Mechanisms of regulation of RNA polymerase II phosphorylation in embryonic stem cell pluripotency and neuronal differentiation
Lead Research Organisation:
Imperial College London
Department Name: Institute of Clinical Sciences
Abstract
Stem cell therapies promise cures for a plethora of complex diseases such as cardiac, infertility and neurodegeneration. Embryonic Stem cells (ESCs) have the potential to differentiate into any of the 200 different cell types that make up a higher organism. Complex regulatory mechanisms are present to maintain genes important for cell commitment silent in ESCs, but in a metastate which is compatible with activation upon differentiation. Towards devising efficient and safe stem cell procedures, we, and others, are interested in understanding the control mechanisms that maintain important developmental regulator genes silent in ESCs, and the dynamic changes that occur during cell differentiation. Our group has recently shown that an important cohort of developmental regulator genes, including genes that orchestrate commitment into neurons, are poised for activation in ESC. The poised state is characterized by association of silent genes with active and repressive chromatin, and with RNA Polymerase II, the enzyme that initiates the cascade of gene expression by transcribing the genetic information contained in the DNA into RNA molecules that serve as template for protein synthesis. We have found that developmental regulator genes, which are silent in ESCs, are actively transcribed but associated with polymerase II in a novel 'poised' conformation which uncouples the process of transcription from downstream events in gene expression. The presence of poised polymerase II complexes at developmental regulator genes maintains these genes silent in ESCs, but primed for induction in response to differentiation signals. With this proposal, we aim to understand the mechanisms of gene poising in ESCs and the dynamic changes in polymerase and chromatin conformation that accompany gene expression reprogramming during neuronal differentiation, from ESCs to neural stem cells, to fully differentiated dopaminergic neurons. At poised developmental regulator genes, we aim to investigate the activity of molecular complexes known to modulate polymerase II conformation, either by hindering its elongation during transcription, by preventing its phosphorylation, or by actively increasing dephosphorylation. Polymerase II is crucial to integrate events in the cascade of gene expression, as it not only transcribes the DNA sequence, but also recruits nuclear machineries which modify chromatin to make it compatible with transcription and RNA processing activities which promote the maturation nascent transcripts and transport out of the nucleus for protein synthesis. This research will provide important insights into the mechanisms of chromatin regulation that maintain the pluripotent program of gene expression in ESCs, to help understand the mechanisms of induced pluripotency and lineage commitment towards the design of cell therapies. We also aim to elucidate the roles of RNA polymerase II modification in integrating transcription with RNA processing and chromatin states, to help understand the basic mechanisms of gene regulation during differentiation.
Technical Summary
The differentiation from ES to specialized cells is one of the most important fields of research in modern cell biology. To unveil the mechanisms by which programs of gene expression are established during differentiation represents a key challenge towards the development of stem cell therapies and differentiation protocols into specific cellular lineages. Recent work has shown that important cohorts of developmental regulator genes are silent in ESCs but primed for activation upon differentiation. We and others have shown that the poised state reflects an equilibrium between Polycomb silencing by histone modification and the presence of poised RNAPII complexes, which elongate through coding regions, but lack Ser2 phosphorylation which uncouples transcription from downstream RNA processing events such as RNA splicing and polyadenylation. Poised RNAPII have not yet been identified in lineage-committed cells, but are likely to occurs since Polycomb silencing is essential during development. The molecular mechanisms that regulate the poised state of RNAPII at developmental regulator genes in ESCs are still unknown. The aims of this proposal are to: 1) investigate dynamic changes in RNAPII phosphorylation during neuronal commitment of ESCs in neural stem cells and post-mitotic neurons, and 2) to address the mechanisms of RNAPII poising regulated by the modulators of RNAPII activity in ESC pluripotency, using a combination of ChIP, siRNA, and transcriptome analyses. The Systems Approach proposed to integrate epigenetic and expression data obtained here during neuronal commitment and upon interference with the regulatory networks that control RNAPII activity in ESCs will provide important insights about the chromatin priming mechanisms that are active in ESCs. The accomplishment of these objectives will clarify important transcriptional mechanisms of outstanding interest in the biology of pluripotent cells and in the basic mechanisms of gene regulation.
Planned Impact
Neurological diseases represent a growing cause of mortality and morbidity in the western world with important socio-economic and welfare impact. Although hereditable mutations on specific genes are often associated with these diseases, most are multifactorial and complex diseases, where the mechanisms underlying the diversity of pathological states are still unknown. A major challenge in devising efficient clinical strategies is still the lack of understanding of the complex molecular interactions associated with normal cell function. Dysregulation of transcriptional mechanisms is observed in several neurological disorders, including Huntington's, Alzheimer's, Amyotrophic Lateral Sclerosis and Rett Syndrome. Our proposal addresses basic aspects of RNAPII regulation at important developmental regulator genes in ESCs and during neuronal differentiation. Understanding gene regulation in stem cells is particularly important towards the development of efficient and safe therapeutic approaches to treat neurological disorders. We propose a systems approach that takes advantage of large whole genome datasets of gene expression and chromatin occupancy by regulatory factors which combines the generation of novel datasets with the integration of publicly available resources, across the whole genome in ESCs, neural stem cells (NSCs) and dopaminergic neurons. The resulting datasets will be of value to academic and industry partners. The research program will establish a pipeline for integrating a whole range of chromatin changes related with gene expression states that occur at multiple scales during normal neuronal development. We and others have interests in studying changes in gene expression and chromatin states during neurodegeneration and so far there is no available data of RNAPII conformation or about the roles of RNAPII modulators in the normal state, before mouse models of disease or human samples can be addressed. The research program combines experimental and computational approaches, which will provide a rich scientific environment for training the new generation of scientists in the current post-genomic era. The proposal also allows us to establish novel collaborations within the Clinical Sciences Division (ICL), with Drs Jesus Gil and Meng Li that bridge between the themes of stem cells and neuronal specification, and make our basic research skills in chromatin and RNAPII regulation relevant in the fields of neuroscience. The immediate beneficiaries of the proposed research will be mostly research scientists engaged in basic academic research. However, our research program on chromatin state and RNAPII conformation will provide important insights into chromatin regulation in ESCs and during neuronal cell commitment, towards improving the design of induced pluripotency and differentiation protocols for stem cell therapies. Our datasets will provide an important resource for the scientific community, but also have an industrial and economical benefit towards developing stem cell therapies in drug screens. We will disseminate the results and conclusions of the research to broader audiences. We will present the work at meetings supported through the grant, and at invited seminars in various institutions. We will publish our findings in international peer-reviewed journals with the best possible impact factors, and make our datasets immediately available to the community at the time of publication. Moreover, we work with science journalists to submit press releases of our most prominent publications in science databases such as AlphaGalileo, which then reach the wider public. Imperial College London, and locally at the MRC-CSC, have in place a press release office which endeavor the public dissemination of the most relevant discoveries. We will use that communication system to reach the goal of engaging a wide public audience.
Organisations
- Imperial College London (Lead Research Organisation)
- Institute of Biology (IBENS) (Collaboration)
- UNIVERSITY OF EDINBURGH (Collaboration)
- McGill University (Collaboration)
- RIKEN (Collaboration)
- Medical Research Council (MRC) (Collaboration)
- Helmholtz Association of German Research Centres (Collaboration)
People |
ORCID iD |
Ana Pombo (Principal Investigator) |
Publications
Arner E
(2015)
Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells.
in Science (New York, N.Y.)
Ferrai C
(2010)
Poised transcription factories prime silent uPA gene prior to activation.
in PLoS biology
Ferrai C
(2017)
RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation.
in Molecular systems biology
Ferrai C
(2010)
Gene Positioning
in Cold Spring Harbor Perspectives in Biology
Fraser J
(2015)
Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.
in Molecular systems biology
Noguchi S
(2017)
FANTOM5 CAGE profiles of human and mouse samples.
in Scientific data
Description | Impact has been mostly fully achieved. A review was published at the onset of the funding period. Four publications have been published. |
Exploitation Route | The findings are of interest for basic research in understanding stem cell biology and neuronal differentiation pathways, mechanisms of gene regulation by Polycomb Repressor Complexes, and provide a collection of datasets that will be openly available to the scientific community. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | BBSRC Project Grant (Mechanisms of regulation of RNA polymerase II phosphorylation in embryonic stem cell pluripotency and neuronal differentiation) |
Amount | £407,817 (GBP) |
Funding ID | BB/H008098/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2010 |
End | 02/2013 |
Description | Imperial College London Incentivizing the Faculty Scheme |
Amount | £60,000 (GBP) |
Organisation | Imperial College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 03/2010 |
End | 02/2013 |
Description | Wellcome Trust, VIP award (to CF) |
Amount | £14,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2009 |
End | 11/2009 |
Title | Datasets published in Ferrai et al 2015 |
Description | We produced genome-wide datasets for Hi-C and CAGE from mouse embryonic stem cells, neuronal precursor cells and in-vitro differentiated day-16 dopaminergic neurons. |
Type Of Material | Technology assay or reagent |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | GSE59027 PMID: 26700852 |
URL | https://www.embopress.org/doi/full/10.15252/msb.20156492 |
Title | Datasets published in Ferrai et al 2017 |
Description | We produced genome-wide datasets for ChIP-seq for RNAPII modi?cations (S5p, S7p), histone modi?cation H3K27me3, and genome wide transcriptomes (total RNA-seq) in mESCs, day1 and day3 differentiation from mESCs, d16 and d30 dopaminergic neurons differentiated from mESCs. |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | PMID: 29038337 GSE94364 |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94364 |
Title | FANTOM5 CAGE profiles of human and mouse samples. |
Description | FANTOM5 CAGE profiles of human and mouse samples. |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | Datasets for transcriptional start sites across a collection of human and mouse samples. |
URL | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574368/ |
Title | Ferrai et al. 2017 ChIP-seq and RNA-seq datasets |
Description | Ferrai et al. 2017 ChIP-seq and RNA-seq datasets |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | Description of promoter activations states and gene expression from pluripotent stem cells to early differentiation, and terminal dopaminergic neurons |
URL | http://msb.embopress.org/content/13/10/946.long |
Title | Fraser et al. 2015 - CAGE and Hi-C datasets |
Description | Raw reads for the ESC, NPC and Neuron Hi-C datasets generated for this study are available online from GEO, accession number GSE59027. CAGE data used in this study were produced as part of the FANTOM5 project, and all FANTOM5 sequence data have been deposited at the DNA Data Bank of Japan (DDBJ) under accession numbers DRA000991, DRA002711, DRA002747 and DRA002748. Additional analysis, documentation and visualizations of the CAGE data are available at http://fantom.gsc.riken.jp/5/tet/ under "ES-46C embryonic stem cells, neuronal differentiation, day00, biol_rep1.CNhs14104.14357-155I1" (ESCrep1); "ES-46C embryonic stem cells, neuronal differentiation, day00, biol_rep2. CNhs14109.14362-155I6" (ESCrep2); "ES-46C derived epistem cells, neuronal differentiation, day05, biol_rep1. CNhs14126.14378-156B4" (NPC) and "ES-46C derived epistemcells, neuronal differentiation, day14, biol_rep1. CNhs14127.14379-156B5" (Neurons). |
Type Of Material | Database/Collection of data |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Manuscript has been cited 27 times. |
URL | http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59027 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | Helmholtz Association of German Research Centres |
Department | Berlin Institute for Medical Systems Biology |
Country | Germany |
Sector | Public |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | Institute of Biology (IBENS) |
Department | Department of Biology |
Country | France |
Sector | Academic/University |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | McGill University |
Department | Department of Neurology and Neurosurgery |
Country | Canada |
Sector | Hospitals |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | Medical Research Council (MRC) |
Department | MRC Clinical Sciences Centre (CSC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | RIKEN |
Department | RIKEN Preventive Medicine & Diagnosis Innovation Program |
Country | Japan |
Sector | Private |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Hierarchical organization of long-range chromatin contacts in stem cells, neuronal progenitor cells and neurons |
Organisation | University of Edinburgh |
Department | Clinical Psychology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We designed the project, set up differentiation of neuronal cells in large scale for analyses of chromatin contacts by chromosome conformation capture (3C) approach Hi-C, validated cell sample purity, produced gene expression data. |
Collaborator Contribution | Partners contributed to the project design, performed experiments to produce Hi-C maps, their bioinformatic and computational analyses, bioinformatic analyses of gene expression data, polymer modelling and fluorescence in situ hybridization. |
Impact | Fraser J*, Ferrai C*, Chiariello AM*, Schueler M*, Rito T*, Laudanno G*, Barbieri M, Moore BL, Kraemer DCA, Aitken S, Xie SQ, Morris KJ, Itoh M, Kawaji J, Jaeger I, Hayashizaki Y, Carninci P, Forrest ARR, the FANTOM Consortium, Semple CA1, Dostie J1,Pombo A1, Nicodemi M1 (2015) Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Molecular Systems Biology 11, 852. (*joint first and 1joint corresponding authors) (1joint corresponding authors) |
Start Year | 2009 |
Description | Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells |
Organisation | RIKEN |
Country | Japan |
Sector | Public |
PI Contribution | We provided RNA samples for early ESC differentiation into neuronal lineage and the corresponding validation. |
Collaborator Contribution | All other work, including other cell samples, extracting RNA and producing CAGE data, and bioinformatics and computational analyses. |
Impact | http://dx.doi.org/10.1126/science.1259418 |
Start Year | 2009 |
Description | Chromatin dynamics during neural differentiation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Internal seminar on 17 Feb 2012, at the Berlin Institute for Medical Systems Biology, Max Delbrueck Centre, Berlin, Germany. No actual impacts realised to date. |
Year(s) Of Engagement Activity | 2012 |
URL | https://www.mdc-berlin.de/events/40551099/11254 |
Description | Conference invited talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | conference presentation |
Year(s) Of Engagement Activity | 2017 |
Description | Dynamic transitions in gene expression states during differentiation of ES cells to functional dopaminergic neurons. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | We obtained feedback from our peers towards our research and its publication in scientific journals. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.embl.de/training/events/2014/TRM14-01/ |
Description | Mechanisms of Polycomb repression are associated with poised RNAPII states during terminal neuronal differentiation |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Carmelo Ferrai presented a poster that shared our work with colleagues at the conference. |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.cph-bioscience.com/conferences/stem-cell-niche-development-disease |
Description | Steady, Ready, Go: mechanisms that prime RNA polymerase II for activation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | We shared our work with our colleagues at the Max Planck Institute for Biomedicine, at Muenster, Germany. |
Year(s) Of Engagement Activity | 2017 |
Description | The differentiating landscapes of RNAPII |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | We shared our work with our colleagues at the institute. We received feedback from our colleagues and our presentation promoted a new collaboration that adds value to the BBSRC funded project. |
Year(s) Of Engagement Activity | 2013 |
URL | https://www.mdc-berlin.de/41794582/en/bimsb/images_and_PDFs/BIMSB_retreat_2013_program.pdf |
Description | Transcriptional and Epigenetic Dynamics during ES cell differentiation in to dopaminergic neurons. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | The presentation sparked interest from our colleagues in the neurosciences field. We received positive feedback about our work and strategy from our colleagues in the neurosciences field.. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.bnf-info.de/general_information |