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GRNHairCell Report Summary

Project ID: 707015
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - GRNHairCell (Understanding the Gene Regulatory Network involved inner ear Hair Cell differentiation)

Reporting period: 2016-03-01 to 2018-02-28

Summary of the context and overall objectives of the project

• What is the problem/issue being addressed?
Understanding the molecular basis for transcription factor (TF) specificity in different developmental contexts is vital to grasp a mindful knowledge of the embryonic development and key for the development of effective regenerative therapies. Our model system is the inner ear hair cell (HC), essential to hearing and balance. Very little is known about the genetic networks regulating HC development. To gain new insights, we developed a new transcriptional programming strategy to promote in vitro HC differentiation, starting from pluripotent stem cells. In vivo Atoh1 is the only TF known to be necessary and sufficient for HC differentiation, but in vitro its overexpression induces neuronal rather than HC differentiation. Previously, we discovered that Atoh1 expression combined with two other TFs (Pou4f3, Gfi1) resulted in efficient HC generation. This work offers a new paradigm to understand the molecular mechanisms governing TF specificity. How do Pou4f3-Gfi1 modulate Atoh1 activity to orchestrate a HC differentiation program? To address these questions, we have interrogated cells undergoing this well-defined transcriptional programming event in order to understand the properties of the gene regulatory network controlling HC differentiation.

• Why is it important for society?
Hearing loss affects millions of people worldwide and is largely caused by the loss of mechanosensory HCs. There is no cure: HCs do not regenerate in mammals (unlike in other vertebrates) and prosthetic devices poorly substitute for lost HCs. bHLH transcription factor, Atoh1, drives HC specification in development and understanding its role is crucial for future therapeutic strategies. Indeed, much effort is expended on Atoh1 research and it is already the subject of gene therapy trials. However, our mechanistic knowledge is incomplete. For instance, why is Atoh1 unable to drive HC regeneration in mammals? Understanding the mechanism of Atoh1 function and its limitations requires determining its place within the gene regulatory network (GRN) of HC development. This knowledge will have a major impact for novel therapeutic approaches that could restore HCs.

• What are the overall objectives?
The overall objectives of this project can be divided in 3 general aims containing different sub-aims:
1) Understand how Atoh1 cooperates with Pou4f3 and Gfi1 to implement the HC differentiation program:
-Determine the precise gene expression changes triggered by Atoh1, Pou4f3 and Gfi1 during HC differentiation.
-Determine the direct target genes of Atoh1, Pou4f3 and Gfi1 during early stage of HC differentiation and identify the main functions of each TF.
2) Understand how Atoh1 function can be modulated to cause a switch from neural to HC fate:
-Determine the precise gene expression changes triggered by Atoh1 alone or in combination with other TFs (Gfi1/Pou4f3).
-Determine how Atoh1’s direct targets differ between HC and neuronal contexts (i.e. with and without Gfi1/Pou4f3).
3) Generate gene regulatory models (GNR) of HC differentiation:
-Test predictions arising from these models in vivo.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work performed during this project can be divided in three main phases
1. Generation and validation of new Dox-inducible ESC lines:
To avoid technical issues related to poor antibody quality and variations in the levels of Dox-inducible transgene, we have engineer new ESCs lines containing a different epitope tag in each TF and a fluorescent reporter (mVenus) in the Dox-inducible locus. These new ESCs lines were characterized and validated in terms of their ability to induce HC and neuronal differentiation, as previously reported (Costa et. al. Development 2015). ESCs lines containing in their inducible locus just one TF (Atoh1, Pou4f3 or Gfi1), combination of these 2 TFs and the 3 TFs combination were generated and validated.
Main results achieved:
-Epitope tagging was successful for Atoh1 and Pou4f3 but not for Gfi1. Therefore, the new Dox-inducible ESCs lines were re-engineered not to contain any tag for Gfi1 only.
-HC differentiation is more efficient when all 3 TFs are induced together, however, up-regulation of several HC markers was detected when Atoh1 was combined with Gfi1 and when Pou4f3 was combined with Gfi1.
-Up-regulation of neuronal but not HC markers was efficiently promoted by overexpression of Atoh1 alone or when Atoh1 was combined with Pou4f3.

2. RNA-seq and ChIP-seq protocol optimization, sample preparation and highthroughput sequencing:
The first step was the optimizations of RNA collection followed by fluorescence activated cell sorting (FACS), as well as, ChIP protocol optimization for each TF (Atoh1, Pou4f3 and Gfi1) assessed by ChIP-qPCR. The second step involved the optimization of the libraries preparation protocol for ChIP-seq and RNA-seq experiments. Finally, these optimized procedures were applied in all the Dox-inducible ESC lines validated in the previous task and highthroughput sequencing was performed for the RNA-seq and ChIP-seq libraries.
Main results achieved:
-RNA-seq revealed that Gfi1 induces a significant transcriptional change in the genes regulated by Atoh1 and Pou4f3.
-ChIP-seq for Gfi1 requires a superior antibody quality to achieve a good signal/noise ratio similar to those obtained in Atoh1 and Pou4f3 ChIP-seq.

3. Bioinformatics training and analyses:
The bioinformatics analyses of the next generation sequencing experiments performed in the previous task were carried out by the beneficiary (Dr. Aida Costa). The beneficiary carried out extensive training which included Unix, Python and R programming and Bioinformatics methods for the whole workflow, ranging from data processing to functional analyses.
Main results achieved:
-During HC differentiation Atoh1 and Pou4f3 regulate distinct enhancers and promoters showing only 20% of binding overlap.
-Despite being a known transcriptional repressor in haematopoiesis, Gfi1 enables the activation of HC genes mediated by Atoh1 and Pou4f3 during HC differentiation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has revealed a new and vital role of Gfi1 in HC development. However, it remains to be discovered the molecular mechanisms by which Gfi1 exerts such a key function during HC differentiation. To gain new insights, we are further analysing the ChIP-seq and RNA-seq datasets by using various computational models. In addition, we are generating new Dox-inducible ESCs lines containing mutations in Gfi1 aiming to discover its functional domains for HC differentiation. Finally, we are exploring the in vitro HC differentiation platform to screen and identify Gfi1 co-players and/or targets by testing various compounds that affect epigenetic and transcriptional pathways known to be important for Gfi1 function in haematopoiesis.
We expect to extend the social-economic impact of this project by I) identifying the function and molecular mechanisms by which Atoh1, Pou4f3 and Gfi1 drive hair cell differentiation. II) highlighting the potential of our HC differentiation platform for basic research as well as for pre-clinical drug discovery and III) Releasing our next generation sequencing datasets to the scientific community. This will allow hundreds of scientists around the world working on a variety of fields to use it, explore it and exploit it, greatly increasing the potential impact of the project.

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