<i>NPEPPS</i> segmental duplication drives position effect expression of <i>TBC1D3</i> in the human brain

In humans, the <i>TBC1D3</i> gene family is thought to play a critical role in the expansion of the frontal cortex by promoting neuronal proliferation during brain development. This gene family shows some of the greatest structural heterozygosity (~97%) with haplotype copy numbers ranging from 3-39 among different human haplotypes. This raises the question as to how a gene so crucial in the evolutionary expansion of the human frontal cortex can be so variable in the human population. Here, we characterize the regulatory architecture that explains this paradox. We show that 45-96% of <i>TBC1D3</i> expression is attributable to a single paralog located at the most telomeric position at the edge of a cluster of <i>TBC1D3</i> genes. We find that its >3-fold higher expression relative to other copies is driven by a 110 kbp segmental duplication that occurred ~8.9 million years ago, relocating a partial duplication of the puromycin-sensitive aminopeptidase gene (<i>NPEPPS</i>), including its promoter, adjacent to this <i>TBC1D3</i> locus. Using neurospheres and comparative transcriptomics of iPSC-derived cultures, we show that expression of <i>NPEPPSP1-TBC1D3</i> increases as neurons differentiate as a result of alternative splicing and differential polyadenylation usage. While the fusion exists in other ape lineages, we show subsequent deletion of the <i>NPEPPSP1</i> promoter in <i>Gorilla</i> and a separate, lineage-specific duplication in the <i>Pan</i> lineage ablated the production of this fusion product, rendering this position effect of <i>TBC1D3</i> specific to humans.