Mechanistic understanding of Cohesin-mediated genome organization
Lead Research Organisation:
University College London
Department Name: Cancer Biology
Abstract
Our view of the regulatory genome has changed dramatically in recent years. We have expanded beyond classical models of gene control to appreciate that the spatial organization of the genome and the manner in which genes and regulatory elements are embedded therein has a critical role in facilitating the regulation of gene expression. How gene control can be dictated by the three-dimensional (3D) organization of chromatin in nuclear space is only just beginning to be addressed, however this exciting new research area is already transforming our understanding of the mechanisms that regulate gene activity.
We use molecular methods (Hi-C, 4C-Seq, ChIP) and computational analysis to investigate the 3D organisation of mammalian chromosomes and the influence of this complex organization on biological processes such as gene regulation and cellular development. Our work has shown that the chromatin proteins, CTCF and cohesin are functionally important to anchor chromatin loops both for the purpose of gene regulation (Hadjur S et al., Nature 2009; Parelho V & Hadjur S et al., Cell 2008) as well as for global chromosome structure (Sofueva S et al. EMBO J 2013). We have also shown that the genome encodes chromosome structure at evolutionarily conserved, directional CTCF motifs in the genome (Vietri Rudan M. et al. Cell Reports 2015).
We use molecular methods (Hi-C, 4C-Seq, ChIP) and computational analysis to investigate the 3D organisation of mammalian chromosomes and the influence of this complex organization on biological processes such as gene regulation and cellular development. Our work has shown that the chromatin proteins, CTCF and cohesin are functionally important to anchor chromatin loops both for the purpose of gene regulation (Hadjur S et al., Nature 2009; Parelho V & Hadjur S et al., Cell 2008) as well as for global chromosome structure (Sofueva S et al. EMBO J 2013). We have also shown that the genome encodes chromosome structure at evolutionarily conserved, directional CTCF motifs in the genome (Vietri Rudan M. et al. Cell Reports 2015).
People |
ORCID iD |
| Suzana Hadjur (Primary Supervisor) | |
| Samuel Weeks (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/R506199/1 | 01/10/2017 | 31/03/2022 | |||
| 1990686 | Studentship | BB/R506199/1 | 01/10/2017 | 31/03/2022 | Samuel Weeks |
| Description | Embryonic Stem (ES) cells represent a pluripotent phase of development, with the capacity to differentiate into all mature cell types. The balance between ES cells maintaining pluripotency and entering differentiation is finely regulated by epigenetic regulation of gene expression. A recently emerging form of epigeneitic regulation is genome looping, whereby chromatin loops within the nucleus bring distal enhancer or silencer elements into proximal 3D space with their target gene promoters to facilitate upregulation or repression of gene expression. This process is partly mediated by the ring-shaped cohesin complex, comprising of Smc3, Smc1a, Rad21 subunits which encircle a strand of DNA to anchor the loop. Genome organisation in ES cells is thought to be very 'fluid' with few genes being repressed as heterochromatin and hence the dynamic cohesin looping between cells and may be greatly responsible for ES cells being able to maintain their pluripotent gene expression profile, while having the propensity to differentiate if their looping structures alter. So far, my research has found that the highly homologous Stag proteins (Stag1 and Stag2) which associate with cohesin in a mutually exclusive manner to facilitate genome organisation, are themselves dynamic in their expression. As soon as ES cells begin to differentiate, Stag1 expression is down-regulated while Stag2 expression is up-regulated, prompting the hypothesis that these highly similar proteins are conducting heterogenous roles in organising the genome to influence different cell fates. We have now confirmed this to be the case as upon knock-down of Stag1 in ES cells, there is global loss of pluripotent gene expression coinciding with upregulation of genes associated with cell fate specification. To further complex this, I have found that a third form of Stag protein, Stag3, is also expressed at significant levels within these cells and is able to associate with cohesin during its role in genome organisation. Previously, Stag3 has only been observed to be expressed as a meiosis-specific Stag protein with roles in facilitating cell division of germ cells. The fact that this protein (which has considerable divergence in its peptide sequence compare to Stag1 and Stag2) is present in ES cells implies a greater variety of Stag-bound cohesins within ES cells that could have (as we have already begun to uncover between Stag1 and Stag2) dramatically different impacts on influencing gene expression and, through this, cell fate. |
| Exploitation Route | ES cells share similar properties to cancer cells- both are able to self-replicate, and both are able to change their cell identity. The main difference between them is that ES cells do this in a controlled manner that relies on environmental stimulus and epigenetic regulation of gene expression profiles, while cancer cells have become deregulated, act independently of their 'natural' environmental niche and instead adhere to Darwinian-style survival. Understanding the role of Stag proteins in correctly co-ordinating cell fate has important implications on our understanding of how cancer cells can become de-regulated in this process. This is particularly important with the fact that Stag2 is one of only 12 genes that is frequently mutated in more than 4 different types of cancer; it is therefore essential to understand how a balanced expression of Stag proteins influence cell identity. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Keystone Symposia Scholarship for attending 'Higher Chromatin Organisation in Space and Time' conference. |
| Amount | $1,200 (USD) |
| Organisation | Keystone Symposia on Molecular and Cellular Biology |
| Sector | Charity/Non Profit |
| Country | United States |
| Start | 03/2020 |
| End | 03/2020 |
| Title | CRISPR-tagged Mouse Embryonic Stem Cells |
| Description | To address my hypotheses, I have generated ES cell lines that are engineered at their Stag1 and Stag3 genes with green and red fluorophores, respectively, using CRISPR technology. I have also added an FKBP subunit to either Stag1 or Stag3 in each cell line which, upon the addition of the dTag drug, recognises this compound and rapid processes the protein it is tagged onto for degradation. The cell lines I have developed in mouse embryonic stem cells are: -Stag1-mNeonGreen-V5-FKBP/Stag3-mScarlet -Stag1-mNeonGreen-V5/Stag3-mScarlet-3xHA-FKBP |
| Type Of Material | Cell line |
| Year Produced | 2019 |
| Provided To Others? | Yes |
| Impact | Using these cell lines, we have been able to visualise differences in the localisation of Stag1 and Stag3 proteins within the nucleus and understand the heterogeneity of their expression in individual cells of a homogenous ES population. We have also been able to observe the effects, both in terms of gene expression change and phenotypic change in cell identity, upon knock-down of the FKBP-tagged Stag protein. |
| Description | Astrazeneca CASE Industrial Partnership |
| Organisation | AstraZeneca |
| Department | Research and Development AstraZeneca |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | As part of a CASE PhD, AstraZeneca are my industrial partner and a member of their research team acts as my point of contact and secondary supervisor. As such, their role is to work with my academic institution in the development of my PhD. My contributions to this partnership is sharing of knowledge, specifically educating my secondary supervisor in the background of cohesin biology, and openly sharing any results from my research. |
| Collaborator Contribution | From this partnership, I have principally received expertise and advise that my secondary supervisor has provided to further my PhD. This advise is usually imparted through email, monthly skype sessions and visits to AstraZeneca. Such advise includes: -Methods and optimisation techniques for generating a cell line with CRISPR-mediated genetic knock-ins for fluorophore tagging of Stag proteins and conditional knock-down of these proteins using dTag. -Mer-FISH methods of visualising and quantifying single cell heterogeneity in RNA expression of multiple cohesin components (specifically Stag1, Stag2, and Stag3) simultaneously. This technique is aimed to be over-laid with visualising RNA markers of cell fate to better understand the relationship between ratio of Stag paralog expression and cell fate preference. My supervisor has also provided access to microscopy tools housed at AstraZeneca that will enable us to visualise fluorescently tagged Stag1/2/3 RNAs. |
| Impact | Through taking on advise in optimising CRISPR knock-in for primary cells, I have now generated my first cell line: mouse Embryonic Stem Cells (mESC) that are tagged with an mNeonGreen fluorophore and a mutant FKBP subunit on the c-terminal end of Stag1 protein. Upon addition of dTag, this drug recognises and binds to the FKBP subunit, which in turn directs degradation of the adjoining Stag1 protein. The role of Stag1 protein expression in maintaining embryonic stem cell fate can as well as the single cell heterogeneity of Stag1 expression (through use of the green fluorophore) can now be assessed. |
| Start Year | 2017 |
| Description | Centre of Regenerative Medicine |
| Organisation | University of Edinburgh |
| Department | MRC Centre for Regenerative Medicine |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We shared our findings and data on cohesin diversity and its potential roles in influencing pluripotent cell fate with the research groups headed by Steve Pollard and Sally Lowell. |
| Collaborator Contribution | We reached out to Pooran Dewari of Steve Pollards group to gain expertise on a protocol for CRISPR Knock-In of large constructs that had been described by Dewari et al. in 'An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein'. Pooran provided us with detailed advise of how best to maximise our knock-in efficiency. He also helped us to design guide RNAs to target the 3 prime coding region of Stag1, Stag2 and CTCF, as well as a simple knock-in construct for a V5 tag. |
| Impact | This collaboration has resulted in developing a simple and effective protocol I use for effective knock-in of both small and large constructs and, as such, has enabled me to produce V5/3xHA/fluorophore-tagged cell lines with CRISPR technology. |
| Start Year | 2019 |
| Description | Karolinska Institute |
| Organisation | Karolinska Institute |
| Country | Sweden |
| Sector | Academic/University |
| PI Contribution | We have shared our data on differing expression of Stag1, Stag2, and Stag3 proteins in pluripotent embryonic stem cells and have proposed to them our hypothesis that the balanced ratio of these proteins is important for influencing cell fate decisions. |
| Collaborator Contribution | Our collaborators have conveyed their expertise in smFISH- a technique using fluorescently labelled oligos to tile along multiple RNAs in fixed cells to quantify mRNA abundance of different genes within a cell, and measure the variation of this abundance within individual cells of a homogenous population. In addition, our collaborators have designed and made an oligo pool set for Stag1, Stag2, Stag3, Smc3, and pluripotent and totipotent markers so that we can investigate the heterogeneity of their mRNA expression within ES cells and try to correlate certain ratios of Stag paralog mRNAs to markers of different cell fates. |
| Impact | We now have the tools and knowledge to implement an smFISH experiment. |
| Start Year | 2019 |
| Description | UCL Cancer Institute Mass Spectroscopy Facility |
| Organisation | University College London |
| Department | UCL Cancer Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have been interested in protein variants for Stag paralog proteins, and also wished to gain a comprehensive overview of their protein interaction partners. Therefore, we reached out to the UCL Cancer Institute Mass Spectroscopy facility to set up a collaboration with them. I shared this information with the facility team and conducted the preparation work for the Mass Spec (cell culture/chromatin protein isolation/immunoprecipitation/coomassie staining/trypsin-mediated peptide digestion). |
| Collaborator Contribution | The Mass Spectroscopy facility shared their expertise in using digestion enzymes to enable us to identify peptides that would be unique to Stag protein variants. They ran our samples on the analyser and presented us with the peptide data for Stag1, and the data for its interacting protein partners that were also pulled down in the Stag1 immunoprecipitation and detected by the Mass Spec. |
| Impact | We were successfully able to identify Stag1 protein variants and found interesting protein interaction partners of Stag1 from the Mass Spectroscopy data that fuelled our hypothesis into Stag1's role in cell fate. This information will be published in a future paper. |
| Start Year | 2020 |
| Description | London Cancer Network- PhD Short Talks |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Termly networking events are arranged between London cancer institutes with each institute nominating a student to present his or her work to the audience in a short 15minute Chalk Talk. I gave a talk for the UCL Cancer Institute in November 2019 that focused on conveying my current research in cohesin biology and the role of Stag proteins in cell fate determination. The aim of these meetings is to expand knowledge of PhD fields, receive feedback from peers of different backgrounds and Institutes, and increase networking opportunities. |
| Year(s) Of Engagement Activity | 2019,2020 |
| Description | London Stem Cell Network Conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | This conference was set up by stem cell researchers in different London institutions to share research from an expansive field of development biology. Expert researchers from a range of institutions present their work in this discipline. There is also a research poster session. The main outcomes are to network and gain further insight into cutting edge stem cell research. |
| Year(s) Of Engagement Activity | 2018,2019 |
| Description | UCL Cancer Institute Annual Conference |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | The day-long event involves presentations from leaders of research within the UCL Cancer institute to convey their current research and findings. In addition, there is an external keynote speaker and discussions on how the institute is progressing with regards to areas such as increasing women in science and development of BSc and MSc courses associated with the institute. There are also poster sessions, which I have participated in to convey my research. The main outcome of this day is to network and keep up-dated on both the running of the institute and the research being conducted within it. |
| Year(s) Of Engagement Activity | 2017,2018,2019 |
| Description | UCL Cancer Institute Poster Day |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | The day-long activity enables PhD students to present research posters within the Cancer Institute to peers and a judging panel. This is followed by an award ceremony. The main outcomes are to network and gain insights into what makes a good poster. |
| Year(s) Of Engagement Activity | 2018,2019 |