expose Variations in Enhancer-Chromatin Modifications during Embryonic Stem Cell Differentiation. BioRxiv: doi: https://doi.org/10.1101/2020.11.25.39728. PLoS Genet. 2021. Impact factor: 5.917.
Limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq that combines clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq).
2.Bavli D*PD, Sun X*S, Kozulin C*S, Ennis DS, Motzik APD, Biran APD, Brielle SS, Alajem ALB, Meshorer ECoPI, Buxboim A*PI, Ram O*PI. CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture. BioRxiv: https://doi.org/10.1101/2020.11.24.395541. Dev Cell. 2021. Impact factor: 12.270. #Cit=NA. Our cover illustration was selected.
3.Sun X*S, Bavli D*PD, Kozulin C*S, Motzik APD, Buxboim ACoPI, Ram O*PI. CloneSeq - single-cell clonal 3D culture and analysis protocol. STAR protocols. 2021. Impact factor: NA.
Cell cycle and differentiation decisions are tightly linked; however, the underlying principles that drive these decisions are not fully understood. We show that while both G1 and G2/M cells have the capacity to differentiate towards Epiblast Stem Cells (EpiSCs), only G2/M ESCs could differentiate into extraembryonic endoderm cells (XENs).
We identified ESRRB, a key pluripotency factor that is upregulated during G2/M phase, as a central driver of XEN differentiation. 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.
4.Herchcovici LS*S, Feldman S*S, Arnon L*S, Alajem ALM, Bavli DPD, Sun XS, Buganim YCoPI, Ram OPI. Esrrb is a cell cycle dependent associated factor balancing between pluripotency and XEN differentiation. Stem Cell Rep. 2021. Impact factor: 7.765. #Cit=NA.
For Aim 1, we have successfully established a robust single-cell RNA-seq using drop based microfluidics in our lab and applied it to a variety of different cell types (ex. mouse ES cells, human lung adenocarcinoma cells, etc.). We also designed and produced polyacrylamide beads carrying double-strand primers which will allow capturing open chromatin from a single cell. Barcoding ligation efficiency was improved by changing from the current PCR-based amplification approach, which requires two-side ligation, to a T7 in-vitro transcription-based-system which needs only one side of the nucleosomal DNA to be ligated.
For Aim 2 and 3, we have generated mouse ES cell lines perturbed in multiple chromatin regulator genes rendering the cells viable and able to differentiate. This was done by cloning of a lentiviral plasmid bearing tet-on Cas9 fused to a GFP reporter (TRE-Cas9-GFP-tetON), it yielded a cell line expressing Cas9 upon Doxycycline activation. By analyzing the resulting genetic background of the perturbed cell lines, we verified the effectiveness of the perturbations, while the robustness of perturbations needed to be refined. We are now working on a manuscript focusing on Phf8 and Kdm4b perturbations in mESCs, which expand our undestanding on bivalncy and ESCs primed states.
During the course of this project we intiated several collaborations with leaders in the field of biophisics and mechanobiology which resulted with publications:
Mesenchymal stem cells assess extracellular elasticity by actively probing the resistance to applied forces via adhesion, cytoskeletal and nuclear mechanotransducers, and the emerging signals direct differentiation towards soft and stiff tissue lineages. Using single-cell transcriptional profiling at naïve, matrix-conditioned and early differentiation states, we identify distinct MSC subpopulations that are distinctively characterized by cellular mechanosensitivity, differentiation capacity and cell cycling.
1. Brielle SS, Bavli DPD, Sun XS, Kozulin CS, Motzik APD, Kan-Tor YS, Avni BCoPI, Ram O*PI, Buxboim A*PI. Tropomyosin mediates matrix-directed differentiation of distinctive subpopulations of primary mesenchymal stem cells. BioRxiv: https://doi.org/10.1101/2020.11.23.394460. PNAS. 2021. Impact factor: 11.205.
Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. We reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. Our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.
2. Meir Mellul*S, Shlomtzion Lahav*S, David B. LukatskyPI, Oren RamPI. DNA repeats apply negative design on gene expression in embryonic stem cells. Biophys J. 2022. Impact factor: 4.033.
For Aim 2 and 3, we have generated mouse ES cell lines perturbed in multiple chromatin regulator genes rendering the cells viable and able to differentiate. This was done by cloning of a lentiviral plasmid bearing tet-on Cas9 fused to a GFP reporter (TRE-Cas9-GFP-tetON), it yielded a cell line expressing Cas9 upon Doxycycline activation. By analyzing the resulting genetic background of the perturbed cell lines, we verified the effectiveness of the perturbations, while the robustness of perturbations needed to be refined. We are now working on a manuscript focusing on Phf8 and Kdm4b perturbations in mESCs, which expand our undestanding on bivalncy and ESCs primed states.
During the course of this project we intiated several collaborations with leaders in the field of biophisics and mechanobiology which resulted with publications:
Mesenchymal stem cells assess extracellular elasticity by actively probing the resistance to applied forces via adhesion, cytoskeletal and nuclear mechanotransducers, and the emerging signals direct differentiation towards soft and stiff tissue lineages. Using single-cell transcriptional profiling at naïve, matrix-conditioned and early differentiation states, we identify distinct MSC subpopulations that are distinctively characterized by cellular mechanosensitivity, differentiation capacity and cell cycling.
1. Brielle SS, Bavli DPD, Sun XS, Kozulin CS, Motzik APD, Kan-Tor YS, Avni BCoPI, Ram O*PI, Buxboim A*PI. Tropomyosin mediates matrix-directed differentiation of distinctive subpopulations of primary mesenchymal stem cells. BioRxiv: https://doi.org/10.1101/2020.11.23.394460. PNAS. 2021. Impact factor: 11.205.
Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. We reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. Our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.
2. Meir Mellul*S, Shlomtzion Lahav*S, David B. LukatskyPI, Oren RamPI. DNA repeats apply negative design on gene expression in embryonic stem cells. Biophys J. 2022. Impact factor: 4.033.
Throughout the project's development, we achieved progress that extended beyond the current state of the art. Single-cell assays have unveiled the significance of heterogeneity in various biological systems. Yet, the challenge of low sensitivity has impeded the differentiation of biological variation from noise. In response, we formulated the CloneSeq concept, a novel approach involving 3D clone-based RNA-seq. Our premise was that clonal cells share greater similarity than randomly chosen cells, leading to enhanced sensitivity and broader coverage compared to conventional single-cell RNA-seq methods. Our results affirm this hypothesis; cells stemming from the same clone exhibit more comparable transcriptional profiles than those from different clones. This phenomenon has facilitated an in-depth exploration of cellular heterogeneity within lung adenocarcinoma PC9 cells. Notably, we delineated distinct cellular states including cancer stem-like cells (CSCs), diverse replicative cancer states, and varying degrees of invasiveness. These nuanced characteristics remain elusive through conventional scRNA-seq due to its limited mapping resolution. Furthermore, our 3D system has demonstrated the capacity to induce embryonic stem cell formation without typical supplements like LIF and MEK/GSK3 inhibitors (2i). Additionally, it has augmented the efficiency of induced pluripotent stem cell (iPSC) production, outperforming conventional ESC culturing methods.