Umbilical cord blood (CB)-derived hematopoietic stem cells (HSCs) are essential in many life saving regenerative therapies, but their low number in CB units has significantly restricted their clinical use despite the advantages they provide during transplantation1. Select small molecules that enhance hematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified2,3, however, in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular pathways that underpin the unique human HSC self-renewal program will facilitate the development of targeted strategies that expand these critical cell types for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs4,5, the post-transcriptional mechanisms guiding HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we determined that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signaling through post-transcriptional downregulation of canonical AHR pathway components in CB HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and give evidence that manipulating such networks ex vivo can provide a novel means to enhance the regenerative potential of human HSCs.

SummaryMSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.

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Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and their long-term integrity. While regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventative machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and when overexpressed endows a 15.6-fold enhancement in the frequency of functional HSC in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127, and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis, and classifies PLAG1 among a rare set of bona fide regulators of mRNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.

The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.