Systematic Transcriptome Analysis Reveals the Crucial Role of Alternative Promoters in Hematopoietic Lineages

Alternative promoters generate transcript isoforms with distinct exon compositions, playing critical roles in the precise regulation of lineage-specific gene expression. Yet it is largely unknown about their usage and functional relevance during hematopoietic development. In this study, we performed a comprehensive transcriptomic analysis of 532 RNA-seq datasets spanning 57 hematopoietic cell types and constructed a high-resolution landscape of alternative promoter activity. Among 1,074 high-impact promoters, Pou2f1 and Ikzf1 were identified as novel examples of lineage-associated alternative promoter usage. Ikzf1 P1 was predominantly active in B cells and monocytes, whereas Ikzf1 P2 exhibited selective activity in T cells. Pou2f1 P1 was active in T cells, NK cells and Monocytes, whereas Pou2f1 P2 was largely restricted to B cells. To assess functional relevance, we employed CRISPRi technology with sequence-specific sgRNAs to repress individual promoters and validate their lineage-specific contributions. Further, we identified the upstream regulatory factors that drive alternative promoter activation: Spi1 for Ikzf1 P1, Foxo1 for Ikzf1 P2, YY1 for Pou2f1 P1 and Pax5 for Pou2f1 P2. Therefore, our findings provide novel insights into the functional landscape of alternative promoters in hematopoiesis, as well as establish a foundation for future therapeutic interventions targeting promoter-specific regulation in hematopoietic-related disorders.