Extended Data Fig. 10: ZBTB18 Binding Motifs Exhibit High Conservation in Putative CREs of Eutherian IT Neurons.

a) Taxonomic tree of the 23 Chordate species ZBTB18 SST protein sequence features of ZBTB18 were examined. For every species, the Pfam architecture of protein domains is indicated to the right. b) The ZBTB18 binding motif, as well as 16 other motifs from the JASPAR database, have consensus sequences enriched in regulatory regions related to genes downregulated in the Zbtb18 KO that are specifically conserved in placental mammals. Fisher’s exact test was used to test for an enrichment of conservation. Red dots represent motifs with a False Discovery Rate corrected P-value < 0.05. For detailed statistics, please refer to Supplementary Table 17. c) Evolutionary divergence of the key enhancers and/or ZBTB18 binding motifs. The evolutionary divergence of ZBTB18 motifs in key enhancers (Zbtb18 P1, Cux2 E1, Cux2 E4, Robo1 E1, Satb2 E1, and Bcl11b P1) are plotted onto a phylogenetic tree that shows the divergence of placental mammals (black line), marsupials (red line), monotremes (green line) and non-mammal vertebrates (purple line). All three motifs in Zbtb18 P1(Zbtb18 P1_motif1, Zbtb18 P1_motif2, Zbtb18 P1_motif3) and Cux2 E1_motif2 (purple) had evolved before the divergence of monotremes from non-mammal vertebrates, and Satb2 E1_motif2 appeared between the divergence of monotremes to the rest of mammals. In contrast, Cux2 E1_motif1, Satb2 E1_motif1, and all motifs in Cux2 E4, Robo1 E1, and Bcl11b P1 appeared after the divergence of placental mammals from marsupials. MYA: million years ago. d) Bootstrap-based strategy to evaluate the robustness of motif enrichments. The percentile occupied by the observed ratio represents an empirical P-value (Red dash line), indicating how likely it is to obtain this ratio by chance among the invariant peaks. The categories IT Placentalia, IT Marsupialia, and KO up Placentalia present significantly extreme observed ratios (P = 0.005, P = 0.003, and P < 0.001, respectively).