"Bivalent Chromatin and cell fate determination" – BIVANDEV

To perform this project we use a key developmental process, the genomic imprinting, as a model as we recently determined that bivalent domains are involved in the control of tissue-specific imprinting. We further identified several imprinted genes controlled by bivalent domains specifically during neural lineage commitment, constituting a panel. To evaluate mechanism involved in the control of this panel of bivalent domain we developed an in vitro differentiation model that resumes events occurring during normal neural lineage commitment in mouse, from embryonic stem cells and upon differentiation to neuronal and glial cells. By this mean we can precisely monitor the outcome of bivalent domain during this differentiation process and investigate the role of both cis and trans acting factors in their control. Role of relevant factors is further evaluated by specifically inactivating and or over-expressing them in several cell lines.
Main outcome of this first part enable us to delineate and focus our analyses aiming to identify the causes of inappropriate regulation of bivalent domains in human pathologies. In line with our work conducted on neural lineage in mouse, we investigate for misregulation of bivalency on samples obtained from a brain tumor bank, that enable us to have detailed clinical and histophatological features for each analyzed tumor detailed. In parallel, we are studying the regulation of bivalent domains at key developmental stages, during germline reprogramming. To ensure the proper development of a newly formed organism, epigenetic program must be erased and reset between generations. This epigenetic reprogramming takes place during development of primordial germ cells of both sexes. To perform this study we are using “scaled-down” molecular approaches that we recently developed and that enable us to monitor the dynamic of bivalent domain during mouse germline commitment in vivo and identify factors involved in their control.

Our work reveals that bivalency is involved in fine-tuned regulation of imprinted genes, particularly in neural lineages. This observation provides a frame to further investigate the influence of imprinted genes on neural development, and, on a clinical point of view , stress that deregulation of bivalency has now to be taken in consideration to apprehend causes of imprinted disorders. In addition, we showed that bivalent structures are the target of alterations in malignant glioma with, as a main consequence, an ectopic expression of the associated genes. This original observation is of importance in the frame of the development and use of chemical that aim to correct epigenetic-based defect in cancer cells.

This project will provide valuable information and original information on mechanism controlling cell fate determination during differentiation. Specifically, results expected from this proposal will represent a significant step forward on our understanding of fine-tune regulation of tissue-specific imprinting. This will have clear implication for human health. Deregulation of imprinted genes has indeed frequently been linked with a wide range of disorders, including those pertaining to growth and cancer, metabolism and behaviour. Investigate for the role and regulation of the newly characterized chromatin bivalent domains in the process of genomic imprinting offers thus a new mean to apprehend events leading to this aberrant regulation, offering new diagnosis opportunities as new therapeutic candidate targets.

The scientific production related to this project is currently of 3 article published in a peer-reviewed journal, 1 book chapter, 1 review article in a french peer-reviewed journal, 4 oral communicatiosn in international meeting, 4 oral communications in national meeting.
Publications are:
Bouschet T, Dubois E, Reynès C, Kota SK, Rialle S, Maupetit-Méhouas S, Pezet M, Le Digarcher A, Nidelet S, Demolombe V, Cavelier P, Meusnier C, Maurizy C, Sabatier R, Feil R, Arnaud P, Journot L, Varrault A. (2016)
“In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression.”
? Cereb Cortex. 2016 Apr 19. pii: bhw102.

Fogli A, Chautard E, Vaurs-Barrière C, Pereira B, Müller-Barthélémy M, Court F, Biau J, Pinto AA, Kémény JL, Khalil T, Karayan-Tapon L, Verrelle P, Costa BM, Arnaud P. (2016)
“The tumoral A genotype of the MGMT rs34180180 single-nucleotide polymorphism in aggressive gliomas is associated with shorter patients' survival.”
?Carcinogenesis. 37(2):169-76.

Maupetit-Méhouas S, Montibus B, Nury D, Tayama C, Wassef M, Kota SK, Fogli A, Cerqueira Campos F, Hata K, Feil R, Margueron R, Nakabayashi K, Court F, Arnaud P. (2016)
“Imprinting control regions (ICRs) are marked by mono-allelic bivalent chromatin when transcriptionally inactive.”
?Nucleic Acids Res. 44:621-35.


Maupetit-Mehouas S, Nury D, Arnaud P (2014)
“Epigenetic reprogramming in the mammalian germline”
In: A.K. Naumova and C.M.T. Greenwood (eds.), Epigenetics and Complex Traits, in press, Springer Science+Business Media New York 2014

Bertille Montibus B., Maupetit-Méhouas S., Arnaud P. (2013)
« L’empreinte Parentale dans les gamètes et sa dérégulation dans les pathologies humaines. »
Médecine Clinique, Endocrinologie et Diabète (MCED), numéro spécial « Epigénétique », n°66, Sept-Oct 2013.

Pluripotent stem cells can differentiate in all cell types of an organism. During differentiation they progressively develop a narrower potential that ultimately results in commitment to specific cell fate with specific gene expression profile and functional property. How this process is regulated is an area of intense research. Changes in gene expression are caused by sequence-specific transcription factors but are also accompanied or caused by epigenetic modifications. One major challenge in developmental biology is to understand how these epigenetic modifications participate in the establishment of cell identity during development. Recent studies uncovered key regulatory histone modifications thought to be important for differentiation decision.
Of interest is the unexpected identification of unusual chromatin domain, named bivalent domain, containing both “active” H3K4me3 and “repressive” H3K27me3 modifications. Bivalent chromatin has been observed at the promoters of many genes in ES cells as well as in some differentiated cells and has been considered to be involved in maintaining key developmental genes in a “permissive” chromatin configuration, while being repressed by H3K27me3. The resolution of these domains into either H3K4me3 or H3K27me3 upon differentiation is believed to stably marks these regions for activation or repression respectively. These peculiar structures offers thus a novel means of chromatin-based gene regulation that is believed to be especially relevant for development and differentiation.
However, how bivalent domains are regulated during cell fate determination remains to be unravelled. This is precisely what we propose to do in this ANR-jeune chercheur proposal by using a key developmental process, the genomic imprinting, as a model as we recently determined that bivalent domains are involved in the control of tissue-specific imprinting.
By developing, in both mouse and human, innovative and complementary molecular approaches in normal and pathological contexts, we will investigate how bivalent domains are controlled during differentiation with the main underlying objective being to establish how are recruited and/or activated H3K27me3 histone demethylase on these domains during lineage commitment.
In detail, by using a panel of bivalent domains associated with imprinted genes we will (1) identify signature predictive of their fate outcome during development. (2) Establish their outcome during epigenetic reprogramming leading to acquisition of totipotency in germline lineage (3) Determine whether inappropriate regulation of bivalent domains could contribute to tumorigenesis in human and identify its causes. (4) Functionally test and validate main outcomes obtained through these three approaches by investigating for the role of the recently characterized H3K27me3 demethylase.
This work will benefit to colleagues in the scientific community, particularly those involved in the field of epigenetic regulation and cell fate determination. Results expected from this proposal will represent a significant step forward our understanding in fine-tune regulation of tissue-specific imprinting and in a broader view the epigenetic control of tissue-specifc expression. In addition it will provide valuable and original information on the role, in this process, of an H3K27me3 demethylase, a recently discovered epigenetic player of which regulation and role in development remain largely uncharacterised. Particularly, one of a main objective or our proposal is to establish how bivalent domains recruit the H3K27me3-demethylase, revealing thus a signature predictive of the fate outcome of these structures, and the associated transcriptional state, during development. This is of main importance in the view of the therapeutic use of pluri- and muti- potent stem cells.