Analysis of gene expression profiling of Alu induction by adenovirus e1a and TFIIIC commitment in hESCs

Alus represent the most abundant class of repetitive elements in the human genome. Despite their abundance, the transcription of these elements is rare, since they are generally repressed. However, some Alus are transcribed and have epigenetic characteristics of active enhancers. Adenovirus 5 has been reported to induce upregulation of Alus transcription. In this thesis, we demonstrate by RNA-seq that the adenoviral small e1a, whose ability to reorganize the epigenome of the host cell through interaction with p300/CBP and pRb has been characterized, is sufficient to trigger Alus transcription, in virtue of its interaction with the chromatin remodeler p400. We thus investigated the molecular mechanism underlying this p400 dependency. By employing genome wide ChIP-seq, we uncovered that the recruitment of TFIIIB is dependent on the interaction between e1a and p400. The epigenetic characterization of the expressed Alus confirmed their enhancer nature as these elements uphold enrichment in their upstream region for p300/CBP, p400 and the YAP/TAZ factors. Moreover, e1a reprograms the Alus in a state reminiscent of a poised enhancer, with an increase in H3K4 mono-methylation (H3K4me1) and a depletion of H3K27 acetylation (H3K27ac). As Alus could serve as platform for 3D genome organization, during our analysis of genome-wide occupancy of the RNA polymerase III machinery in cell infected by adenoviruses, we identified a cluster of genomic regions associated with genes involved in embryonic development, that were depleted of TFIIIC upon e1a expression (e1a is a known de-differentiation factor). This finding led us to further investigate this and will be described in the second part of this thesis. TFIIIC associating with Alus linked to embryonic genes agrees with the identification of the largest TFIIIC subunit (TFIIIIC220) in several CRISPR-Cas9 screenings performed on hESCs to unveil genes involved in the maintenance of stemness and differentiation capabilities. Human TFIIIC has been shown to deposit H3K18ac on Alus in serum-starved conditions and more recently was shown to have specific HAT activity against H3K18 (normally acetylated by the p300/CBP HATs). As hESCs are routinely grown in serum-free medium we, therefore, hypothesized that TFIIIC could play a role in the deposition of the H3K18ac mark in hESCs. The findings described in this thesis strengthen this hypothesis. The genome-wide comparison of H3K18ac and H3K27ac profiles with the occupancy of p300 performed in hESCs, NECs (partially differentiated cells) and IMR90 (fully differentiated cells) showed that in undifferentiated cells most acetylated regions are not occupied by p300. Thus, we produced ChIP-seq of TFIIIC and detected the regions that were bound by TFIIIC in the absence of p300 in hESCs. In these regions H3K18ac and H3K27ac were still enriched, supporting the idea that TFIIIC might be involved in the acetylation of at least H3K18. Gene Ontology and Cistrome dbToolkit score analysis showed that the TFIIIC-bound regions are involved in stemness maintenance and neurodifferentiation. Thus, we suggest that TFIIIC could play an important role in hESCs biology.