Figure 4. Autism-linked HAR variant increases human CUX1 promoter activity in all cortical layers.

Two unrelated consanguineous families, a) AU-20400 and b) AU-13200 with c) homozygous mutation within long distance regulatory element of CUX1 within a methylated CpG marked by the presence of H3K4me1 in neuronal cell types. ChIA-PET (Fullwood et al., 2010; Li et al., 2010) data demonstrates interaction of HAR with promoter of CUX1. The mutation alters d) TF motifs in the reference genome by e) adding additional motifs. f) The G>A CUX1 interacting mutation results in a 2-fold increase in CUX1 promoter activity in N2A cells (neural precursor-like condition) and 2.5-fold increase in the presence of dominant-negative (DN) REST (neuronal-like condition). g) Dendritic filopodia and spines of control and Cux1 overexpressing neurons co-transfected with GFP plasmid. Bar represents 2μm. Quantifications show that Cux1 overexpression results in i) increased spine density than compared to controls and that this increase is markedly higher when treated with 4AP/BIC. Cux1 overexpression also results in h) significant increase in spine head surface area. Student’s t test p-value * ≤ 0.01; ** ≤ 0.0001 compared to control. j) Mutant A allele (Mt-A) HAR increases transcriptional activity of human CUX1 promoter linked to GFP reporter, compared to wild-type G allele (Wt-G) HAR, in E16.5 transgenic mice. k) Both Wt-G and Mt-A mutations drive expression of CUX1 across all layers of the cortical plate, with increased expression due to the mutation. See also Figure S4.