A Conserved Cis-Regulatory Retinoic Acid Responsive Element Is Essential for Maintenance of Primitive Hematopoietic Stem Cells through Regulation of Hoxb Cluster

Hematopoietic stem cells (HSCs) sustain lifelong production of multiple blood cell types through a finely-tuned balance between stem cell maintenance and activation to prevent bone marrow exhaustion or overgrowth. The highly conserved Hox family of homeodomain containing transcription factors have been identified as key regulators and contributors in both normal hematopoiesis and leukemogenesis. Most previous work has focused on individual Hox genes; however, it remains largely unknown whether and how multiple Hox genes in a cluster are regulated and function in hematopoiesis. We initiated a study to perform systematic, high-throughput transcriptome analysis in the following 17 cell types from the bone marrow (BM) of C57BL/6J mice: 4 hematopoietic stem and progenitor cells (CD49b^lo long-term (LT)-HSC, CD49b^hi intermediate-term (IT)-HSC, short-term (ST)-HSC, and MPP); and 4 committed progenitors (CLP, CMP, GMP and MEP); and 9 mature lineage cells (B cell, T cell, NK cell, dendritic cell, monocyte, macrophage, granulocyte, megakaryocyte and nucleated erythrocyte). Intriguingly, as part of a unique fingerprint observed in the most primitive CD49b^lo LT-HSCs, we detected expression from the Hoxb cluster. Further analysis on all the four Hox clusters revealed that most of the genes from the Hoxb cluster, and not from the other Hox clusters, were predominantly expressed in the CD49b^lo LT-HSCs. This suggests that they might function as a cluster to maintain CD49b^lo LT-HSCs. A previous study has shown that one cis -regulatory retinoic acid responsive element (RARE), is conserved among vertebrate species and regulates multiple Hoxb gene expression in central nervous system development. Thus, we asked whether RARE is essential for maintenance of primitive CD49b^lo LT-HSCs by regulation of Hoxb cluster. To test this hypothesis, we utilized a RARE^Δ knockout mouse model and assayed for HSC numbers in BM. We observed that homozygous deletion of RARE led to 2-fold reduction in both the frequency and absolute number of CD49b^lo LT-HSCs. Functionally, we first conducted limiting dilution, competitive repopulating unit (CRU) assays by transplanting 2.5×10⁴, 7.5×10⁴ or 2×10⁵ of BM cells from RARE^Δ mutants and their control littermates, together with 2×10⁵ recipient BM cells derived from the Ptprc mutant strain, into lethally irradiated recipient mice. Our data showed a 2.5-fold decrease in functional HSCs in RARE^Δ HSCs (1/20,326) compared to control (1/50,839). To further evaluate the long-term effect of RARE on HSCs, we performed serial BM transplantation and observed a 12.9-fold reduction of reconstitution ability after secondary transplantation. These data indicate that deletion of RARE compromised HSC long-term reconstitution capacity. Collectively, our work provides evidence showing that RARE is essential for maintenance of the primitive HSCs by regulation of Hoxb cluster genes.