Computational analysis of nucleosome assembly and cis-regulatory DNA elements
by Oleksandr Dovgusha
Date of Examination:2024-10-28
Date of issue:2025-11-12
Advisor:Dr. Ufuk Günesdogan
Referee:Dr. Ufuk Günesdogan
Referee:Prof. Dr. Argyris Papantonis
Persistent Address:
http://dx.doi.org/10.53846/goediss-11612
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Abstract
English
Induced pluripotent stem cells (iPSCs) represent embryonic stem cells (ESCs) that are reprogrammed from somatic stem cells. iPSCs can be directed to differentiate into any cell type in the human body, therefore, holding great potential for regenerative medicine and drug discovery. In addition, iPSCs overcome ethical issues such as embryo damage during conventional isolation of ESCs, limited cell source and immunogenicity. However, the pluripotency and differentiation potential of human iPSCs can vary due to genetic background, somatic mutations and epigenetic variation. Gene expression is regulated by cis-regulatory regions called enhancers, which are bound by transcription factors. One way to predict the stability and differentiation capacity of iPSCs is to understand how pluripotency-related genes are regulated by enhancers. Enhancer activity is known to be associated with specific histone modifications such as lysine residue acetylation of histone H3 (H3K27ac) (Creyghton et al., 2010a). However, recent studies suggest that H3K27ac may be dispensable for an active enhancer (Pradeepa et al., 2016a; T. Zhang et al., 2020). Thus, relying on chromatin screening for H3K27ac alone could lead to missing out on non-canonical enhancer regions and enrichment of false negative results. In the first project, we applied STARR-seq to human iPSCs, which allows comprehensive interrogation of enhancer activity genome-wide. Additional ATAC-seq (assay for transposase-accessible chromatin with sequencing, (Buenrostro et al., 2013a), ChIP-seq (chromatin immunoprecipitation followed by sequencing) (Park, 2009), and PCHi-C (promoter capture Hi-C) (Mifsud et al., 2015a) datasets led to the identification of active non-canonical enhancers, which are devoid of the H3K27ac mark. Importantly, this class of enhancers does not gain H3K27ac in differentiated cell types. We performed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) experiments to functionally validate the effect of non-canonical enhancers on target genes, which lead to their inactivation or activation, respectively. We also found unique motif features of non-canonical enhancers. Finally, we verified the presence of putative non-canonical enhancers in other human cell types and model organisms. Taken together, our results suggest the existence of a separate class of active enhancers that are not marked by the H3K27ac mark but are interacting with highly expressed genes. In the second project, we studied how epigenetic histone marks are re-established during DNA replication, when nucleosomes are dis- and reassembled. To study this, we made use of a Drosophila histone mutant as a model organism (Günesdogan et al., 2010a), in which the assembly of nucleosomes relies only on pre-existing (or parental) histones in embryonic S phase 15 since new histones synthesis is lacking (Günesdogan et al., 2010, 2014). We found that the lack of histone synthesis reduces the occupancy of nucleosomes, which may be partially assembled as subnucleosomes. However, the original positions of parental histones carrying modifications were preserved, suggesting that these harbour a ‘positional memory’, which allows the reestablishment of the epigenetic landscape during DNA replication.
Keywords: iPSC; enhancer; bioinformatics; chromatin; Drosophila melanogaster; Homo sapiens
