Periodic Reporting for period 4 - ReachingCompleteness (The Molecular Basis of Somatic Nuclear Reprogramming)
Reporting period: 2020-09-01 to 2021-08-31
Background and Significance: The capability to convert skin cells into clinically-relevant cell types such as neurons, heart cells, liver cells and embryonic stem-like cells (induced pluripotent stem cells (iPSCs)) provide an invaluable resource of cells for disease modelling, drug screening, and patient-specific cell-based therapy known as regenerative medicine. However, many of the directly converted cells are not stable, and the vast majority of iPSCs exhibit poor developmental potential. This suggests that the prevailing method of reprogramming is not ideal and leads to incomplete conversion. To improve the quality of the converted cells, efforts should be focused on the molecular mechanisms that characterize the nuclear reprogramming process.
Goal: the main objective of this grant proposal is to shed light on the molecular mechanisms that dictate successful reprogramming events in two somatic cell conversion models by employing high throughput and single cell analyses. The central hypothesis behind this objective is that looking at the transcriptional, and epigenomic profile of induced cells in two somatic cell conversion models will illuminate the general criteria that must be fulfilled to reach complete reprogramming.
Methodologies: In this grant proposal we intend to establish a sophisticated fluorescent knock-in reporter system using the CRISPR/Cas9 method to capture the early rare reprogrammable cells and to employ single-cell RNA-seq, ATAC-seq, RRBS and ChIP-seq for major histone marks on cells during reprogramming. To identify common and more global elements that facilitate nuclear reprogramming, we will trace in parallel, induced cells from two different somatic cell conversion models (i.e. iPSCs and iTSCs) that reach high degree of nuclear reprogramming.
Conclusions: Our analysis revealed that cells undergoing reprogramming to pluripotency and TSC state exhibit specific trajectories from the onset of the process, suggesting ‘V’-shaped model. Using these analyses, not only we could describe in detail the various trajectories toward the two states, but we also identified previously unknown stage-specific reprogramming markers, reprogramming blockers and facilitators and TSC subpopulations. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these specific regions are initially open but then retain inactive by the elimination of the histone mark, H3K27ac.
Goal: the main objective of this grant proposal is to shed light on the molecular mechanisms that dictate successful reprogramming events in two somatic cell conversion models by employing high throughput and single cell analyses. The central hypothesis behind this objective is that looking at the transcriptional, and epigenomic profile of induced cells in two somatic cell conversion models will illuminate the general criteria that must be fulfilled to reach complete reprogramming.
Methodologies: In this grant proposal we intend to establish a sophisticated fluorescent knock-in reporter system using the CRISPR/Cas9 method to capture the early rare reprogrammable cells and to employ single-cell RNA-seq, ATAC-seq, RRBS and ChIP-seq for major histone marks on cells during reprogramming. To identify common and more global elements that facilitate nuclear reprogramming, we will trace in parallel, induced cells from two different somatic cell conversion models (i.e. iPSCs and iTSCs) that reach high degree of nuclear reprogramming.
Conclusions: Our analysis revealed that cells undergoing reprogramming to pluripotency and TSC state exhibit specific trajectories from the onset of the process, suggesting ‘V’-shaped model. Using these analyses, not only we could describe in detail the various trajectories toward the two states, but we also identified previously unknown stage-specific reprogramming markers, reprogramming blockers and facilitators and TSC subpopulations. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these specific regions are initially open but then retain inactive by the elimination of the histone mark, H3K27ac.
Overall most of our initial aims were completed successfully.
We have completed the characterization of our newly-developed iTSC reprogramming paradigm and publish this new model in the prestigious journal cell stem cell (Benchetrit et al. 2015).
Next, we have proposed to generate a quadruple fluorescence knock-in reporter system to be able to sort out reprogrammable cells for transcriptional analysis and completed this task successfully. Currently we have a system that contains four reporters (Nanog-2A-EGFP- specific for iPSC reprogramming, Elf5-2A-EYFP-NLS- specific for TSC reprogramming, and Utf1-2A-tdTomato and Esrrb-2A-TagBFP- shared between TSC and iPSC reprogramming). This system will refer hereafter as “BYKE” system.
We examined the transcriptome (bulk RNA-seq and single cell RNA-seq), methylome (RRBS), chromatin accessibility (ATAC-seq), chromatin activity (ChIP-seq for H3K4me2 and H3K27ac) and genomic stability (CNVs) of BYKE cells undergoing reprogramming to induced pluripotent stem cells (iPSCs) and induced TSCs (iTSCs) along the reprogramming process. Using single-cell analysis, we revealed unique and previously unknown stage-specific markers, as well as markers for faithful (Tdgf1 for OSKM and Cd82 for GETM) and failed (Anx3 for OSKM) reprogramming process.
Chromatin accessibility and activity analyses identified many reprogramming blockers, such as Usf1/Usf2, Nrf2 and MafK, along with other oxidative stress response genes that significantly hinder both reprogramming systems but with different dynamics. These results suggest a massive occupancy on fibroblastic identity regions even after 9 days of reprogramming, especially in GETM reprogramming.
In accordance with that, the AP1/CREB/ATF binding sites, which their corresponding protein members act as somatic cell identity safeguards that block the reprogramming process to pluripotency are significantly more enriched in GETM reprogramming peaks compared to OSKM reprogramming peaks. In contrast, in regions that are open in fibroblasts and closed upon reprogramming, the binding sites of the AP1/CREB/ATF family of proteins are significantly more enriched in OSKM reprogramming compared to GETM reprogramming. These results explain the potent ability of OSKM to erase somatic cell identity, as well as the continued presence of MEF-like cells in GETM reprogramming, even at day 12 of the process, as we observed in the SC-RNA-seq analysis.
Lastly, by integrating all the data together, we illuminated key aspects that characterize each fate at all levels of regulation. Remarkably, by comparing both systems we show that from the onset of the reprogramming process, OSKM define regions that are important for the development of the heart and brain, the two most essential organs for the developing embryo. Mechanistically, we show that while GETM shut off the embryonic programs by DNA methylation, OSKM open these regions but retain them as inactive by eliminating the histone mark H3K27ac.
In conclusion, besides providing the first multi-layer characterization of cells undergoing reprogramming to the TSC state, our approach of conducting concomitant and comparative multi-omics analysis of cells acquiring both pluripotent and TSC states yield knowledge that cannot be revealed otherwise, such as when each reprogramming process is analyzed separately. We show that this unique approach of comparative parallel multi-omics analysis is a powerful tool to illuminate features of the nuclear reprogramming process as well as general and broad cellular plasticity principles.
We have completed the characterization of our newly-developed iTSC reprogramming paradigm and publish this new model in the prestigious journal cell stem cell (Benchetrit et al. 2015).
Next, we have proposed to generate a quadruple fluorescence knock-in reporter system to be able to sort out reprogrammable cells for transcriptional analysis and completed this task successfully. Currently we have a system that contains four reporters (Nanog-2A-EGFP- specific for iPSC reprogramming, Elf5-2A-EYFP-NLS- specific for TSC reprogramming, and Utf1-2A-tdTomato and Esrrb-2A-TagBFP- shared between TSC and iPSC reprogramming). This system will refer hereafter as “BYKE” system.
We examined the transcriptome (bulk RNA-seq and single cell RNA-seq), methylome (RRBS), chromatin accessibility (ATAC-seq), chromatin activity (ChIP-seq for H3K4me2 and H3K27ac) and genomic stability (CNVs) of BYKE cells undergoing reprogramming to induced pluripotent stem cells (iPSCs) and induced TSCs (iTSCs) along the reprogramming process. Using single-cell analysis, we revealed unique and previously unknown stage-specific markers, as well as markers for faithful (Tdgf1 for OSKM and Cd82 for GETM) and failed (Anx3 for OSKM) reprogramming process.
Chromatin accessibility and activity analyses identified many reprogramming blockers, such as Usf1/Usf2, Nrf2 and MafK, along with other oxidative stress response genes that significantly hinder both reprogramming systems but with different dynamics. These results suggest a massive occupancy on fibroblastic identity regions even after 9 days of reprogramming, especially in GETM reprogramming.
In accordance with that, the AP1/CREB/ATF binding sites, which their corresponding protein members act as somatic cell identity safeguards that block the reprogramming process to pluripotency are significantly more enriched in GETM reprogramming peaks compared to OSKM reprogramming peaks. In contrast, in regions that are open in fibroblasts and closed upon reprogramming, the binding sites of the AP1/CREB/ATF family of proteins are significantly more enriched in OSKM reprogramming compared to GETM reprogramming. These results explain the potent ability of OSKM to erase somatic cell identity, as well as the continued presence of MEF-like cells in GETM reprogramming, even at day 12 of the process, as we observed in the SC-RNA-seq analysis.
Lastly, by integrating all the data together, we illuminated key aspects that characterize each fate at all levels of regulation. Remarkably, by comparing both systems we show that from the onset of the reprogramming process, OSKM define regions that are important for the development of the heart and brain, the two most essential organs for the developing embryo. Mechanistically, we show that while GETM shut off the embryonic programs by DNA methylation, OSKM open these regions but retain them as inactive by eliminating the histone mark H3K27ac.
In conclusion, besides providing the first multi-layer characterization of cells undergoing reprogramming to the TSC state, our approach of conducting concomitant and comparative multi-omics analysis of cells acquiring both pluripotent and TSC states yield knowledge that cannot be revealed otherwise, such as when each reprogramming process is analyzed separately. We show that this unique approach of comparative parallel multi-omics analysis is a powerful tool to illuminate features of the nuclear reprogramming process as well as general and broad cellular plasticity principles.
We have made a lot of progress beyond the state of the art.
1. We developed a new reprogramming model (from skin cells to iTSCs). This is currently the only model that reaches high degree of nuclear reprogramming.
2. We established a very attractive and complicated quadruple fluorescent knock-in reporter system that allows the discrimination between cells undergoing reprogramming to iPSCs and iTSCs.
3. We identified a combination of reporters that can successfully detect reprogrammable cells that will become iPSCs or iTSCs.
Moreover, we understood that probing the transcriptome both at the population and single cell level will not be sufficient to get a clear picture of the reprogramming process and thus we decided to probe also the chromatin structure (ATAC-Seq), DNA methylation (RRBS) and CNVs of induced BYKE cells.
We believe that the elements that we have detected between the two models during this grant period will be relevant also in other reprogramming models and will aid in achieving high quality converted cells for future clinical use.
1. We developed a new reprogramming model (from skin cells to iTSCs). This is currently the only model that reaches high degree of nuclear reprogramming.
2. We established a very attractive and complicated quadruple fluorescent knock-in reporter system that allows the discrimination between cells undergoing reprogramming to iPSCs and iTSCs.
3. We identified a combination of reporters that can successfully detect reprogrammable cells that will become iPSCs or iTSCs.
Moreover, we understood that probing the transcriptome both at the population and single cell level will not be sufficient to get a clear picture of the reprogramming process and thus we decided to probe also the chromatin structure (ATAC-Seq), DNA methylation (RRBS) and CNVs of induced BYKE cells.
We believe that the elements that we have detected between the two models during this grant period will be relevant also in other reprogramming models and will aid in achieving high quality converted cells for future clinical use.