Elgood Hunt, E.; Vivori, C.; Mitter, R.; Hannah Johnkingsly Jebaraj, J.; Agnadottir, V.; Delas, J.; Serna Morales, E.; Frith, T.; Skehel, M.; Elosegui-Artola, A.; Briscoe, J.; van Werven, F.

Transcription start site (TSS) selection diversifies the transcriptome and proteome, yet how alternative TSSs regulate development remains unclear. We show that the chromatin regulator ASH2L undergoes developmentally regulated alternative TSS switching in differentiating mouse cells, generating distinct mRNA and protein isoforms: a full-length ASH2L and a truncated form lacking an intrinsically disordered region (IDR). While both ASH2L isoforms are conserved across mammals, their regulation in mouse pluripotent stem cells is uniquely driven by a mouse-specific retrotransposon. This element suppresses transcription from the downstream TSS through a transcription interference mechanism involving SETD2-directed histone H3 lysine 36 methylation. The resulting stem cell-specific truncated ASH2L isoform primes developmental gene promoters for histone H3 lysine 4 methylation, establishing a chromatin state required for embryogenesis and motor neuron differentiation. These findings show that co-option of a mouse-specific retrotransposon rewires Ash2l TSS and protein isoform usage to control developmental timing and cell fate decisions.