The Methylation-Sensitive Enhancer Derare Maintains Hematopoietic Stem Cells through Regulation of Hoxb Cluster

The balance between self-renewal and multilineage differentiation in hematopoietic stem cells (HSCs) is orchestrated by genetic regulatory networks, which are constituted by hundreds of thousands of cis-regulatory elements, such as promoters, enhancers, insulators, etc. Aberrant mutations in these cis-acting modules and their trans-acting factors have been frequently found in hematopoietic malignancies including leukemia. Although next-generation sequencing technologies have identified comprehensive maps of these modules, much remains unknown about their physiological functions and underlying mechanisms in HSC maintenance and leukemogenesis. Our transcriptome analysis in 17 murine hematopoietic cell types, including HSCs, committed progenitors and lineage cells, showed that most of HoxB cluster genes were predominantly enriched in the permanently reconstituting long-term (LT) HSCs. Interestingly, one of the two putative enhancers within HoxB cluster, identified by H3K27ac ChIP-Seq analysis, shared the same sequence as the retinoic acid responsive element DERARE, which was recently reported to regulate multiple HoxB gene expression in the central nervous system. To test whether DERARE is required for normal hematopoiesis, we utilized the DERARE knockout mouse and found that homozygous deletion of DERARE led to 2-fold reduction in both the frequency and absolute number of LT-HSCs. Functionally, limiting dilution, competitive repopulating unit assays showed a 2.5-fold decrease in functional HSCs of DERARE^Δ mice compared to wildtype control. We further performed serial transplantation and observed a 4.3-fold reduction of repopulation rate after secondary transplantation of DERARE^Δ HSCs, indicating long-term reconstitution capacity was impaired. Mechanistically, RNA-Seq in LT-HSCs from DERARE^Δ mice exhibited significantly enriched apoptosis pathway in DERARE^Δ compared with that from Wt control. Furthermore, DNA methylation analysis showed gradually gained methylation on DERARE during HSC differentiation, which is negatively correlated with HoxB cluster gene expression, suggesting DERARE might be a methylation-sensitive enhancer to control HoxB genes. Moreover, HoxB gene expression is markedly upregulated in Dnmt3a KO HSCs, indicating that Dnmt3a is responsible for the DERARE methylation in HSCs. Finally, the analysis of clinical data from acute myeloid leukemia 200 patients in the Cancer Genome Atlas project revealed that lowly methylated DERARE was significantly correlated with overexpression of HoxB genes, high cytogenetic risk, and poor prognosis, suggesting abnormal regulation of DERARE contributes to leukemogenesis. Collectively, our study demonstrates the essential roles of the cis-regulatory element DERARE in both maintenance of LT-HSCs and contribution to leukemogenesis through regulation of HoxB cluster genes.