The Trithorax Group Protein Ash1l Is An Essential Epigenetic Regulator of Adult Hematopoietic Stem Cell Maintenance

Abstract 387

The Trithorax family of epigenetic regulators is intimately linked to normal and malignant hematopoiesis. While substantial work indicates that Mixed lineage leukemia (Mll) is required for hematopoietic stem cell (HSC) homeostasis, the functions of many other Trithorax family members have not been evaluated. We have discovered that the mammalian Trithorax group gene absent, small, or homeotic 1-like (ash1l) is required for the maintenance of adult, but not fetal HSCs. Mice homozygous for a gene trap insertion into the first intron of ash1l (GT/GT) had a ca. 90% reduction in ash1l transcripts. These animals had normal numbers of phenotypically defined fetal liver HSCs (CD150⁺CD48⁻Lin⁻Sca-1^hic-Kit^hi cells), but a 10-fold reduction in adult bone marrow HSCs already apparent by 6 weeks after birth. GT/GT bone marrow HSC depletion began in the first three weeks of life, the period during which HSCs turn off their fetal homeostasis program and enter a state of increased quiescence in the bone marrow. Cell cycle analysis revealed that GT/GT HSCs had an increased cycling fraction with a profound reduction in quiescent HSCs in the G0 phase of the cell cycle. This suggested that ash1l is essential to establish and/or maintain a quiescent population of adult-type HSCs. Furthermore, competitive and non-competitive transplantation assays showed that both fetal liver and adult bone marrow were incapable of providing long-term reconstitution in lethally irradiated recipients, indicating that neither compartment could sustain long-term HSC activity after reaching the recipient's bone marrow. To understand this profound HSC defect, we next sought to analyze the in vivo function of Ash1l. Like MLL, Ash1l is a SET domain-containing histone methyltransferase. Although MLL functions as an H3K4 methyltransferase, the in vivo specificity of the Ash1l SET domain has not been described. Transcriptional analysis of GT/GT HSCs indicated that expression of Hoxa9, a known target of MLL, was reduced by 50%. This suggested that Hoxa9 could be a useful locus at which to evaluate the biochemical activity of Ash1l by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). ChIP-qPCR revealed that GT/GT bone marrow cells had reduced H3K36 dimethylation but preserved H3K4 trimethylation at the Hoxa9 locus. This was consistent with previous in vitro analysis of Ash1l SET domain activity and computer predictions showing a high degree of conservation between Ash1l and SET2, an H3K36 methyltransferase. Thus, Ash1l and MLL are both required for Hoxa9 expression although their enzymatic activities differ, suggesting that Ash1l and MLL may act cooperatively at Hoxa9 and other target loci. To evaluate the functional consequences of this interaction, we studied mice deficient for ash1l and lacking Menin, a factor required for proper MLL targeting to target loci. Strikingly, these animals rapidly progressed to hematopoietic failure with a complete obliteration of the hematopoietic stem and progenitor compartment, a phenotype not observed in either genetic background independently. These data indicate that Ash1l and MLL/menin work cooperatively in hematopoiesis. Together, our findings reveal an essential function of ash1l in the maintenance of adult HSCs. Furthermore, they indicate that Ash1l functions in vivo as an H3K36 methyltransferase and suggest that different Trithorax family members can coregulate target genes by providing distinct activating histone marks. Future work will reveal the full extent and mechanisms of these cooperative effects in normal and malignant hematopoiesis.