Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells.

To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro.

Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage⁻Sca-1⁺Kit⁺(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing.

In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs.

Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation.

Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis.