HOXA9 Recruitment of Methyltransferase Complexes Alters Enhancer Landscape during Leukemic Transformation

HOXA9 is a homeodomain-containing transcription factor that regulates hematopoietic stem cell renewal and differentiation and is commonly over expressed in acute leukemia, including acute myeloid leukemia (AML), and T- and B-precursor acute lymphoblastic leukemia (B-ALL and T-ALL). Together with its co-binding factor MEIS1, HOXA9 has been shown to play a causal role in leukemic transformation; however, the mechanism through which HOXA9 promotes leukemogenesis is poorly understood.

Previously, we showed that HOXA9 primarily binds to promoter-distal regions of the genome that show histone H3 lysine 4 (H3K4) monomethylation and histone H3 and H4 acetylation, epigenetic signatures indicative of active enhancers. HOXA9 cobinds with other lineage specific transcription factors such as C/EBPα, which we previously showed to be essential for leukemic transformation. This suggests that HOXA9 functions in a multi-subunit complex including lineage-specific transcription factors as well as chromatin modulators, but the role of HOXA9 in promoting the formation of these "enhanceosomes" and how HOXA9 alters the enhancer landscape remains unknown.

In these studies, we found that in both myeloid and lymphoid murine leukemia models, HOXA9 alters the enhancer landscape through creation of de novo enhancers, many of which are active in other cell lineages in early embryogenesis. RNA expression analysis revealed that these de novo enhancers drive a leukemia-specific transcription program, whose up regulation is significantly impaired upon either HOXA9 inactivation or CRISPR-mediated deletion of specific HOXA9-bound enhancer sequences. Protein and chromatin immunoprecipitation studies showed that HOXA9 physically interacts with the MLL3/MLL4 histone methyltransferase complex and colocalizes with MLL3/MLL4 at many sites in vivo . HOXA9 is required for the recruitment of C/EBPα, the MLL3/MLL4 complex and histone H3 lysine 4 monomethylation at de novo enhancers. This activity of HOXA9 is essential for the activation of genes regulated by de novo enhancers and is associated with increased interaction of these enhancers with promoters as assessed by chromosome conformation capture (4C). In contrast, HOXA9 is dispensable for both C/EBPα and MLL3/MLL4 binding and H3K4 monomethylation at enhancers active in normal hematopoietic cells. Genetic disruption of components of the MLL3/MLL4 complex abrogates the active epigenetic profile of de novo enhancer regions, and significantly delays leukemia progression driven by HOXA9/MEIS1 in vivo . Together these findings show that HOXA9 reprograms the enhancer landscape of hematopoietic progenitors in leukemic cells, including formation of many de novo enhancers active during early embryonic development. This mechanism involves HOXA9-dependent recruitment of MLL3/MLL4 methyltransferase complexes, suggesting that targeting this methyltransferase complex could be an effective strategy for malignancies associated with HOX deregulation.