Humans differ from other primates in various traits, despite nearly identical protein-coding sequences. Understanding the evolution of these differences requires studying transcriptional regulation. Here, we examine ZEB2, a transcription factor crucial for immune and neural development, to explore its regulatory divergence across great apes. Using B-lymphoblastoid cells, chosen as experimental model system due to the availability of biological replicates for three great ape species, we show that, in addition to conserved ZEB2 targets, human ZEB2 is distinct in regulating a larger repertoire of genes implicated in neuronal development. ZEB2 knock-down in human, chimpanzee, and orangutan B-lymphoblastoid cells followed by transcriptome profiling uncovered human-specific regulatory differences, especially in nervous system-related genes. Additional analysis using single-cell RNA-Seq and brain organoid data identified cell-type-specific differences in ZEB2 expression and regulated genes between humans and other apes, most pronounced in ventral progenitors and neurons. Moreover, human-specific ZEB2 targets are enriched in non-coding genes, suggesting an expanded and possibly rewired regulatory network. Our study demonstrates that species differences in ZEB2 regulation can be detected in a controlled cell system and validated in neural contexts. More broadly, we provide new insights into the functional divergence of TFs across closely related species and how regulatory shifts can contribute to phenotypic evolution.
