2023 – TWO DISTINCT ISOFORMS OF MLLT3 BALANCE HUMAN HSC FATE DECISIONS

The MLLT3 is a critical regulator of human hematopoietic stem cell (HSC) self-renewal. Analysis of RNA-seq data and epigenetic marks associated with MLLT3 gene in human HSCs uncovered a novel short MLLT3 isoform (MLLT3-S) that is expressed from a second TSS. MLLT3-S was predicted to encode a truncated protein that retains the C-terminal AHD domain responsible for protein-protein interactions, but lacks the N-terminal chromatin-binding YEATS domain. Accordingly, MLLT3-S overexpressed in cell lines could interact with known MLLT3 protein partners, but was unable to bind chromatin. Whereas overexpression of MLLT3-L in cord blood HSCs promoted their expansion, overexpression of MLLT3-S suppressed it, implying distinct functions for the two isoforms in HSCs. Conversely, while knockdown of MLLT3-L in CB HSCs abrogated the most undifferentiated HSPC and led to premature differentiation, knockdown of MLLT3-S resulted in immediate relative expansion of immunophenotypic HSCs. Strikingly, MLLT3-S deficient HSCs were unable to engraft in immunodeficient mice, showing that MLLT3-S is required for proper HSC function. scRNA and bulk RNA seq analysis of human developmental tissues revealed that MLLT3-L is expressed already in hemogenic endothelium in the embryo, whereas MLLT3-S first appears in AGM HSPCs, and increases in maturing fetal liver HSCs. RNA-seq analysis also revealed that overexpression of MLLT3-S suppresses IGFBP2, a regulator associated with HSC expansion and highly proliferative fetal HSCs, whereas MLLT3-L promotes its expression. These data suggest that by suppressing MLLT3-L-driven expression of IGFBP2 and HSC expansion, MLLT3-S may promote maturation of HSCs in the fetal liver and transition to homeostatic state. The interplay between two distinct isoforms of MLLT3 may provide a key mechanism by which mature HSCs balance between expansion and maintenance modes.