Epigenetic Regulation of Hematopoietic Stem and Progenitor Cells By the Eset Histone Methyltranferas

Methylation of histone H3 at lysine 9 (H3K9) mediates heterochromatin formation by forming a binding site for HP1. ERG-associated protein with a SET domain (Eset) or Setdb1E is a methyltransferase which trimethylates H3K9 in the euchromatic region. In mice, homozygous mutations of Eset result in peri-implantation lethality. However, its role in hematopoiesis remains to be addressed. In this study, we performed detailed analyses of mice in which Eset was deleted only in adult hematopoietic cells.

We first harvested bone marrow (BM) cells from Cre-ERT;Eset^+/+ (WT) or Cre-ERT;Eset^flox/flox mice, and then transplanted them into lethally irradiated recipient mice. At 4 weeks post transplantation, we deleted Eset by injecting tamoxifen. All the Eset knockout mice died by 21 days post-deletion due to severe hematopoietic failure. Competitive BM repopulating assays revealed severely impaired repopulating capacity of Eset-deficient hematopoietic stem cells (HSCs). These findings clearly indicate that Eset is essential for the maintenance of hematopoiesis.

Next we examined BM of Eset knockout mice at 2 weeks post-deletion. Interestingly, Lin⁻Sca-1⁺c-Kit⁺ (LSK) hematopoietic stem/progenitor cells (HSPCs) were profoundly decreased upon loss of Eset. In addition, Eset knockout LSK cells gave rise to significantly smaller colonies compared with the control cells. Eset knockout LSK were actively cycling compared to the control HSPCs and underwent massive apoptosis. Then, we made use of the myeloid leukemia model induced by the leukemic fusion gene MLL-AF9. Interestingly, deletion of Eset significantly prolonged the survival of recipient mice. These results suggest that Eset has an essential role of not only in normal HSCs but also in leukemic stem cells.

To understand the molecular mechanisms by which Eset regulates function of HSPCs, we performed comprehensive gene expression and epigenomic analyses. Microarray analysis revealed 1666 up-regulated and 1125 down-regulated genes more than 2-fold in Eset-deficient LSK cells compared with the control LSK cells. Unexpectedly, ChIP-sequence analysis of H3K9me3 identified only 193 genes showing reduced H3K9me3 levels greater than 2-fold upon deletion of Eset on the region from ±4.0 kb of transcription start sites (TSSs) of reference sequence genes in myeloid progenitor cells. Correspondingly, western blot analysis revealed no significant changes in H3K9me3 levels in Eset-deficient HSPCs. It has been reported that Eset-mediated H3K9me3 is essential to repress transcription of retrotransposons in ES cells. Of interest, however, the vast majority of retrotransposons were kept repressed in the absence of Eset in HSPCs. Among 193 genes with reduced H3K9me3 levels greater than 2-fold upon deletion of Eset, only 16 genes became transcriptionally activated more than 10-fold. Among these, 3 genes (Fbp2, Rpl39l, Akap5) showed loss of DNA methylation at their promoters upon deletion of Eset. Fructose-1,6-bisphosphatase (Fbp2) is a muscle-specific gluconeogenesis enzyme which converts fructose-1,6-bisphosphate to fructose 6-phosphate. Fbp2 was indeed drastically upregulated in a HSPC-specific manner, but not in other organs or ES cells. Therefore, we tested its contribution to the Eset-deficient phenotypes of HSPCs. Metabolome analysis confirmed severely compromised glycolysis and reduction in levels of ATP by 20% in Eset-deficient HSPCs. Forced expression of Fbp2 in HSCs caused growth retardation and significant reduction in levels of ATP.

We conclude that Eset plays a critical role in the epigenetic regulation of normal as well as leukemic stem cell functions such as energy metabolism.