Satb1 Determines Hematopoietic Stem Cell Differentiation Toward the Lymphoid Lineages

Abstract 388

There is accumulating evidence that combinations of transcription factors coordinately and sequentially regulate lymphopoiesis. Five transcription factors, PU.1, Ikaros, E2A, EBF, and Pax5 are known to be hierarchically involved in early steps in B-lineage differentiation. However, it remains unclear whether the initiation of lymphoid differentiation is regulated entirely by transcription factors in a hierarchical manner. A major goal of our study was to find key genes involved in specification of lymphoid fates. For this purpose, we compared gene expression profiles between hematopoietic stem cells (HSC) and early lymphoid progenitors (ELP), which were sorted from E14.5 fetal liver of Rag1/GFP knock-in heterozygous embryos. As a result, we found that the expression of Satb1, a global chromatin regulator, was significantly increased along the differentiation of HSC to ELP. To explore roles of Satb1 in early lymphoid specification, we performed transplantation experiments, injecting HSC isolated from bone marrow of 2 weeks old Satb1-null mice into wild-type (WT) mice. We observed that Satb1-null HSC could not reconstitute CD3⁺ T cells in lethally irradiated WT recipients. Indeed, CD3⁺ T lineage recoveries from Satb1-null HSC were decreased approximately 10-fold compared with WT HSC. On the other hand, we observed varied levels of reconstitution of the B lineage and no reduction in reconstitution of the myeloid lineage resulted from Satb1 ablation. These results demonstrate that expression of Satb1 in HSC is indispensable for lymphopoiesis, but not for myelopoiesis. Furthermore, our data indicate that abnormalities of lymphoid development observed in Satb1-null mice are intrinsic to Satb1-deficient HSC. Next we conducted over-expression experiments to define the role of Satb1 in lineage fate decisions of HSC. Limiting dilution assays in MS5 co-culture condition revealed 1 in 41 Satb1-transduced Flt3⁻Lineage⁻Sca1⁺c-Kit⁺ (LSK) cells produced B cells. However, only 1 in 143 control ones were lymphopoietic under these conditions. These results suggest that Satb1 affects the lineage fate of HSC and promotes their commitment to lymphoid cells. Next we examined if the exogenous expression of Satb1 promotes B lymphocyte growth from ES cells in the OP9 co-culture system. We established ES cell clones, which can be induced to express Satb1/GFP on removal of tetracycline (Tet) from the culture medium. Eight days after Tet deprivation, 22 % of GFP⁺ cells expressed CD45 and CD19. On the other hand, only 1% of GFP⁻ cells expressed same cell-surface markers. A majority of the CD19⁺ cells in Satb1/GFP⁺ ES-derived cells were positive for CD11b/Mac1 and/or CD5, suggesting that B1-B lineage cells were produced. In addition, Igh rearrangement assay revealed that D_H-J_H recombination occurred in the Satb1/GFP+ ES-derived cells. These results indicate that Satb1 over-expression directs even ES cells to differentiate toward the lymphoid lineage.

Lymphopoietic activity becomes compromised during aging. Accumulating evidence suggests that the earliest lymphoid progenitor pools proximal to HSC are deficient in aged bone marrow. It is likely that the down-regulation of genes mediating lymphoid specification and function is involved as a major cause. Satb1 has been listed in microarray panels as one of the genes that are down-regulated in aged HSC (Rossi et al., 2005). To confirm this observation, we sorted CD150⁺ LSK cells from bone marrow of 6-weeks or 2-years old mice and examined expressions of Satb1. As a result, the aged CD150⁺LSK cells showed reduced expression (about 6-fold) of Satb1 compared with these cells from young mice. We then examined whether Satb1 expression could restore the lymphopoietic activity of progenitors derived from aged mice. Satb1-transduced Rag1/GFP⁻ LSK cells produced significantly higher percentages of B220⁺ cells than control cells. With respect to the recovered B-lineage cell counts, about 3-fold more B220⁺ Rag1/GFP⁺ Mac1⁻cells were obtained by Satb1transduction than mock transduction. These results demonstrate that Satb1 can at least partially restore the lymphopoietic activity of aged hematopoietic progenitors. In conclusion, our results indicate that Satb1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. Such activity in generating lymphoid cells may be of clinical significance and useful to overcome immuno-senescence.