Background Mutations, both activating and inactivating ones, continue to be identified in human diseases, including systemic lupus erythematosus (SLE). One emphasis is on mutations in transcription factors and their impact on the genome, leading to disease. Here I discuss experimental approaches, genome-editing coupled with next-generation sequencing-based technologies, that provide a framework to provide insight into the function of mutant proteins in disease. The JAK/STAT (Janus kinase/Signal Transducer and Activator of Transcription) signaling cascade transduces cytokine signals in normal development and disease. Dysregulation of cytokine action on immune cells plays a key role in the initiation and progression of autoimmune diseases including systemic lupus erythematosus (SLE). Elevated levels of phosphorylated (activated) STAT5 are detected in conventional CD4 T cells and activated regulatory T cells of SLE patients. However, the contribution of protein-altering mutations in STAT5 in the etiology of the disease have not been investigated. Mutations in the SH2 domain of STAT5B, which is essential for its dimerization and biological activation, have been identified in patients with T cell leukemias.

Methods The Y665F mutation has been identified in patients with T cell large granulocyte lymphocytic leukemia (T-LGLL) using whole genome sequencing. To understand the molecular consequences of this mutation we introduced it into the mouse genome using CRISPR/Cas9 genome editing and deaminase base editing.

Results Using ChIP-seq and RNA-seq, we identified activity and formation of STAT5-dependent regulatory elements and expression of target genes that are altered by mutations. These findings provide insight into the initiation and progression of hematopoietic disease. Single cell RNA-seq and flow cytometry explored the impact of the STAT5B mutation on different immune cell populations and their genetic programs.

Conclusions Our study offers insights into pathogenic molecular immune mechanism elicited by STAT5 mutations. Our experimental approach provides a blueprint to investigate and understand mutations in autoimmune disease including lupus.