Project description:Gene expression analysis on purified murine hematopoietic stem cells (HSCs) deficient for Special AT-rich sequence-binding protein 1 (Satb1) compared to wild-type HSCs.
Project description:Gene expression analysis on purified murine hematopoietic stem cells (HSCs) deficient for Special AT-rich sequence-binding protein 1 (Satb1) compared to wild-type HSCs. Analysis of gene expression of bone marrow-derived CD45.2+ CD150+cKit+Sca-1+CD4-CD8a-CD19-B220-Gr-1- HSCs from congenic recipient animals transplanted >20 weeks with either wild-type or Satb1-deficient hematopoeitic cells.
Project description:How hematopoietic stem cells (HSCs) produce specific lineages is not well understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was significantly induced with lymphoid lineage specification. HSCs from Satb1-null mouse were defective in lymphopoietic activity in culture, and failed to reconstitute T-lymphopoiesis in wild-type recipients. Furthermore, Satb1-transduction in HSCs, as well as in embryonic stem cells, robustly promoted their differentiation toward lymphocytes in culture. We prepared RNA samples from control or Satb1-transfected Lin- c-kitHi Sca-1+ Flt3- cells derived from WT mouse bone marrow.
Project description:How hematopoietic stem cells (HSCs) produce specific lineages is not well understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was significantly induced with lymphoid lineage specification. HSCs from Satb1-null mouse were defective in lymphopoietic activity in culture, and failed to reconstitute T-lymphopoiesis in wild-type recipients. Furthermore, Satb1-transduction in HSCs, as well as in embryonic stem cells, robustly promoted their differentiation toward lymphocytes in culture.
Project description:Hematopoietic stem cells (HSCs) are now recognized as a heterogeneous population in self-renewing and differentiation capabilities. However, fundamental mechanisms governing the heterogeneity remain uncertain. We here show that special AT-rich sequence-binding protein 1 (SATB1), a global chromatin organizer, is involved in the mechanisms. Analyzing hematological lineage-restricted SATB1 knock out mice proved that SATB1 is indispensable for both self-renewal and normal differentiation of adult HSCs. Using SATB1/Tomato knock-in mice, we subdivided HSCs according to SATB1 intensity. Culture experiments and RNA-sequencing revealed essential differences between SATB1- and SATB1+ HSCs regarding lineage potential.
Project description:Our data show Satb1 deficiency leads to alterations in DNA cytosine methylation and a commitment-primed epigenetic state in HSCs. Examination of DNA cytosine methylation in wild type HSC and differentiation-committed progenitors as well as in wild type HSC and HSC lacking Satb1 (n=2 each).
Project description:Hematopoiesis requires the interaction of hematopoietic stem cells (HSCs) with various stromal microenvironments. Here, we examine the role of early B cell factor 2 (Ebf2), a transcription factor expressed in a subset of immature osteoblastic cells. Ebf2-/- mice show decreased frequencies of HSCs and lineage-committed progenitors. This defect is cell nonautonomous, as shown by the fact that transplantation of Ebf2-deficient bone marrow into wild-type hosts results in normal hematopoiesis. In coculture experiments, Ebf2 Ebf2-/-osteoblastic cells have reduced potential to support short-term proliferation of HSCs. Expression profiling of sorted Ebf2-/- osteoblastic cells indicated that several genes implicated in the maintenance of HSCs are downregulated relative to Ebf2+/- cells, whereas genes encoding secreted frizzled-related proteins are upregulated. Moreover, HSCs from Ebf2-/- mice and wild-type HSCs cocultured with Ebf2-/- osteoblastic cells show reduced Wnt responses. Thus, Ebf2 acts as a transcriptional determinant of an osteoblastic niche that regulates the maintenance of hematopoietic progenitors, in part by modulating Wnt signaling.
Project description:The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFkB pathway (the NFkB inducing kinase, NIK, and the downstream molecule NFkB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation), however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, i.e., either NFkB2 or c-Rel, only NFkB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFkB pathway for the recovery of normal levels of hematopoiesis after stress.
