Transcriptional control of cell fate decisions by chromatin remodelling proteins
Lead Research Organisation:
University of Cambridge
Department Name: Wellcome Trust - MRC Cam Stem Cell Inst
Abstract
A cell's identity is defined by its genes. A cell located in the heart, for example, will express heart genes, but not genes important for liver or brain function. Conversely, liver cells express liver genes but not heart or lung genes. During embryonic development animals are formed by different groups of stem cells, which are able to give rise to the myriad of different cell types found in an adult organism. When stem cells undergo the process of differentiating into a new cell type, it is critically important that they turn on genes appropriate for the new cell type, but also turn off the genes that define a 'stem cell' identity and also keep off any genes associated with other cell types. Failure to properly regulate expression of all these different kinds of genes can result in the wrong decisions being made, or the inability to make any decision when required. The consequences of such failure can be developmental abnormalities or cancer. Unsurprisingly, then, our cells contain many proteins whose jobs are to ensure the proper control of gene expression. This proposal focusses on two such proteins, called Chd4 and Brg1. These proteins control the way genes get packaged within the cell. Though at a mechanistic level these two proteins appear to have similar activities, a number of studies have found that they actually tend to work in opposition. We know that loss of either protein results in failed development from very early stages, and both proteins are often found to be deleted or mutated in cancer. We propose that it is precisely this balance of opposing activities between Chd4 and Brg1 that fine tunes gene expression patterns, and that this balance is crucial for cells to successfully make developmental decisions. Yet exactly how the biochemical activities of these proteins controls gene expression, and whether the two really directly control the expression of the same genes during development is not well understood. In the work proposed herein we will use cutting edge technologies to very accurately define the function of these proteins in stem cells and in early embryonic cells as they make their very first decisions. We will show how loss of one either protein impacts the function of other key regulatory proteins, and how this leads to developmental failure. We will then examine how these proteins function during the very first cell fate decision in mammalian embryogenesis. Together this project will reveal how gene expression is controlled as cells make decisions, and how the cells use these proteins to precisely control cell fate choices during normal development. This will inform the creation of new methods for controlling gene expression in mammalian cells. Such methods could be of considerable benefit in efforts to prevent the progression of cancer, and to enable more efficient and effective protocols to use stem cells in regenerative medicine.
Technical Summary
A cell's identity is defined by its gene expression patterns, and gene expression potential is encoded in chromatin. Changes to cell state which occur during normal development require very precise changes in chromatin structure and gene expression. Failure to properly regulate gene expression patterns can prevent successful execution of developmental decisions, but can also lead to tumourigenesis. Chd4 and Brg1 are two mammalian chromatin remodelling proteins which use energy derived from ATP hydrolysis to alter the structure of chromatin and thereby influence gene expression patterns. Both proteins play essential roles in early mammalian development, and both proteins have been found to be mutated in a wide variety of different tumour types. A body of evidence indicates that these two proteins often act in opposition. I hypothesise that these two proteins normally act in opposition to precisely correlate gene expression patterns. This proposal will test this hypothesis in pluripotent cells undergoing cell fate decisions both in culture and during early mouse embryogenesis. We will provide precise molecular details of how control of enhancer chromatin structure by Chd4 and Brg1 impacts the binding and subsequent activities of enhancer-binding transcription factors, and how this then influences the activity of RNA polymerase at target promoters. Using super resolution microscopy, we will determine how chromatin remodellers control transcription factor binding kinetics in live cells in culture and in preimplantation stage embryos undergoing the first cell fate decision. We will further define how these chromatin remodelling systems are used by embryonic cells to drive developmental transitions. This project combines cutting edge imaging and single cell biology to reveal fundamental transcriptional mechanisms at unprecedented molecular detail, greatly increasing the resolution of our understanding of how cell fate decisions are enacted in mammalian cells.
Planned Impact
The work proposed herein will define at a molecular level how chromatin remodelling proteins impact gene expression, and how this activity is finely balanced to enable lineage decisions. We will also develop reagents and methodologies to study transcription factor behaviour in live cells. What sets our work apart from what has been done in the past is the inclusion of cutting edge imaging methodology (the development of which in the Laue lab was funded largely by the MRC) and validation of our in-depth molecular analyses in an in vivo system, the early mouse embryo. This will be of direct relevance to a broad range of researchers interested in chromatin biology, stem cells, development, and oncogenesis (See 'Academic Beneficiaries' section). More broadly, improved understanding of the complexities of gene expression, cellular differentiation and cancer progression is potentially relevant to specialists in different fields including history, archaeology and anthropology.
We will ensure the impact of our work is felt as widely as possible by promoting it at national and international meetings, and on social media, which can immediately reach thousands of scientists worldwide. Exposure and impact will be maximised by depositing manuscripts on the BioRxiv preprint server prior to publication, and by ensuring all published work is open access from the date of publication.
The work proposed is to investigate the functions of protein complexes known to play important roles in cancer initiation and/or progression, and therefore will contribute towards our understanding of how cells become tumourigeneic. This will provide mechanistic insights which could be exploited in the design of cancer therapeutics, thereby benefitting the commercial sector. Similarly, insights gained into the control of developmental decisions in pluripotent cells will be useful to those working to increase the utility of pluripotent stem cell-based applications. The indirect and long-term impact on therapeutics and patient care could be considerable.
Aside from our potential impact on future healthcare, we also plan for our research to have a more immediate impact on the wider public through a commitment to effective communication and public engagement. Many different non-specialists have a thirst for the latest information about gene expression; be they families with a history of a genetic condition, schools teaching developmental biology, or groups interested in genealogy. On the broadest level, public understanding of genetics is a wide-reaching social, cultural and political issue. It is important that as a society we have access to the latest research that may influence our thinking. The details of our commitments to sharing our research in an accessible and appropriate way are outlined in the attached 'Pathway to Impact'.
We will ensure the impact of our work is felt as widely as possible by promoting it at national and international meetings, and on social media, which can immediately reach thousands of scientists worldwide. Exposure and impact will be maximised by depositing manuscripts on the BioRxiv preprint server prior to publication, and by ensuring all published work is open access from the date of publication.
The work proposed is to investigate the functions of protein complexes known to play important roles in cancer initiation and/or progression, and therefore will contribute towards our understanding of how cells become tumourigeneic. This will provide mechanistic insights which could be exploited in the design of cancer therapeutics, thereby benefitting the commercial sector. Similarly, insights gained into the control of developmental decisions in pluripotent cells will be useful to those working to increase the utility of pluripotent stem cell-based applications. The indirect and long-term impact on therapeutics and patient care could be considerable.
Aside from our potential impact on future healthcare, we also plan for our research to have a more immediate impact on the wider public through a commitment to effective communication and public engagement. Many different non-specialists have a thirst for the latest information about gene expression; be they families with a history of a genetic condition, schools teaching developmental biology, or groups interested in genealogy. On the broadest level, public understanding of genetics is a wide-reaching social, cultural and political issue. It is important that as a society we have access to the latest research that may influence our thinking. The details of our commitments to sharing our research in an accessible and appropriate way are outlined in the attached 'Pathway to Impact'.
People |
ORCID iD |
Brian Hendrich (Principal Investigator) | |
Ernest Laue (Co-Investigator) |
Publications
Ragheb R
(2020)
Differential regulation of lineage commitment in human and mouse primed pluripotent stem cells by the nucleosome remodelling and deacetylation complex.
in Stem cell research
Montibus B
(2023)
The Nucleosome Remodelling and Deacetylation complex coordinates the transcriptional response to lineage commitment in pluripotent cells
in Biology Open
Title | Chromatin remodeller-inducible deletion cells |
Description | ES cell line in which specific chromatin remodelling proteins can be degraded at will in living cells. |
Type Of Material | Cell line |
Year Produced | 2019 |
Provided To Others? | No |
Impact | These cell lines will be made available upon request. |
Title | Inducible degron system plasmids |
Description | Plasmids have been created to rapidly degrade specific proteins in living cells, using published systems. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | No |
Impact | These reagents in instrumental to our ongoing work. Following a recent closed meeting with Japanese colleagues, we received a request for these reagents. Though we have not published these reagents we have provided them to our Japanese collaborators. They will be made available for distribution on Addgene when we are preparing for publication. |
Title | Monitoring protein behaviour in live cells |
Description | Mouse embryonic stem cell lines in which genes of interest have been edited to express endogenous alleles as fusions to SNAP or HALO proteins have been created. These are being used to monitor protein activity in live cells using single molecule imaging. Cell lines also have conditional alleles of NuRD complex components so that protein behaviour can be monitored with and without the chromatin remodeller. |
Type Of Material | Cell line |
Year Produced | 2018 |
Provided To Others? | No |
Impact | These cell lines are integral to the goals of our grant and are the subject of active research. They will be made generally available upon publication. |
Title | Monitoring transcription in live cells |
Description | Mouse embryonic stem cell lines have been edited to allow us to monitor transcription of specific genes in live cells. These lines also have conditional alleles for chromatin remodellers so that we can assess how these remodellers impact transcriptional dynamics. |
Type Of Material | Cell line |
Year Produced | 2018 |
Provided To Others? | No |
Impact | These novel reporters will be of considerable interest to the wider community and will be made available upon publication. |
Title | Targeting constructs - SNAP or HALO |
Description | We have created cloning vectors and targeting constructs to allow us to fuse HALO or SNAP to endogenous alleles of genes of interest |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | No |
Impact | These constructs will be of use to others wishing to use this technology and will be made available upon publication |
Title | Targeting constructs - transcription reporters |
Description | We have made cloning vectors and targeting constructs used to target transcription reporters to endogenous alleles of genes of interest. |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | No |
Impact | These will be of use to anyone wanting to use this technology and will be made available upon publication. |
Description | Anne Ferguson-Smith |
Organisation | University of Cambridge |
Department | Department of Genetics |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We will investigate transcriptional control in hybrid ES cell lines |
Collaborator Contribution | Provision of F1 intercross hybrid ES cell lines |
Impact | This collaboration has only just started so no outputs yet. |
Start Year | 2019 |
Description | David Rueda |
Organisation | Imperial College London |
Department | MRC London Institute of Medical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Targeting aptamer sequences in mouse ES cells for live cell imaging trials |
Collaborator Contribution | Providing RNA Aptamers for live cell imaging |
Impact | Vectors, targeting constructs, and targeted ES cell lines are being made |
Start Year | 2018 |
Description | Greg Findlay |
Organisation | University of Dundee |
Department | College of Life Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Joint grant application to the Wellcome Trust to expand and extend on findings from the MRC-funded project |
Collaborator Contribution | Will proivde expertise in phosphoproteomics and signalling in ES cells. |
Impact | Joint grant application |
Start Year | 2021 |
Description | Sabine Dietmann |
Organisation | Washington University in St Louis |
Department | School of Medicine |
Country | United States |
Sector | Academic/University |
PI Contribution | Joint grant application to the Wellcome Trust expanding and extending from findings made in current project |
Collaborator Contribution | Bioinformatics and machine learning applications |
Impact | Joint grant application |
Start Year | 2021 |
Description | Conference Participation |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Invited speaker. My talk sparked a lot of discussion, including an offer of collaboration from a colleague in Japan |
Year(s) Of Engagement Activity | 2022 |
Description | Genomic Medicine Optional Module 5: Epigenetics and Epigenomics 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | Gave a lecture to Healthcare Professionals about our work. |
Year(s) Of Engagement Activity | 2019 |
Description | Invited Seminar - Babraham Institute |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Invited seminar at the Babraham Institute. Included meetings with a range of scientists. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited seminar- Naples |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited seminar at IGB Naples. Roughly 80-100 attendees as well as informal visits to different labs and discussions with various scientists. |
Year(s) Of Engagement Activity | 2018 |
URL | http://www.igb.cnr.it/ptimages/SEMINARLIST2018.pdf |
Description | Seun Filming with BBC |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Seun Ogundele: Filming with the BBC at the Institute to talk about #dishlife, an interactive game that allows players to see what it's like to work with stem cells. Behind the scenes pictures uploaded to IG: https://www.instagram.com/p/B8_atuzJqFw/ |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.instagram.com/p/B8_atuzJqFw/ |
Description | SeuninScience Youtube Channel |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Undergraduate students |
Results and Impact | Group member Oluwaseun Ogundele launched a You Tube channel to make videos about her experiences being a BAME woman in science, hoping to inspire younger BAME people to consider a career in academic research |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.youtube.com/channel/UCeky9avt81tiHJuqE_RGzrg/about |
Description | Short videos produces about stem cells |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | In recognition of #stemcellawarenessday on the 9th October 2019. #openupstemcells was a digital engagement action designed and led by public-facing organisation, EuroStemCell, of which Cambridge Stem Cell Institute is a member. Worked with the Institute to film stem cell researchers answering public questions. The films were released consecutively each week during the month of October on EuroStemCell social media channels, and shared by partner Institutions across the network. A repository of all the films is now freely available on eurostemcell.org, a hub for people looking for reliable information on stem cell therapies and current research https://www.youtube.com/watch?v=45S4sN1ApcE https://www.youtube.com/watch?v=xSgsMbEmvmA https://www.youtube.com/watch?v=K4xb2ETUIJM https://www.youtube.com/watch?v=PIC2U_T76yY |
Year(s) Of Engagement Activity | 2019 |
Description | TRR81 Symposium 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Attended and presented at the 5th International Symposium on "Chromatin Changes in Differentiation and Malignancies" |
Year(s) Of Engagement Activity | 2019 |
Description | Virtual Seminar with Fragile Nucleosome |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Seminar presentation online |
Year(s) Of Engagement Activity | 2023 |
URL | https://harvard.zoom.us/webinar/register/2416746614019/WN_QNhI5xhHQOa3oVQ-DxRk0w |
Description | Women in STEM |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Seun Ogundele featured on on the University's website as part of the Women in STEM series. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.cam.ac.uk/research/news/women-in-stem-oluwaseun-ogundele?ucam-ref=read-this-next |
Description | Work experience Student |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | We hosted a 6th Form student for a week of work experience in the lab. This involved him becoming familiar with lab life, culturing mammalian cells, performing simple experiments and attending seminars. |
Year(s) Of Engagement Activity | 2018 |