Understanding human disease mutations through gene editing and genome-wide next generation investigations

Abstract

Mutations, both activating and inactivating ones, continue to be identified in human diseases. 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, offering a framework to gain insights 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. Notably, elevated levels of phosphorylated STAT5, an activated form of the protein, are observed in conventional CD4 T cells and activated regulatory T cells of autoimmune patients. However, the contribution of protein-altering mutations in STAT5 in the etiology of the disease remains unexplored. 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. The Y665H and Y665F mutations have been identified in patients with T-cell large granulocyte lymphocytic leukemia (T-LGLL) using whole genome sequencing. To understand the molecular consequences of these mutations, we introduced them into the mouse genome using CRISPR/Cas9 genome editing and deaminase base editing. Using ChIP-seq and RNA-seq, we identified alterations in the activity and formation of STAT5-dependent regulatory elements, as well as changes in the expression of target genes caused by these mutations. These findings shed light on the initiation and progression of hematopoietic diseases. To explore the impact of the STAT5B mutation on various immune cell populations and their genetic programs, we employed single-cell RNA-seq and flow cytometry. Our study offers insights into pathogenic molecular immune mechanisms elicited by STAT5 mutations, providing a blueprint for investigating and understanding mutations in autoimmune diseases.