Lincrna Hotset Regulates Hox Gene Transcription and Promotes Early Hematopoietic Fates

Mammalian genomes encode tens of thousands of long intergenic noncoding RNAs (lincRNAs). Accumulated evidence suggests that many of lincRNAs are expressed in a development- or tissue-specific manner. However, it remains largely unknown how lincRNAs reprogram lineage-specific gene expression and regulate lineage-specific differentiation processes during early embryonic development. It is also important to identify specific lincRNA(s) that is involved in early hematopoietic lineage formation and differentiation. Here, we reported a HoxB locus-associated lincRNA HOTSET that is conservatively expressed in both mouse and human hematopoietic stem and progenitor cells (HS/PCs). During embryonic development, HOTSET RNA is dramatically induced upon embryoid body (EB) hematopoietic differentiation. Expression of HOTSET RNA is required for development of the mesoderm-derived Flk1⁺ bipotential precursors of blood and endothelium lineages and CD41⁺/c-Kit⁺ hematopoietic stem and progenitor cells. We demonstrated that HOTSET RNA directly interacts with the SET domains of Setd1a and MLL1 histone methyltransferases in a sense-strand dependent manner. HOTSET RNA directly binds to the HoxB1-6 gene promoters and recruits the Setd1a/MLL1 complexes to the anterior HoxB locus upon differentiation. Furthermore, HOTSET RNA mediated H3K4 methylation is essential for long-range chromatin loops that bring anterior HoxB genes into a close proximity with HOTSET RNA locus and for subsequent activation of anterior HoxB gene expression. In addition, knockdown of HOTSET RNA disrupts the recruitment of the Setd1a/MLL1 complexes, H3K4me3 levels, the three-dimensional chromatin architecture, and HoxB gene transcription at the HoxB gene cluster. Depletion of HOTSET RNA also impairs differentiation of the CD41⁺/c-Kit⁺ HS/PC population and hematopoietic transcription factor and signaling networks required for early hematopoietic differentiation. Importantly, re-expression of HoxB2-4 genes in the HOTSET-depleted embryoid bodies rescues the expression of core hematopoietic transcription factors as well as the Flk1⁺ mesoderm-derived precursors and CD41⁺/c-Kit⁺ HS/PCs that undergo hematopoietic differentiation. Thus, HOTSET plays an important role in early hematopoietic lineage commitment by shaping the three-dimensional chromatin landscape in the HoxB locus to activate anterior HoxB genes that subsequently modulate lineage-specific transcription networks during hematopoietic development.