An MLL-dependent network sustains hematopoiesis

Abstract

The histone methyltransferase Mixed Lineage Leukemia (MLL) is essential to maintain hematopoietic stem cells and is a leukemia protooncogene. Although clustered homeobox genes are well-characterized targets of MLL and MLL fusion oncoproteins, the range of Mll -regulated genes in normal hematopoietic cells remains unknown. Here, we identify and characterize part of the Mll -dependent transcriptional network in hematopoietic stem cells with an integrated approach by using conditional loss-of-function models, genomewide expression analyses, chromatin immunoprecipitation, and functional rescue assays. The Mll -dependent transcriptional network extends well beyond the previously appreciated Hox targets, is comprised of many characterized regulators of self-renewal, and contains target genes that are both dependent and independent of the MLL cofactor, Menin. Interestingly, PR-domain containing 16 emerged as a target gene that is uniquely effective at partially rescuing Mll -deficient hematopoietic stem and progenitor cells. This work highlights the tissue-specific nature of regulatory networks under the control of MLL/Trithorax family members and provides insight into the distinctions between the participation of MLL in normal hematopoiesis and in leukemia.

The histone methyltransferase activity of mll1 is dispensable for hematopoiesis and leukemogenesis

“…Depletion of Wdr82 reduced Hoxa9 expression slightly in both WT and ΔSET MEFs ( Figure S1E). Similar trends were observed with the hematopoietic-specific (Artinger et al, 2013) target gene Mecom in lin neg bone marrow cells (Figures S1F and S1G), although less effective knockdown in this cell type reduced the statistical significance. Thus, ΔSET cells do not exhibit an increased dependence on potential compensatory HMTs or C-terminal subcomplex components.…”

“…We examined Hoxa9, Evi1, and Prdm16 TSS regions and found a remarkable consistency in H3K4me3 and H3K4me1 enrichment comparing control and Mll1 Δ/Δ samples ( Figure 4A), which was true genomewide at all enriched loci (Figures 4B and 4C). Despite the downregulation of over 300 genes (Artinger et al, 2013), the changes in H3K4me enrichment vary only within the range observed between replicate samples ( Figures S4C and S4D). These data demonstrate that the HMT activity of MLL1 is not responsible for the maintenance of target gene expression in HSPCs.…”

“…To identify potentially subtle gene expression defects in ΔSET cells, we performed real-time quantitative PCR (qPCR) analyses using sorted LSK cells. We found that the strongly MLL1-dependent target genes Hoxa9, Mecom (locus encoding MDS-Evi1 and Evi1 transcripts), and Prdm16 (Artinger et al, 2013) were expressed at normal levels in ΔSET cells ( Figure 1G). Similarly, H3K4me3 and H3K4me1 enrichment around the transcription start site (TSS) of these loci was also unchanged in ΔSET HSPCs ( Figure S1B).…”

“…Using 4-hydroxytamoxifen (4-OHT) inducible cre recombinase (ER T2 -cre) animals in which ex vivo deletion of Mll1 resulted in rapid target gene downregulation (Artinger et al, 2013), we determined transcript levels and chromatin modifications over a period of 3 days, during which lin neg bone marrow cells remain phenotypically undifferentiated (B.P.M., data not shown). ER T2 -cre;Mll1 F/+ cells were used as controls because target gene expression is indistinguishable between WT and heterozygous cells (Figures S3A and S3B) and induction of cre recombinase can affect cell viability and gene expression.…”

“…The fact that these large proteins function within multiprotein complexes and frequently lack satisfactory in vivo structure-function assays has limited our understanding of gene regulatory mechanisms used in the tissues in which they function. To determine MLL1 function in vivo, we employed inducible Mll1 loss-of-function alleles and demonstrated that Mll1 plays a unique and essential role in HSCs and developing B cells (Artinger et al, 2013;Jude et al, 2007;Li et al, 2013). These studies also revealed MLL1 target genes that were MLL1 dependent only in hematopoietic cells, presumably due to tissue-specific mechanisms of recruitment (Artinger et al, 2013).…”