A Chromatin Modifier SATB1 Promotes Lymphocyte Production From Primitive Hematopoietic Stem/Progenitor Cells

Abstract 836

Although hematopoietic stem cells (HSC) are capable for reconstituting all hematopoietic lineages in serial transplantation experiments, they undergo substantial age-related changes. Indeed, many studies have suggested that the immune system qualitatively and quantitatively changes along aging, including the selective loss of lymphopoietic potential. Lymphocyte progenitors explosively expand in fetal liver, but after birth their production sifts to bone marrow (BM) and becomes stable. Thereafter, replenishment of the adaptive immune system declines with age. If key inducers priming lymphoid lineage in HSC can be identified, they would be practical for expanding lymphocytes in culture for clinical purposes. Additionally, exogenous manipulation of their expression might boost the immune systems of immuno-compromised and/or elderly people.

A primary aim of the present work was to describe molecular signatures of early stages of lymphopoiesis. Our previous studies developed a method to isolate Rag1⁺ early lymphocyte progenitors (ELP) from the Lineage marker^- Sca-1⁺ c-kit^Hi (LSK) HSC-enriched fraction using Rag1/GFP reporter mice. We exploited the method and performed microarray analyses to compare gene expression patterns between HSC and ELP. While many molecules specific for the lymphoid lineage including T cell receptor and immunoglobulin heavy chain were highly induced at the ELP stage, some lymphoid-related genes were already expressed in the HSC-enriched fraction. In addition, our search identified many signal transduction kinases of whose function in lymphopoiesis is unknown. We then sought candidate genes governing the early lymphoid program with the goal of discovering molecules involved in the HSC to lymphoid lineage transition. Chromatin modifiers particularly drew our attention because of their ability to control spatial and temporal expression of essential genes. As a result, we identified Special AT-rich Sequence Binding 1 (SATB1) whose expression was faint in HSC and markedly up-regulated in ELP. SATB1, originally identified as a protein binding to the enhancer region of the IgH gene, is recently attracting considerable attention as a global transcription organizer for various genes.

Our real-time RT-PCR analyses revealed that Satb1 expression remarkably increases along the differentiation of HSC to lymphoid-primed multipotent progenitors and ELP. In contrast, it was significantly down-regulated in the Lineage marker^- Sca-1^- c-kit^Hi myeloid progenitors. Interestingly, our analyses also found approximately 2-fold reduction of Satb1 transcripts in the Rag1/GFP^- LSK cells of aged BM. In functional assays, adult BM-derived Flt3^-LSK cells were retrovirally transduced with SATB1 and cultured in various conditions. In stromal cell co-cultures that support the growth of B lymphocytes and myeloid cells, the SATB1 transduction gave rise to 50–300 fold more CD19⁺ CD45R/B220⁺ Mac1^- B lineage cells. In stromal-free cultures containing SCF, Flt3-ligand and IL7, SATB1 transduction also strongly induced B lineage cell production. On the other hand, methylcellulose colony assays showed 2-fold reduction of CFU-GM colony formation from SATB1-transduced cells compared to Mock-transduced cells. Next we examined if the exogenous expression of SATB1 promotes B lymphocyte growth from embryonic stem (ES) cells. SATB1 overexpression effectively promoted B1-B cell growth from mouse ES cells in OP9 co-culture system. Furthermore, we found that exogenous SATB1 expression partly restored the lymphopoietic potential in aged BM HSC that are compromised in lymphopoietic activity. Finally, we performed microarray analyses comparing SATB1- and Mock-transduced Flt3^- LSK cells to seek candidate genes involved in the SATB1 function. While neither Pu.1, Ikaros, E2a, nor Notch1 was affected, some lymphoid-related genes, such as Sp4, Maf and Rag1, were increased in SATB1-overexpressed HSC. In conclusion, our results indicate that SATB1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. The findings are providing important information regarding how the earliest step of lymphopoiesis is epigenetically regulated and how modulation of the process might be useful to induce or rejuvenate the immune system.