... / ... / ... / ... / Stefan Barakat, MD, PhD / Brief summary of previous research

Brief summary of previous research

During my PhD I discovered the X-linked Rnf12 gene as an essential activator of X-chromosome inactivation (XCI), a crucial epigenetic mechanism during female embryonic development (Cell 2009, PLoS Genetics 2011). Subsequently, our studies focussed on unravelling molecular mechanisms behind Rnf12-mediated XCI-activation, and identified a network between Rnf12 and the transcription-factor (TF) Rex1 during XCI-initiation (Nature 2012, and submitted work). In addition, using extensive genetic-engineering approaches in embryonic stem cells (ESCs) and by using heterokaryons, I could demonstrate that XCI is regulated by RNF12 as a trans-acting factor, in an inter-play with other cis-acting elements, independent of X-pairing (Molecular Cell 2014). Together, these results provided answers to longstanding questions in the field of dosage compensation, explaining female-specific XCI-initiation. The generation of an Rnf12-knockout mouse-model (Barakat et al, unpublished) has further proven the essential role of Rnf12 for female development in vivo, and has revealed a peculiar X-reactivation phenotype with bi-allelic gene-expression in heterozygous females, which might have possible implications for future therapies for X-linked disorders (recently acknowledged with ASHG Charles J. Epstein Award, 2015).

My postdoctoral work has focused on understanding the pluripotency-network in embryonic stem cells, from a perspective of enhancer regulation by pluripotency TFs and histone modifications. Enhancers are non-coding elements that are crucial for a correct spatiotemporal regulation of gene expression, but identifying sequences with enhancer activity remains a challenge and is prone to mistakes. By using an innovative approach,  I have developed a method that enables the genome-wide identification of functional enhancers, in a quantitative manner. Using this method, which combines chromatin immunoprecipitation (ChIP) with a massively-parallel reporter assay, we have dissected the functional repertoire of enhancers in human embryonic stem cells, generating the largest resource of functional validated human enhancers existing to date (Barakat et al., Cell Stem Cell 2018 (https://doi.org/10.1016/j.stem.2018.06.014) and supplementary data available at our resource website: http://hesc-enhancers.computational-epigenetics.org). This study has provided new insights in the regulation of enhancer sequences, by showing specific combinatorial enrichment of TFs at active enhancers, whereas many other sites that are also marked by several TFs or histone modifications where found to be functionally inactive. Deletion of only these active sequences at the endogenous genome loci affects gene expression of target genes. This shows that a direct functional readout is superior to identify functional enhancer sequences compared to predictions based on ChIP-seq experiments, and can further help to decipher the functional parts of the non-coding genome essential for correct gene regulation. We have applied a similar approach to study co-dependency on TFs in enhancer regulation in mouse embryonic stem cells (manuscripts in preparation).

I recently returned to the Erasmus MC to start my own laboratory at the department of Clinical Genetics. Here my research focusses on deciphering the role of the non-coding genome in genetic disorders, with a particular focus on brain disorders. Using functional genomics and various other approaches, including induced pluripotent stem cells and cerebral organoids ("mini-brains"), we are studying the enhancer landscape in cells representing neurodevelopment. As many genetic disorders of brain development can at present not be explained by routine genetic diagnostics, we anticipate that many of these disorders are caused by alterations of non-coding elements, including enhancers. We plan to sequence the enhancers identified through our genome-wide functional enhancer activity assays in patients, to directly test the hypothesis that alterations of non-coding genome regions can be causal in the pathogenesis of brain disorders. We can then apply disease modelling for the patients harboring identified mutations, using patient specific induced pluripotent stem cells and differentiation to cerebral organoids, thereby simulating brain development in a dish. Our studies will facilitate the identification of functional enhancer sequences in the non-coding genome, and will allow novel innovative approaches for patient diagnostics using functional genomics.