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Safeguarding Cell Identities: Mechanisms Counteracting Cell Fate Reprogramming

Periodic Reporting for period 4 - REPROWORM (Safeguarding Cell Identities: Mechanisms Counteracting Cell Fate Reprogramming)

Reporting period: 2019-09-01 to 2021-02-28

Using cellular reprogramming to regenerate tissues is a 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 studies and tissue replacement therapies.
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 neurons. Testing a number of the newly identified factors in human fibroblasts revealed that the essential chromatin regulator FACT is an evolutionarily conserved barrier for cell fate reprogramming by transcription factors in nematodes, as well as in human cells (Kolundzic et al., 2018 DevCell).
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. This system allowed the identification of the chromatin-regulating factor MRG-1 (MRG15 in humans) as a barrier for cellular reprogramming in C. elegans (Hajduskova et al., 2019 Genetics). Biochemical characterization revealed that MRG-1 interacts with the methyltransferase SET-26 in order to safeguard germ cells (Hajduskova et al., 2019 Genetics). 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/RBBP4) and the chromatin remodeler FACT exemplify the high level of functional conservation from nematodes to human for reprogramming barriers. By continuing to study our first identified reprogramming barrier LIN-53, we revealed an unexpected antagonistic relationship of the conserved NOTCH signaling system with the repressive chromatin regulator 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).
Furthermore, we revealed that LIN-53 is required for normal lifespan and muscle homeostasis in C. elegans. We could show that LIN-53 regulates metabolism and the levels of the sugar Trehalose, which is essential for normal lifespan. While metabolism regulation by LIN-53 occurs via the histone-deacetlyase complex SIN3, LIN-53 maintains muscle integrity during aging via the chromatin-remodeling complex NuRD (Müthel et al., 2019 Aging Cell). Strikingly, there is evidence that the human counterpart of LIN-53, CAF-1p48/RBBP4, has a conserved role in regulating lifespan and preventing myopathy in humans. 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.
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. We recently discovered a novel in vivo reprogramming phenomenon allowing the direct Reprogramming of hepatic Coelomocytes (CCs) to intestinal cells or glutamatergic neurons. CCs scavenge by endocytosis macromolecules from the animal’s body cavity. Remarkably, Reprogramming of CCs to intestinal cells causes a dramatic reorganization of membranous organelles to form de novo an intracellular lumen. Dissecting the dynamics of gene expression changes and structural reorganization of CCs during aging will allow unique insight into the Reprogramming trajectories of different molecular processes.
We are continuing to reveal the mechanisms that safeguard cell fates and maintain tissue identities in C. elegans. The overall 60% homology of the genome between C. elegans and humans may lead to the identification of highly conserved mechanisms that act as barriers to cellular reprogramming and play important roles during lifespan and tissue homeostasis. 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.
Graphical Abstract of REPROWORM