Suzanne de Bruijn

182 Chapter 4 Wildtype GDPD1 SMG8 YPEL2 LN 01476 > < NL-SV1 GDPD1 SMG8 YPEL2 01476 GDPD1 SMG8 LN 01476 GDPD1 YPEL2 LN YPEL2 SMG8 GDPD1 Boundary CTCF orientation B Enhancer interaction > UK-SV2 Retinal enhancer YPEL2 YPEL2 GDPD1 > < > > < < A B D GDPD1 CTL NL-SV1 0.0 0.5 1.0 1.5 2.0 Fold changes SMG8 CTL NL-SV1 0.0 0.5 1.0 1.5 2.0 Fold changes YPEL2 CTL NL-SV1 0.0 0.5 1.0 1.5 2.0 Fold changes GDPD1 SMG8 GDPD1 CTL UK-SV2 0.0 1.0 2.0 3.0 Fold changes SMG8 CTL UK-SV2 0.0 1.0 2.0 3.0 Fold changes YPEL2 CTL UK-SV2 0.0 1.0 2.0 3.0 Fold changes C E ** ** ** **** Retinal organoid expression Photoreceptor precursors expression Neo-TAD Neo-TAD Novel TAD domain Gene transcription B B B B B B B B B B B B B B B B B B B B B B B B B YPEL2 TAD Figure 5. Convergent mechanism of ectopic retinal enhancer- GDPD1 interaction caused by RP17 SVs. (A) In wildtype genomic context, YPEL2 expression in retina is driven by retinal enhancers in a TAD with CTCF boundaries. Neighboring genes are insulated from retinal enhancer activation. (B) The NL-SV1 duplication creates a neo-TAD with a full-length copy of YPEL2 , GDPD1 and the retinal enhancers. This enables retinal-specific enhancers to ectopically interact with GDPD1 , that drives its misexpression (C) qPCR analysis of photoreceptor precursor cells (PPCs) revealed a significant upregulation of GDPD1 in NL- SV1 PPCs compared to controls. (D) The UK-SV2 duplication and inversion creates a neo-TAD with a full-length copy of GDPD1 and SMG8 and the retinal enhancers bounded by CTFC sites (E) qPCR analysis ROs revealed a significant upregulation of GDPD1 expression in UK-SV2 ROs compared to controls. (n=3 independent ROs, mean ± standard error of the mean, **p≤0.01, ****p≤0.0001).