Studying potential interplay between active demethylation and WT1-dependent transcriptional regulation during glial differentiation.

Lead Research Organisation: University of Nottingham
Department Name: School of Medicine

Abstract

Cellular differentiation is governed by the switching on or off of certain genes. Methylation of cytosine contributes to the regulation of gene activity. Old patterns of DNA methylation (5-methylcytosine, 5mC) are erased and new ones are established during differentiation but it is still unclear how 5mC is removed from DNA. We showed that 5mC is being specifically modified during early brain development, which leads to its subsequent removal from certain regions of DNA in neurons and glial cells, suggesting a specific mechanism for DNA demethylation. This, most likely, contributes to the reprogramming of undifferentiated cells into mature neurons and glial cells. WT1 (Wilms' tumour 1) is a protein, which, according to several reports and our preliminary data, may interact with this modified form of 5mC and may be important for brain development. The aim of this proposal is to understand the biological significance of potential interplay between WT1 and modified 5mC taking place in embryonic brain. We propose to study the mechanisms of this process using mouse embryonic stem cells (mESCs) as a model. We will differentiate mESCs into glial lineages and will identify the genes which are being modified during glial differentiation. Moreover, we will compare these genes with the genes which are regulated by WT1 during glial differentiation. Finally we will test how the depletion of WT1 and proteins involved into the 5mC modification affect glial differentiation. The proposed research program would have multiple implications for basic science, cancer research and regenerative medicine.

Technical Summary

Our previous results suggest that both active demethylation and 5caC-dependent transcriptional regulation are involved in epigenetic reprogramming taking place during lineage specification of NSCs. WT1 protein has also been recently implicated into both recruiting Tet proteins to their target sequences and specific recognition of 5caC-containing DNA substrates. Moreover, our pilot experiments revealed that WT1 regulates the same key glial markers which transiently accumulate 5caC in differentiating NSCs. There may be 2 main modes of WT1 interaction with the DNA methylome in embryonic brain: (1) WT1 may participate in recruiting TET2 and/or TET3, which both are expressed in embryonic brain, to their respective target sequences and/or (2) WT1 may directly bind to 5caC-enriched promoters causing activation or repression of corresponding genes.

The aim of this proposal is to acquire a mechanistic insight into the roles of Tet-dependent DNA demethylation and WT1 transcriptional regulation and their potential interplay during glial differentiation.

Specifically, we propose (1) to determine the distribution of oxi-mCs during glial differentiation and potential dependence of active demethylation on recruiting Tet proteins by WT1, (2) to compare the patterns of genomic distribution of WT1 with those of oxi-mCs in differentiating NSCs addressing the possibility that this transcriptional factor directly interacts with 5caC-enriched DNA and (3) to identify potential impacts of Tet-dependent 5mC oxidation and WT1 transcriptional regulation on glial differentiation.

Understanding the roles of both DNA demethylation and WT1 in glial differentiation would have multiple implications for basic research in the fields of epigenetics, developmental biology, neuroscience, regenerative medicine and cancer biology.

This work will directly address the BBSRC priorities of "Healthy ageing across the lifecourse", "Data driven biology" and "Systems approaches to the biosciences".

Planned Impact

Our work will contribute to advancing the areas of regenerative medicine, health science, pharmaceutical industry and biotechnology. We identify the following groups of stakeholders who will directly benefit from the outcomes of our research:

1. Stem cell technology and biotechnology companies. Our research will get an insight into epigenetic reprogramming taking place during lineage specification in embryonic brain. This will expand understanding of the mechanisms of neural and glial lineage specification in mammlian brain, which will lead to modification and refinement of existing protocols of ESCs differentiation and potentially will provide new strategies for differentiation of ESCs into specific lineages.

2. Clinicians working in the field of cancer, particularly in the field of pathogenesis of brain tumours. Since both the levels of oxi-mCs and expression of WT1 are altered in a number of cancers, including gliomas, our findings will facilitate the development of novel cancer treatments and diagnostic approaches for brain tumours.

3. Clinical neurologists and psychiatrists. As emerging evidence suggests important roles for 5hmC and Tet proteins in the development of certain psychiatric conditions (e. g. schizophrenia, bipolar disorder) our work will contribute to decoding the mechanisms of the pathogenesis of these disorders and will advance the establishment of novel approaches for their identification and therapy.
 
Description 1. Our results indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals. 2. We demonstrated that both 5caC enrichment and elevated TET1 expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin. 3. We developed methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy. 4. We have generated the genome wide datasets of WT1 and 5caC and are in the process of their analysis. 5. We proposed a new model of the interplay between TETs and WT1 that may be operational in brain tumors and during embryogenesis.
Exploitation Route Our findings may be of interest for epigenetics, stem cell and developmental biology fields leading to refinement of stem cell differentiation protocols to be potentially used in regenerative medicine. Our results may also contribute to development of novel approaches for diagnosis and therapy of the brain tumours.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description MRC IMPACT DTP PhD Studentship
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Medical PhD Scholarship
Amount £75,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 10/2020
 
Title Methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy 
Description We developed methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy. Specifically,these are the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact These techniques may be operational in evaluating the levels and localization of DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans. 
URL https://www.jove.com/video/56318/immunostaining-for-dna-modifications-computational-analysis-confoca...
 
Description LCMS detection of modified DNA bases 
Organisation Nicolaus Copernicus University in Torun
Department Department of Clinical Biochemistry
Country Poland 
Sector Academic/University 
PI Contribution We provide DNA samples isolated from differentiating neural stem cells to our partners.
Collaborator Contribution Our partners perform mass spec detection of modified DNA bases including oxidised forms of 5mC in the DNA.
Impact We now have an opportunity to analyse our samples for the presence of 5caC/5hmC/5mC using high sensitive LCMS developed by the group of Professor Ryszard Olinski.
Start Year 2017
 
Description Mass spec detection of oxidized forms of 5mC 
Organisation New England Biolabs
Country United States 
Sector Private 
PI Contribution access to DNA samples isolated from cells at various stages of differentiation
Collaborator Contribution mass spec detection of DNA modifications
Impact Research paper: Lewis LC, Lo PCK, Foster JM, Dai N, Corrêa IR Jr, Durczak PM, Duncan G, Ramsawhook A, Aithal GP, Denning C, Hannan NRF, Ruzov A*. (2017). Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification. Epigenetics, In press.
Start Year 2016