Blanc SVSE 6 - Sciences de la vie, de la santé et des écosystèmes : Génomique, génomique fonctionnelle, bioinformatique, biologie systémique

Formation, dynamics and epigenetic functions of 5-hydroxymethylcytosine residues in DNA. – EPIGENOME

Study of the molecular mechanisms of epigenetic regulation

Study of the role of TET proteins and enzymes involved in DNA repair on the formation and dynamics of chemical markers present in the DNA such as 5-methylcytosines and 5-hydroxymethylcytosines. Implications for epigenetic reprogramming towards pluripotency and epigenetic changes in cancer.

Understanding of the mechanisms of active DNA demethylation

This project aims to study the function of 5-hydroxymethylcytosine (5hmC) residues ??in epigenetic regulations and genetic instability. Our objectives are structured in four tasks: i) Role of TET2 protein, oxidative stress and enzymes involved in DNA repair on the formation and dynamics of the 5hmC when present in DNA. This study may provide insights on the nature of the molecular mechanisms involved in the active DNA demethylation which at present remains poorly understood; ii) To identify and characterize human proteins that interact specifically with 5hmC residues in DNA. This study will map the proteins interacting with 5hmc in DNA and define the DNA repair pathways involved in demethylation, iii) To study the role of TET proteins and 5hmC residues on genetic instability and DNA repair. This study will provide insight on why mutations of TET2 are found in many malignancies; iv) To study the role of TET proteins on gene expression in human cell lines in particular human ES cells. Epigenetic control in these cell lines is of crucial interest since recently, the AID protein involved in the deamination of cytosine has been implicated in the reprogramming towards pluripotency via demethylation process.

To study the mechanisms of active DNA demethylation we used four complementary approaches: (i) a biophysical approach using mass spectrometry to quantify 5-hydroxymethylcytosines residues in cellular DNA to elucidate the role of the TET2protein and DNA repair enzymes involved in active DNA demethylation; (ii) a biochemical approach in vitro, by reconstitution of a system with purified DNA repair proteins and oligonucleotides containing 5mC, 5hmC, 5hmU and 5caC residues. This will allow us to analyze and measure the kinetic constants of the elimination of epigenetic markers in DNA, (iii) an approach known as «ligand fishing« using DNA probes containing 5hmC residues and attached to magnetic beads. The retained proteins are identified using proteomics tools. Successful candidates are validated by complementary approaches such as the electrophoretic gel mobility shift assay; (iv) an in situ approach using technology called «microarray« to identify deregulated genes in cells over-expressing or not the TET2 protein to modulate the levels of genomic 5hmC residues.

- We have demonstrated that the DNA mismatch repair system (MMR) is involved in the elimination of methylated cytosines in DNA;
- We have obtained the three-dimensional structure of the complex formed by human DNA glycosylase MBD4 (implicated in the active DNA demethylation) and DNA containing 5-hydroxymethyluracil (an intermediate product of DNA demethylation);
- We have shown that the level of 5hmC residues fall in the presence of an shRNA directed against TET2;
- We have shown that TET2 is a key gene involved in the development of haematopoiesis in human ES cells;
- We have shown that ionizing radiation has a limited impact on the formation of 5hmC residues in DNA;
- We have shown that the transcriptional factor ZBTB2 has a decreased affinity to DNA containing 5-hydroxymethylcytosine (5hmC) residues as compared to DNA containing 5-methylcytosine (5mC) residues.

In perspectives:
- We will study whether the TET2 and AID proteins can initiate the erasure of 5hmC residues in DNA via DNA mismatch repair system (MMR); - we are planning to resolve the three-dimensional structure of the full-length hTDG protein in complex with its DNA substrates; - we are planning to study the mutagenesis induced by over-expression of TET2 in the cells deficient in DNA base excision repair system; - we are planning to investigate whether the hematopoietic phenotype is dependent or independent of 5-hydroxyméthylcytosines (5hmC) residues; - we are planning to optimize the protocol for HPLC-MS-MS method to measure 5-carboxydeoxycytidine, another oxidation product of 5hmC and potential intermediate of active DNA demethylation; - we are planning to validate the results obtained with ZBTB2 and search for other proteins interacting with 5hmC and 5mC by screening other cell types (UT7 cells expressing or not TET2 - provided by partner 2).

1. Pronier, et al., Ravanat JL, et al., Plo I, Delhommeau F. et al. (2011) Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors. Blood, 118, 2551-2555. (This work demonstrates that the deficiency TET2 induced either by shRNA or by genetic mutations in patients leads to lower level of 5hmC residues in DNA well as to bias in differentiation of granulocyte-monocyte. This work was done in collaboration between partner 2 and 3).
2. Quivoron, C., et al., Plo, I., et al., (2011) TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 20, 25-38. (This work describes the phenotype of knockout mice TET2 which exhibit chronic myelomonocytic leukemia).
3. Zlatanou, A., et al., Ishchenko, A. A. and Kannouche, P. L. (2011) The hMsh2-hMsh6 Complex Acts in Concert with Monoubiquitinated PCNA and Pol eta in Response to Oxidative DNA Damage in Human Cells. Mol Cell 43, 649-662. (This work demonstrates that the DNA mismatch repair pathway (MMR) is involved in active DNA demethylation).
4. Morera, S., et al., Saparbaev, M., Ishchenko, A.A. (2012) Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil (5hmU)-containing DNA. Nucleic Acids Res., (in press). (In this work we have elucidated the recognition mechanism of the deamination products of 5-methylcytosine and 5-hydroxymethylcytosine residues in DNA).

Methylation of cytosine at the 5-position (5mC) is a common form of post-replicative DNA modification found in both prokaryotes and eukaryotes. DNA methylation of cellular genome holds essential epigenetic information and regulates crucial aspects of organism’s functions. Methylation patterns of regulatory regions and gene bodies in differentiated cells are very stable and maintained during cell divisions. In mammals during developmental cycles, methylation patterns in germ cells and in embryos are genome-wide reprogrammed resulting in cells with a broad developmental potential. Erasure of DNA methylation during reprogramming is essential for creation of pluripotency in development. Stem cells might also undergo epigenetic reprogramming during differentiation. Moreover, changes in DNA methylation patterns are generally associated with epigenetic changes in human cancers. Many human cancers commonly demonstrate global DNA hypomethylation concomitant with specific hypermethylation of tumor-suppressor genes. Alterations of methylation patterns may lead to chromosomal instability due to chromatin remodelling. Understanding dynamics of DNA methylation during development and oncogenesis requires studies of DNA demethylation processes.
The rapid loss of DNA methylation that occurs within a single cell cycle argues for the existence of enzymes that eliminate or modify 5mC residues in DNA. Recently, the work by Partner 2 and other laboratories have identified mutations in 1 or 2 alleles of TET2 gene in myeloproliferative disorder (MPD), myelodysplastic syndromes (MDS), secondary or sporadic leukemia, chronic myelomonocytic leukemia (CMML) and mastocytosis. TET2 belongs to a three-member family including TET1 and TET3 homologues. Recently, using bioinformatics approach Tahiliani and co-workers identified the TET1 protein as a 2-oxoglutarate Fe dependent enzyme and demonstrated that this enzyme can convert 5mC to 5-hydroxymethylcytosine (5-hmC) both in vitro and in cultured murine embryonic stem (ES) cells. These findings indicate a new unexpected mechanism to control epigenetic information via oxidation of 5mC to 5-hmC, which may be an intermediate in active DNA demethylation through DNA repair. Among possible candidate DNA repair systems are the base excision repair (BER) and nucleotide incision repair (NIR) pathways that could repair oxidized cytosine residues.
The aim of the present study is to evaluate the formation and regulation of 5-hmC (oxidized variant of 5mC) delineating the partners of 5-hmC. Consequently, our goals aim at investigating the function of 5-hmC in epigenetic changes and genetic instability. The proposal will be structured as follows with 4 different tasks
(i) Role of the TET proteins, oxidative stress conditions and DNA repair in formation and dynamic of 5-hmC residues in DNA. Data from this study might give some clues about the molecular mechanism of active DNA demethylation in mammals, which is still unclear and controversial.
(ii) Identification and characterization of human proteins specifically interacting with 5-hmC residues in DNA. This study might allow us to cartography the proteins interacting with 5-hmdC and decipher mechanism of DNA repair.
(iii) Study of the role of TET proteins and 5-hmC residues in genetic instability and DNA repair. This study would enable us to understand the role of 5-hmC in genetic instability and explain why mutations of TET2 are found in various hematological diseases.
(iv) Study of the role of TET proteins in epigenetic regulation of gene expression in ES and other cell lines. The control of epigenetic by 5-hmC is of major interest since very recently, proteins involved in the deamination of cytosine such as activation-induced cytidine deaminase (AID) have been involved in reprogramming towards pluripotency through demethylation process.

Project coordinator

Monsieur Murat SAPARBAEV (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR EST) – smurat2003@mail.ru

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR EST
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PARIS XI
INAC/SCIB UNIVERSITE GRENOBLE I [Joseph Fourier]

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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