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ERC

REPROWORM Report Summary

Project ID: 637530
Funded under: H2020-EU.1.1.

Periodic Reporting for period 2 - REPROWORM (Safeguarding Cell Identities: Mechanisms Counteracting Cell Fate Reprogramming)

Reporting period: 2016-09-01 to 2018-02-28

Summary of the context and overall objectives of the project

Using cellular reprogramming to regenerate tissues is a highly promising prospect for therapeutic approaches that aim to replace lost tissues in patients suffering from injury or degenerative diseases such as Alzheimer’s Disease (AD) or Muscular Dystrophy. According to the EU Joint Programme of Neurodegenerative Disease Research (JPND), over 7 million patients in Europe suffer from AD or related diseases, which will double within the next 20 years. In one very much anticipated scenario, patients suffering from lost tissues due to disease or injury will be replenished with healthy cells in order to regenerate the affected organs. However, most tissues such as neurons have little or no ability for self-regeneration. Therefore, efficient ways of generating the required cell types for prospective tissue replacement treatments need to be developed. Differentiated cells can be converted to other cell types by cellular reprogramming which makes use of forced expression of cell fate-inducing transcription factors. However, most cells are refractory to reprogramming due to inhibitory mechanisms that block their efficient conversion to other cell types. The main objective of REPROWORM is to reveal the mechanisms that restrict cellular reprogramming by using the nematode Caenorhabditis elegans (C. elegans). C. elegans is as a powerful genetic model organism suitable for large-scale screens to identify genes that play a role in the process of cellular reprogramming. Similar to humans, C. elegans has 20.000 genes of which more than 50% have human homologs. A previously identified reprogramming barrier exemplifies the remarkable conservation of a reprogramming barrier from C. elegans to humans. The histone chaperone LIN-53, homolog of human CAF-1p48, protects germ cells in C. elegans from being directly reprogrammed into neurons and was first discovered by Dr. Tursun in 2011. In an analogous study, the group of Konrad Hochedlinger showed in 2015 that the LIN-53-containing CAF-1 complex acts as a barrier for cellular reprogramming of mouse embryonic fibroblasts. Such striking conservation from worm to mouse implies that additional reprogramming barriers might be shared among C. elegans and humans. Therefore, findings derived from ongoing research can facilitate the generation of specific tissues from different cellular contexts for future biomedical approaches.

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

We performed a whole-genome RNAi screen against all 20.000 genes of C. elegans and identified around 160 novel factors, whose depletions allow reprogramming of cells into specific types of neurons. Testing a number of the newly identified factors in human fibroblasts revealed that the essential chromatin regulator FACT (Facilitates Chromatin Transcription) is an evolutionarily conserved barrier for cell fate reprogramming by transcription factors in nematodes, as well as in human cells. Strikingly, reprogramming of human fibroblasts to induced Pluripotent Stem cells and neurons is strongly enhanced by almost 100% in FACT-depleted fibroblasts. Using CRISPR/Cas9-mediated gene editing technology in C. elegans we revealed that FACT has tissue-specific isoforms, which safeguard the intestinal and germ cell fates to prevent their conversion into neurons. Such alternative forms of the essential epigenetic regulator FACT have not been reported in any other species so far.
In order to enhance the identification of reprogramming barriers we set up a novel high-throughput genetic screening system and identified additional genetic factors that inhibit reprogramming of cell fates. Building on our successful validation of the chromatin remodeler FACT as a reprogramming barrier in C. elegans, as well as in human cells, we will continue to study whether more of our newly identified factors have evolutionarily conserved functions. Besides chromatin factors, metabolome regulators such as dehydrogenases were also identified as reprogramming barriers, which will be further characterized with respect to molecular mechanisms and conservation in mammals. The histone chaperones LIN-53 (CAF-1p48) and the chromatin remodeler FACT exemplify the high level of functional conservation from nematodes to human for reprogramming barriers.

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)

While most reprogramming studies make use of tissue cultures, the nematode C. elegans provides the possibility to study reprogramming regulation in vivo. Moreover, C. elegans is a powerful model organism to perform unbiased genetic screens for factors that were not identified in other ex vivo research systems, such as tissue cultures. The chromatin regulator FACT, which was identified as part of REPROWORM using C. elegans, was a highly unexpected reprogramming barrier, since it is predominantly known as a positive gene expression regulator. In contrast, all other previously identified genes that block cellular reprogramming are factors that repress gene expression. Until the end of REPROWORM, the characterization of additional unexpected reprogramming barriers, such as genes encoding for mitochondrial proteins, will reveal unique insights into how cell identities are protected by linking distinct biological processes including epigenetics and metabolomics in an intact organism.
Furthermore, by continuing to study our first identified reprogramming barrier LIN-53, we revealed an unexpected antagonistic relationship of the highly conserved cell signaling system known as the ‘Notch pathway’ with the repressive chromatin regulator Polycomb Repressive Complex 2 (PRC2). Increased activity of the Notch signaling pathway counteracts PRC2-mediated chromatin repression, thereby enhancing the reprogramming of germ cells into neurons (Seelk et al., 2016, Elife).
Additionally, our ongoing study in the context of Aging revealed that the epigenetic reprogramming barrier LIN-53 is required for normal lifespan and muscle homeostasis. In collaboration with Prof. Spuler (Charité) we discovered that expression and localization of the human homolog CAF-1p48/RBBP4 is impaired in primary myoblasts of human myopathy patients. By introducing the respective mutations of the patients we have now established C. elegans as a genetic disease model to systematically investigate the impact of impaired chromatin regulation on muscular homeostasis in myopathy patients.
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