Characterisation of the epigenetic enzyme TET: an integrative approach – EpiTET

By using a combination of state-of the-art developmental, molecular, genomic and bioinformatic approaches, we will tackle several key questions related to TET mode of action.
To characterize TET function in hematopoiesis and transposable element silencing (in the ovary), we will use in vivo genetic approaches with a combination of TET mutant alleles and issue-specific RNAi-mediated knock-down. Confocal imaging with known markers and transgenic reporters as well as RNA-seq experiments will be used to assess the consequences of TET loss and identify the down-stream pathways that are affected.
In parallel, we will use cutting edge NGS technologies (ChIP-seq, TRAP, RIP-seq, Nanopore sequencing..) to identify TET targets at the DNA or RNA level and to characterize its impact on different epigenetic and/or epitranscriptomic modifications.
Finally, to identify TET partners and regulators, we will use two complementary strategies: a proteomic approach to purify TET partners and a genetic screen for modifiers of TET knock-down associated phenotypes.

First, we developed a number of new tools to study TET expression and function in Drosophila. In particular, using CRISPR/Cas9-mediated genome editing in vivo and in cell culture, we generated a new GFP-tagged form of the endogenous TET, which allows to label both TET long (TET-l) and TET short (TET-s) isoforms. Using a similar strategy we generated a catalytic dead mutant allele of TET, which will be complementary to the different null allleles we already had. In parallel, we generated and tested different antibodies against TET.
Our analysis of TET expression by RT-qPCR and immunostaining showed that TET-s is expressed in all the blood cells but downregulated in a particular lineage, and that it is expressed in the follicular cells but not in the germ cells in the ovary. In contrast, TET-l is not expressed in these tissues, indicating that TET CXXC DNA binding domain, which is present only in TET-l, is dispensable for its function in hematopoiesis and transposable element silencing.
Our analysis of TET null alleles show that TET is required for the homeostasis of the larval lymph gland, which is the main larval hematopoietic organ. Based on RNA-seq results, we identified a number of genes regulated by TET specifically in the lymph gland, the wing disc or the brain. We are currently analyzing RNA-seq and small-RNA-seq data for the ovaries to assess the impact of TET on piRNA production and transposable element silencing.
Interestingly, we found that TET expression but not its enzymatic activity is required for fly viability, indicating that an important part of its function is enzymatic-independent in Drosophila.
Finally, we performed a large scale genetic interaction screen that allowed us to map several loci that enhance or suppress the wing phenotypes associated with TET knock down in this tissue. Some of the genes responsible for these genetic interactions have now been identified.

On the one hand, we will pursue the caracterization of TET function in blood cell development and transposable element silencing notably by digging into our RNA-seq data, and we will seek to determine more precisely in which cells TET is required using tissue/lineage-specific knock-down experiments. In order to assess the impact of TET on DNA modifications, we will employ 3rd generation sequencing strategies which allow to probe at genome wide level and with single nucleotide precision the nature of epigenetic modifications. These experiences will be coupled with ChIP-seq analyses on TET to identify its direct DNA targets.
On the other hand, we will pursue the comparative analysis of TET null and catalytic dead mutant alleles in order to determine TET enzymatic-dependent and independent functions in vivo.
Finally, we will assess the nature of functional interaction between TET and the genetic interactors we identified in our screen and we will try to improve our protocol of purification of TET to be able to identify its direct partners by proteomic analysis.

Until now, one publication and one book chapter supported by this ANR grant were published in international peer-reviewed journals:
Duc C et al., Genome Biology, 2019 Jun 21;20(1):127.
Boulet Met al., Advances in Experimental Medicine and Biology, 2018 vol. 1076, pp195-214.

Our project was also presented to 6 national or international congresses.

A precise regulation of gene expression is critical for proper development and cell homeostasis. It is thus essential to decipher the conserved principles governing genome metabolism and underlying several pathologies in Humans. One important layer of regulation is exerted at the transcriptional level notably by epigenetic enzymes that modify DNA. Moreover, RNA modifications have come out as new players in the post-transcriptional regulation of gene expression. Therefore, a deep understanding of the function(s) and mechanism(s) of action of enzymes controlling the catalytic modification of DNA or RNA is needed.
Thanks to their capacity to oxidize methyl-Cytosine (mC), a prevalent epigenetic modification in many genomes, enzymes of the Ten Eleven Translocation (TET) family have emerged as key actors in DNA demethylation and in the control of the ensuing processes. In mammals, the three TET genes have been implicated in different physio-pathological processes, notably in the hematopoietic system. While most studies focused on their action on mC in genomic DNA, they can also oxidize mC in RNA, and some of their functions are independent of their enzymatic activity. Moreover, how TET proteins control cell fate, what are their partners, and how their activity is regulated remain largely unknown. Our project is aimed at tackling these different issues.
Drosophila genome, which encodes a single TET gene, is largely devoid of mC in genomic DNA. Thus this model organism offers a unique opportunity to study TET function beyond mC DNA regulation. The global objective of our project is to provide a comprehensive analysis of TET role and mode of action in Drosophila during two paradigmatic processes (hematopoiesis and transposable element silencing) by integrating a combination of state-of-the-art genetic, developmental, molecular, genomic and bioinformatics approaches. Taking advantage of the tools available in fly and of the complementary expertise within our consortium, we will assess whether TET controls cell fate and genome stability at the transcriptional and/or post-transcriptional level, and identify its targets, partners and regulators.
Our recent unpublished works show that TET plays an important role in the regulation of Drosophila blood cell development, which shares many features with hematopoiesis in mammals. Thanks to Team 1 expertise, we will characterize TET function in fly hematopoiesis, decipher its mechanism of action and define the gene regulatory network it controls. In addition, Drosophila TET has been implicated in the silencing of transposable elements, which is essential to maintain genome stability, and our data suggest that it impinges on the piRNA pathway, a conserved pathway regulating mobile element expression. Together with Team 3, we will explore if and how TET controls the genomic loci or the RNA implicated in transposable element silencing. In parallel, we will use cutting edge NGS and bioinformatic approaches mastered by Team 2 to assess first in cell culture and then in vivo whether TET acts at the transcriptional or post-transcriptional level, and we will identify TET targets at the DNA and/or RNA level. Furthermore, we will endeavor to identify TET partners and regulators by two complementary strategies: a proteomic-based approach and a genetic screen. Finally, key TET targets, partners and regulators will be further studied in the hematopoietic system and/or in transposable element silencing.
This ambitious and timely project will bring deep insights into TET mode of action and regulation. We anticipate that our discoveries will open new avenues of research on this conserved family of DNA and RNA-modifying enzyme. They will also help understand the regulation of blood cell development and mobile element silencing. Thus our findings will have far-reaching implications concerning the control of gene expression, genome stability or cell fate, with direct implications for several health-related topics.