Figure 5: Patient variants in HAR3091 and HAR3094 likely regulate IL1RAPL1 expression in multiple brain regions.

(A) The genomic region containing IL1RAPL1, HAR3091, and HAR3094. (B) Constructs containing either the human or chimpanzee version of HAR3091 and HAR3094 cloned upstream of a minimal promoter driving lacZ expression were randomly integrated into mice and analyzed at E14.5. HAR3091 has enhancer activity predominantly in the telencephalon and olfactory bulb (filled arrowheads), and the human version of HAR3091 is a weaker enhancer than the chimpanzee version. In contrast, HAR3094 has enhancer activity predominantly in the midbrain (asterisks), and the human version of HAR3094 is a stronger enhancer than the chimpanzee version. Representative embryos are shown (all embryos are in Fig. S14). In situ hybridization of IL1RAPL1 at E14.5 from the Eurexpress database (Diez-Roux et al., 2011) is shown for comparison. (C) CRISPRi targeting the IL1RAPL1 TSS and HAR3094 significantly decrease IL1RAPL1 expression compared to the non-targeting control (NTC) gRNAs in iPSC-derived neurons induced by NGN2 expression. Multiple gRNAs were tested per target region. (D) Patient variants in HAR3091 and HAR3094 were tested for luciferase expression in N2A cells. HAR3091 patient variants significantly increased luciferase expression, whereas HAR3094 patient variants significantly decreased luciferase expression. Statistical analyses are detailed in Materials and Methods. Coordinates are in hg19.