Effects of CTCF binding site deletions on genome architecture and gene expression in the Epha4 locus [thesis]

Niklas Gerhards, Universitätsbibliothek Der FU Berlin
Enhancers and promoters are DNA sequences whose physical proximity, through chromatin looping, is a necessary condition to initiate gene transcription. Functional chromatin interactions are limited by topologically associating domains (TADs), structural regulatory units of the genome that constrain enhancer-promoter crosstalk. Recent studies have elucidated that an important role of the CCCTC-binding factor (CTCF) regulates in theT processes of TAD and loop formation. However, it remains
more » ... whether CTCF is indispensable to mediate enhancers-promoter interactions and to achieve precise spatio-temporal patterns of gene expression during development. To investigate this, we systematically deleted CTCF-binding motifs at the Epha4 locus in mice using CRISPR-Cas9 and evaluated the effects on chromatin organization and gene expressionfunctional consequences in vivo. We focused on two CTCF sites associated to an enhancer-promoter interaction that occurs during mouse limb development. Analysis of genome architecture showed that CTCF binding site deletions induce a complete depletion of enhancer-promoter interactions. Analysis of Epha4 expression levels revealed that while the enhancer-promoter loop is disrupted, up to 50% of physiological expression levels are be retained. Our results suggest that the baseline proximity generated by topologically associated domains (TADs can be sufficient to fail-safe expressional output during developmentis sufficient to yield a certain amount of expressional output. Previous research on TADs has stressed their insulating properties - highlighting their function of restricting enhancer-promoter contacts over TAD boundaries. Our results stresshighlight an additional function of TADs in supporting enhancer-promoter communication and in sustaining developmental gene expression.
doi:10.17169/refubium-35463 fatcat:crj3f5py7fdllfud5n32oimo7y