Zebrafish Ortholog of Human DOT1L Regulates Primitive and Transient Definitive Hematopoiesis and Controls hoxa9 and meis1 Expression

DOT1L is a H3K79 methyltransferase implicated in multiple biological processes including embryonic development, cell proliferation, DNA damage repair and hematopoiesis. Recently, it was reported that DOT1L interacts with various transcription factor MLL partner proteins, and that aberrant DOT1L methyltransferase activity is essential for the form of leukemogenesis mediated by MLL fusion oncoproteins. These findings led to current efforts in therapeutic targeting of DOT1L for MLL-rearranged leukemias. However, the role of DOT1L in hematopoiesis is incompletely understood, largely because all prior studies were conducted using mice where Dot1L inactivation leads to early embryonic lethality due in part to the failure of primitive hematopoiesis, precluding analysis Dot1L during the transition from primitive to definitive hematopoiesis. In this study we took advantage of a unique attribute of zebrafish to survive for several days without blood cells, which offers new possibilities to study effects of loss of function of genes that cause embryonic lethality in mice from hematopoietic defects. Here, we report a functional characterization of the zebrafish dot1l gene during development, with particular emphasis on its role in hematopoietic regulation.

We identified a single ortholog of human DOT1L on zebrafish chromosome 22 using the NCBI HomoloGene resource, with a predicted protein that is 66% identical to human DOT1L overall and has greatest similarity (96%) restricted to its methyltransferase domain. Highly conserved syntenic genes surrounding zebrafish dot1l and genes in chromosomal regions containing human DOT1L (chr. 19) and mouse Dot1L (chr. 10) suggested functional similarity and formed the basis to further investigate the zebrafish ortholog. By whole-mount in situ hybridization (WISH) dot1l was found as early as the 2-cell stage before zygotic gene expression starts, indicating maternally supplied transcripts in the embryo. Zygotic dot1l expression was detected at 20 hpf in the posterior intermediate cell mass (ICM) where primitive erythropoiesis occurs in zebrafish. Two different splice blocking morpholinos were used to inhibit dot1l pre-mRNA splicing in either wild type or fluorescent reporter lines to determine the consequences of zebrafish dot1l depletion. The morphants showed impaired growth, defective angiogenesis and cardiac dilatation, consistent with developmental defects in Dot1L^−/−mice. Although Dot1L^−/−murine embryos are anemic, o-dianisidine staining of the morphants at 48 hpf showed reduced circulating red cells during definitive hematopoiesis beyond when Dot1L^−/− mice are viable. Reduced in vivo fluorescence in Tg(gata1:dsRed) dot1l morphants at 48 hpf also suggested an erythroid defect. WISH analysis of 24 hpf dot1l morphants revealed significant up-regulation of the myeloid marker pu.1 in the anterior lateral plate mesoderm (ALPM) where primitive myelopoiesis occurs in zebrafish, persistent and ectopic pu.1 expression in the ICM and reduced expression of the early progenitor marker gata2 and the erythroid marker gata1. Additionally at 20 hpf, Tg(pu.1:gfp) dot1l morphants suggested increased pu.1 expressing cells in the ALPM, yolk and ICM. These results indicate that dot1l plays essential roles in primitive erythropoiesis and primitive myelopoiesis and in the erythromyeloid cell fate decision during transient definitive hematopoiesis. Monitoring of hematopoietic and developmental marker genes by qRT-PCR in 24 hpf embryos confirmed the expansion of myelopoiesis and impairment of erythropoiesis. Interestingly, qRT-PCR analysis also revealed an entirely new finding that two key target genes in MLL-fusion-mediated leukemogenesis, hoxa9 and meis1, were downregulated in dot1l morphants.

This work demonstrates a critical role of dot1l in zebrafish primitive erythropoiesis, in agreement with previous observations in Dot1L^−/− mice, but reveals a new role of dot1l in erythromyeloid progenitor differentiation in transient definitive hematopoiesis. Furthermore, based on reduced hoxa9 and meis1 expression with dot1l depletion, we report for the first time that dot1l is a key regulator of hoxa9a and meis1 gene expression, human orthologs of both of which are key upregulated target genes in MLL leukemogenesis. These discoveries also have important implications for DOT1L directed therapies.

No relevant conflicts of interest to declare.