Periodic Reporting for period 1 - OCTOBER (Control of cell identity in pluripotent and germline cells by OCT4 orchestration of chromatin binding and enhancer regulation)
Período documentado: 2019-10-28 hasta 2021-10-27
Mouse embryonic stem cells (ESCs) are characterized by two defining features: the ability to undergo limitless cell divisions in which the daughter cells posses exactly the same properties as the mother cell (a process referred to as self-renewal), and the capacity to change properties to give rise to all the cells of the developing mouse embryo (a process referred to as differentiation). Although ESCs have been studied since they were first reported in 1981, the molecular mechanisms underlying the choice between self-renewal and differentiation have not been completely described. OCTOBER is a multidisciplinary project that aims to provide new insight into this unresolved problem, focusing on the differentiation of ESCs into primordial germ cells (PGCs), progenitors of gametes. One of the main characters in this pathway is OCT4, a transcription factor (TF) that can bind specific regulatory regions in the genome to modulate gene expression of specific targets. Interestingly, OCT4 has been reported to be essential for the ESCs to maintain their pluripotency but it is highly expressed both in ESCs and PGCs at comparable levels. This observation leads to the question of whether the same protein – OCT4 – may exert specific functions in distinct contexts, possibly by interacting with different regulatory regions in the genome. A potential OCT4 binding protein that may contribute to widespread differential localisation of OCT4 to regulatory sites in chromatin during the differentiation of ESCs is OTX2. This protein has previously been reported to re-direct OCT4 to bind at one specific location (the Fgf5 locus) but whether OTX2 acts more broadly than this is not known. OCTOBER uses recently developed techniques to identify binding sites of OCT4 and OTX2 at chromatin while modulating the expression level of either of the two TFs. As an additional aim, the specific role of OTX2 in the choice of somatic vs germline differentiation has been studied.
The overall objectives of OCTOBER were to generate new knowledge on the mechanism of gene expression and regulation in the differentiation of ESCs to PGCLCs, with a focus on the role of TFs, and to further develop and/or acquire technical and transferrable skills that will help me become an independent leader in the field of genomics and stem cell biology. Although the results are not published yet, the action was successful regarding both objectives.
While the focus of OCTOBER is mainly on basic molecular mechanisms, the results will generate insights into the mechanisms of early germline formation. This will provide new information that may assist development of future approaches to treat both infertility and germ cell tumours.
In conclusion, OCTOBER is a successful action that generated and will continue to generate new knowledge in the regulation of gene expression in mammalian stem cells and, at the same time, provided me with essential skills for my next steps toward an independent career.
The overall objectives of OCTOBER were to generate new knowledge on the mechanism of gene expression and regulation in the differentiation of ESCs to PGCLCs, with a focus on the role of TFs, and to further develop and/or acquire technical and transferrable skills that will help me become an independent leader in the field of genomics and stem cell biology. Although the results are not published yet, the action was successful regarding both objectives.
While the focus of OCTOBER is mainly on basic molecular mechanisms, the results will generate insights into the mechanisms of early germline formation. This will provide new information that may assist development of future approaches to treat both infertility and germ cell tumours.
In conclusion, OCTOBER is a successful action that generated and will continue to generate new knowledge in the regulation of gene expression in mammalian stem cells and, at the same time, provided me with essential skills for my next steps toward an independent career.
OCTOBER has been strongly affected by the COVID19 pandemic that burst at the beginning of 2020 and is still ongoing. The British government, like many other governments in Europe, implemented a national lockdown that lasted more than 3 months. The University of Edinburgh closed for the period of the lockdown, blocking any progress of OCTOBER which was still in its initial phase. Moreover, the global situation lead to a delay in reagents and materials that limited the work in the lab.
To overcome these issues, an additional aim was added to OCTOBER, to take advantage of reagents and cell lines that were already present in the lab. This involved expanding the study to include assessment of the role of OTX2 in the choice of somatic vs germline differentiation. Overall, the action generated a large volume of reagents, implemented protocols and pipelines for NGS analysis. The results generated by this part of the action are unpublished but are extremely promising. The data will be confirmed and expanded in the near future and soon published. Reagents from OCTOBER will be shared with the community upon publication, the implemented protocols will be published on my website and the pipelines for NGS analysis are already available on my GitHub page (https://github.com/ElisaB85/NGS-pipelines/tree/main(se abrirá en una nueva ventana)). Once the results and data will be shared with the community, publications will be advertised on my website (www.elisabarbieriphd.wordpress.com/) with an emphasis on the role played by the Marie Curie and Horizon2020 programmes.
One other important aspect of OCTOBER was the focus on personal development and transferrable skills. During the course of the action, I acquired skills of NGS data analysis, learnt how to culture embryonic stem cells and how to adapt known techniques to this model. On the other hand, I further develop my presentation and writing skills. Finally, I directly supervised two Honours and one Masters student and currently co-supervise a PhD student. This, together with many workshops and courses I attended during the past two years, strongly implemented my ability to supervise lab members. I am now ready to become an independent researcher in the field.
I presented OCTOBER at the Edinburgh Chromatin, Epigenetics & Transcription Regulatory Network in 2020 and took part in the European Researcher's Night in 2021. In both situations, I acted as an MSCA ambassador. I attended virtual conferences in 2020 and 2021, where I had the chance to interact with our fellow scientists and discuss the OCTOBER project. I will actively participate to at least 2 conferences in 2022 where I will present the results I obtained so far for OCTOBER.
To overcome these issues, an additional aim was added to OCTOBER, to take advantage of reagents and cell lines that were already present in the lab. This involved expanding the study to include assessment of the role of OTX2 in the choice of somatic vs germline differentiation. Overall, the action generated a large volume of reagents, implemented protocols and pipelines for NGS analysis. The results generated by this part of the action are unpublished but are extremely promising. The data will be confirmed and expanded in the near future and soon published. Reagents from OCTOBER will be shared with the community upon publication, the implemented protocols will be published on my website and the pipelines for NGS analysis are already available on my GitHub page (https://github.com/ElisaB85/NGS-pipelines/tree/main(se abrirá en una nueva ventana)). Once the results and data will be shared with the community, publications will be advertised on my website (www.elisabarbieriphd.wordpress.com/) with an emphasis on the role played by the Marie Curie and Horizon2020 programmes.
One other important aspect of OCTOBER was the focus on personal development and transferrable skills. During the course of the action, I acquired skills of NGS data analysis, learnt how to culture embryonic stem cells and how to adapt known techniques to this model. On the other hand, I further develop my presentation and writing skills. Finally, I directly supervised two Honours and one Masters student and currently co-supervise a PhD student. This, together with many workshops and courses I attended during the past two years, strongly implemented my ability to supervise lab members. I am now ready to become an independent researcher in the field.
I presented OCTOBER at the Edinburgh Chromatin, Epigenetics & Transcription Regulatory Network in 2020 and took part in the European Researcher's Night in 2021. In both situations, I acted as an MSCA ambassador. I attended virtual conferences in 2020 and 2021, where I had the chance to interact with our fellow scientists and discuss the OCTOBER project. I will actively participate to at least 2 conferences in 2022 where I will present the results I obtained so far for OCTOBER.
The results of OCTOBER will be confirmed and further expanded in the next months with publication anticipated in 2022. Moreover, I plan on participating in at least one conference on epigenetics and chromatin that will be held in 2022 where I will present the results of OCTOBER acknowledging MSCA and Horizon2020 for their support during the last two years. Details will be also shared on my website, under either the OCTOBER or the blog section.
By the end of the project, OCTOBER will drastically contribute to the field, by providing insights into the mechanisms of choice between somatic and germline differentiation of embryonic stem cells, a subject that is still largely unknown. Moreover, it will determine the distinct roles that TFs like OCT4 have in different cell types, generating new knowledge on the mechanisms of gene expression and regulation.
By the end of the project, OCTOBER will drastically contribute to the field, by providing insights into the mechanisms of choice between somatic and germline differentiation of embryonic stem cells, a subject that is still largely unknown. Moreover, it will determine the distinct roles that TFs like OCT4 have in different cell types, generating new knowledge on the mechanisms of gene expression and regulation.