DOT 1L methylates histone H3K79 and is aberrantly regulated in MLL ‐rearranged leukemia. Inhibitors have been developed to target DOT 1L activity in leukemia, but cellular mechanisms that regulate DOT 1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC 1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT 1L dosage. Furthermore, cell lines derived from Hdac1 Δ/Δ thymic lymphomas were sensitive to a DOT 1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC 1 and DOT 1L with impact in murine thymic lymphoma development.

IntroductionHematopoiesis is a tightly regulated process of proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) toward mature blood cells. Lineage commitment and differentiation of HSPCs are orchestrated by transcription factors that are expressed at specific developmental stages. For example, CCAAT/ Enhancer-Binding Protein-Alpha (C/EBPA) is a master regulatory transcription factor that is not expressed in hematopoietic stem cells (HSCs), but starts to be expressed in a small fraction of multipotent progenitor (MPP) cells and increases steeply during the transition from the common myeloid progenitor (CMP) toward the GMP. C/EBPA drives granulopoiesis by controlling the expression of myeloid-specific genes. 1-2 miRNAs belong to a class of small (approximately 22 nt) noncoding RNAs. The RNA-induced silencing complex-bound miRNAs bind to complementary sequences that are predominantly located in the 3Ј-untranslated regions of target mRNAs and regulate gene expression by transcript destabilization and inhibition of protein translation. 3 Recently, the function of miRNAs in myeloid cells has been investigated using mouse models. For example, miRs-17/20/93/106 promote progenitor cell expansion by targeting Sequestosome-1-regulated pathways. 4 In addition, miR-223 negatively regulates myeloid progenitor proliferation and fine-tunes granulocyte differentiation and activity. 5 In addition, miR-146a inhibits the activity of both myeloid and lymphoid cell lineages and plays key roles in the regulation of inflammation. 6 DICER1 is an evolutionarily conserved member of the RNase III family of endoribonucleases that is critical for processing of specific precursor hairpin sequences, the so-called pre-miRNAs, into miRNAs. 7 Genetic deletion of Dicer1 in mice results in early embryonic mortality due to depletion of the Oct-4-positive pluripotent embryonic stem cell pool at embryonic day 6-7 (E6-E7). 8 A floxed Dicer1 allele (Dicer1 fl ) has been generated that allows conditional deletion of Dicer1 in a cell type-and developmental stage-specific fashion. 9 Hematopoietic lineage-specific conditional deletion of Dicer1 has revealed the involvement of miRNAs in the survival, maturation, and homeostasis of peripheral T lymphocytes and in Ab diversity and survival of B lymphocytes. [10][11][12] In addition, conditional Dicer1 deletion in osteoprogenitors using mice that have Cre recombinase under the transcriptional control of the osterix promoter (Osx-GFP-Cre) results in myeloid dysplasia and acute myelogenous leukemia with acquired genetic abnormalities but intact Dicer1. 13 Mouse primary HSCs are impaired by Dicer1 loss and are unable to reconstitute hematopoiesis. 14 In addition, conditional deletion of Ars2, another gene required for miRNA biogenesis, in HSCs results in BM failure and increased apoptosis of hematopoietic cells in thymus and spleen. 15 Therefore, the overall contribution of miRNAs to myeloid-lineage specification remains elusive. To address this issue, we generated a myeloid-specif...

SUMMARY Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Key Points miR-139-3p and miR-199a-3p, induced by ICL-induced damage, respectively, cause a loss and gain of hematopoietic progenitors. miR-199a-3p is an onco-microRNA (onco-miR) causing AML in a Cebpa-deficient mouse model. Target genes of miR-199a-3p include PRDX6, RUNX1, and SUZ12.

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

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MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.

Cytotoxic T-cell differentiation is guided by epigenome adaptations but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8 + T cells. T-cell specific ablation of Dot1L resulted in loss of naïve CD8 + T cells and premature differentiation towards a memory-like state, independent of antigen exposure and in a cellintrinsic manner. Without DOT1L, the memory-like CD8 + cells fail to acquire full effector functions in vitro and in vivo. Mechanistically, DOT1L controlled T-cell differentiation and function by ensuring normal T-cell receptor density and signaling, and by maintaining epigenetic identity, in part by indirectly supporting the repression of developmentally-regulated genes. Through our study DOT1L is emerging as a central player in physiology of CD8 + T cells, acting as a barrier to prevent premature differentiation and supporting the licensing of the full effector potential of cytotoxic T cells.