The authors applied Perturb-seq to dissect how genetic regulators coordinate basal cell-biological programs in hematopoietic stem and progenitor cells (HSPCs). Although many regulators of high-level HSPC function are known, how HSPCs coordinate foundational processes such as cell-cycle control, stress responses and lineage priming and how these relate to stem-cell phenotypes remained incompletely understood. The study used perturbation-derived transcriptional responses to create an interpretable framework linking genetic perturbations, cell-biological processes and functional phenotypes.
The experimental approach profiled transcriptional consequences after targeting 520 genes by CRISPR interference (CRISPRi) in primary mouse HSPC cultures. The screen generated perturbation-level transcriptional signatures across heterogeneous HSPC cell states. The authors used these data as the basis for downstream decomposition into co-regulated programs.
To extract interpretable perturbation effects the team developed an analytical workflow designed to distinguish three types of perturbation-induced signals: changes in overall cell-state abundance, perturbation-driven clonal heterogeneity, and local, cell-state–specific transcriptional changes. This separation was necessary to isolate perturbation signatures that reflect direct transcriptional regulation within a given cell state rather than secondary shifts in population composition.
Using the local perturbation signatures derived from their analysis, the authors defined 19 gene regulatory programs (GRPs). These GRPs were identified by co-regulation across genetic perturbations — that is, genes that responded similarly when different regulators were perturbed — rather than by co-expression patterns or manual curation. The resulting programs represent sets of genes that are co-regulated in response to specific perturbations and thus capture functional relationships among regulators and targets.
The 19 GRPs align well with known cell-biological processes relevant to stem and progenitor cell biology, including processes tied to cell-cycle regulation, stress response pathways and lineage priming. Because GRPs are derived from perturbation co-regulation, they provide a functional readout of how genetic regulators orchestrate these cellular processes in HSPCs.
To test the wider relevance of the perturbation-derived programs, the authors decomposed gene expression data from independent functional studies and retrospective clinical cohorts into GRP activity scores. This approach assessed whether activity of the perturbation-defined programs could explain or predict phenotypes measured outside the original screen.
GRP activity scores associated with functional stem-cell phenotypes such as clonal output following transplantation. In retrospective acute myeloid leukemia (AML) cohorts, GRP activities also associated with clinical outcomes including patient survival and drug response. These associations suggest that perturbation-derived programs capture biologically and clinically meaningful variation and may help link specific genetic regulators to clinically relevant phenotypes.
The authors declared that supporting data and code are available via multiple figshare links and a GitHub repository. Reported resources include the following public links as provided in the source: https://doi.org/10.6084/m9.figshare.31276981, https://doi.org/10.6084/m9.figshare.31277494, https://doi.org/10.6084/m9.figshare.32531301, and https://github.com/veltenlab/hsc19. The authors declared no competing interests.
This work establishes perturbation-derived co-regulation programs as an interpretable framework to connect genetic regulators, cell-biological processes and stem-cell-associated phenotypes. By targeting 520 genes in primary mouse HSPCs and defining 19 gene regulatory programs, the study provides a perturbation-based map that links regulator perturbation to downstream transcriptional programs and to functional and clinical phenotypes, including transplantation output and AML outcomes. The provided data and code enable reuse and further investigation of the identified programs and their relevance to hematopoietic biology and disease.