The mammalian genome is finely regulated to ensure precise gene expression in each cell type. This is achieved through epigenetic mechanisms which dynamically control gene expression without perturbing the underlying genetic code. Two well-characterized types of epigenetic marks are histone modifications and DNA (Deoxyribonucleic acid) methylation. Epigenetic modifier proteins are responsible for either ‘writing’, ‘erasing’, or ‘reading’ these modifications, which can contribute to gene activation or repression.
Epigenetic regulation of gene expression controls differentiation throughout all stages of hematopoiesis, and its disruption can result in disease. In the context of acute myeloid leukemia (AML), epigenetic dysregulation promotes aberrant gene expression in hematopoietic stem and progenitor cells, resulting in defective differentiation and leukemic transformation. The disruption of the epigenetic code can occur through the acquisition of genetic mutations in the genes encoding epigenetic regulators or chromosomal translocations that cause epigenetic defects.
2. Histone Modifications and Their Functions in the Hematopoietic System
Histones can be post-translationally modified with chemical tags such as methylation and acetylation, which are associated with either gene activation or repression. Histone modifications can be used to denote active or repressed cis-regulatory elements, such as enhancers, promoters, and gene bodies 
. Active gene promoters are characterized by both H3K4me3 and H3K27ac, deposited by MLL proteins and p300/CBP (Cyclic adenosine monophosphate response element binding protein), respectively 
. Conversely, active enhancer regions are defined by an enrichment of H3K27ac and H3K4me1 (and, in some cases, H3K79me2/3) 
. Active gene bodies are demarcated by a non-overlapping pattern of H3K79me2/3 and H3K36me3 at the 5′ and 3′ ends, respectively 
. In contrast, H3K27me3, deposited by EZH2, is observed in repressed genomic regions 
. During hematopoiesis, the balance between repressive and activating histone modifications in both enhancer and promoter regions influence gene expression changes, which can drive differentiation programs.
One histone modification that is particularly important and has been extensively studied for its functional role in the hematopoietic system is H3K79me2/3 (Figure 1
. The H3K79 residue is positioned within the histone octamer core and is methylated in humans by the methyltransferase DOT1L (Disrupter of telomeric silencing like-1) 
. DOT1L is critical for normal hematopoiesis, with Dot1l
-deficient mice displaying severe anemia and death at E10.5-13.5 
deficiency most notably impairs erythroid development, which is coupled with reduced H3K79me levels and the downregulation of the erythroid-specific genes Gata2
and Spi1 
. DOT1L can mono-, di-, or tri-methylate H3K79 (H3K79me1/2/3), with H3K79me2/3 being the most abundant forms that are highly correlated with gene activity 
. In a normal cellular context, H3K79me2/3 is linked to transcriptional elongation due to both its position within the 5′ end of the active gene bodies and its ability to directly interact with the components of the super elongation complex (SEC), such as AF9 and ENL (Figure 1
. Importantly, AF9 and ENL act as histone acetylation readers which further stabilize DOT1L to chromatin at the gene targets 
. In addition to transcription elongation, H3K79me is observed at a subset of active enhancers, where it plays an important role in maintaining enhancer–promoter interactions in leukemia models 
3. DNA Methylation and Its Function in the Hematopoietic System
DNA methylation takes place through the deposition of a methyl group on the 5′ carbon of the cytosine bases (5mC) at CpG dinucleotides (Figure 1
a). DNA methylation primarily regulates gene expression by recruiting or blocking the binding of proteins involved in transcription in the regulatory regions in the genome. DNA methylation is a crucial regulator of normal hematopoietic differentiation, balancing the concerted inactivation of stem cell-associated genes whilst governing the stepwise activation of lineage-defining transcription factors that trigger differentiation 
Figure 1. Mechanisms of epigenetic regulation in normal and leukemic hematopoiesis mediated by histone modifications and DNA methylation. (a) The de novo DNA methylation machinery dynamically regulates the methylation status of cis-regulatory elements, such as promoters and enhancers, to modulate gene expression. Epigenetic modifier proteins such as MLL or DOT1L catalyze histone methylation in the promoter (H3K4me3) and gene body (H3K79me2/3) regions, respectively, so as to promote gene expression. (b) During leukemogenesis, disruption of the DNA methylation machinery, such as TET2 loss of function, leads to the hypermethylation of enhancers and transcriptional repression. The fusion of epigenetic modifier proteins, such as MLL::AF9, results in the aberrant stabilization of DOT1L and elevated levels of H3K79me2/3, which drives abnormal gene expression signatures.
In promoter and enhancer regions, DNA methylation inhibits transcription by preventing transcription factor binding and RNA polymerase II activity, resulting in the stable silencing of gene expression 
. During hematopoietic differentiation, the promoters of lineage-specifying genes and transcription factors are demethylated and transcriptionally activated (such as POU2AF1
, implicated in B-cell differentiation), whereas the genes required for hematopoietic stem cell self-renewal, such as MEIS1
, are methylated and silenced 
. DNA methylation also regulates the higher-order 3D (Three-dimensional) chromatin structure by preventing CTCF (CCCTC-binding factor) binding, leading to new topologically associated domain formation, which, in some cases, results in enhancer hijacking and transcriptional activation 
, as in the context of AML 
DNA methylation is regulated in mammalian cells by de novo and maintenance pathways, which take place independently of cell division or during DNA replication, respectively. DNA methylation is actively modified in mammalian cells by de novo DNA methyltransferases 3A and 3B (DNMT3A and DNMT3B) and TET enzymes (Figure 1a). DNMT3A/B catalyze the transfer of a methyl group to cytosines, whereas TET proteins catalyze the stepwise oxidation of 5-methylcytosine (5mC) to 5hmC (5-hydroxymethylcytosine) and the subsequent intermediates, which are then converted to unmodified cytosines through the base excision repair (BER) pathway.
DNA methylation is maintained during cell division through the activity of DNA methyltransferase 1 (DNMT1). DNMT1 preferentially recognizes the hemimethylated CpG dinucleotides generated during DNA replication and copies the methylation patterns of the parental strand 
. Despite this, DNA methylation maintenance is imperfect, especially in genomic regions with a low CpG density 
. This results in a gradual passive loss of DNA methylation over time, a process that is linked to the replicative history and aging of hematopoietic stem cells 
4. Gene Regulatory Function of Histone Modifications and DNA Methylation in Leukemia
Several genetic mutations in histone residues, or in the enzymes which modify them, are observed in AML. Examples of this include mutations in the histone-modifying proteins EZH2, Additional sex combs like-1, LSD1, or MLL3 
. Furthermore, H3K27M/I mutant histones predominantly occur in pre-malignant hematopoietic stem cells (HSCs), where they promote self-renewal and leukemogenesis 
. One specific type of leukemia in which a histone modification, H3K79me2/3, has been extensively studied, is MLL-rearranged (MLL-r) leukemia, which is the focus of a review 
. Many of the cutting-edge technologies that have been used to study H3K79me2/3 and MLL-r AML can be applied to further understand how other histone modifications function in different AML settings.
MLL-r leukemia arises following the chromosomal translocation between the N-terminus of the MLL gene, containing the DNA-binding domain, and the C-terminus of over 100 identified fusion partner genes, with the most common being ALL1-fused gene from chromosome 9, ALL1-fused gene from chromosome 4, ALL1-fused gene from chromosome 10, and ENL 
. This creates an aberrant, functional MLL fusion protein (MLL::FP). Very few co-operating mutations are observed alongside MLL::FPs, indicating that leukemogenesis is driven solely by the MLL::FP itself. MLL::FPs recruit DOT1L to target genes such as the HOXA
cluster and MEIS1
. This causes abnormal transcriptional upregulation primarily through the deposition of H3K79me2/3 in their gene bodies and the acquisition of leukemic stem cell properties (Figure 1
. The catalytic inhibition of DOT1L leads to transcriptional downregulation of these target genes and, ultimately, the abrogation of leukemia, indicating that H3K79me plays a critical role in maintaining MLL-r leukemias 
In the clinic, MLL translocations give rise to both AML and acute lymphoid leukemia (ALL), which have a poor prognosis 
. MLL-r leukemia is more common in childhood, as well as those who develop therapy-induced leukemias, particularly those previously treated with topoisomerase II inhibitors 
. In infants, MLL-r ALL or AML accounts for over 70% of cases acute leukemia 
. Interestingly, different types of MLL::FPs are more strongly associated with AML, while others are more closely associated with ALL (MLL::AF9 and MLL::AF4, respectively). The mechanisms behind this process are still under investigation, but both the cell of origin and the specific fusion partner function are likely key players. This has been demonstrated in murine knock-in models, in which the Cre-LoxP
(Cyclization recombinase, locus of x-over P1) system is used to create in vivo translocations in different cellular contexts. The induction of Mll::Af9
in primitive progenitor cells using Lmo2-Cre
gives rise to AML, but not when Mll::Af9
is induced in other cell types, such as T-cells, or following the induction of different Mll::FPs, such as Mll::Af4 
. This indicates that the cell of origin and the type of MLL::FP expressed are key determinants driving MLL-r AML. Other in vivo models of MLL::AF9 AML rely on retroviral transduction to overexpress human MLL::AF9 in murine granulocyte and macrophage progenitors (GMP) and Lin-Sca1+kit+ (LSK) cells 
. The patterns of gene expression and epigenetic landscapes in these models have been shown to faithfully replicate what is observed in MLL-r AML patients. Specifically, elevated levels of H3K79me2/3 have been observed at the MLL::FP gene targets, suggesting that detailed molecular studies based on these models can be used to gain important mechanistic insights into MLL::FP biology 
In addition to the major role of histone modification in driving aberrant epigenetic states, DNA methylation is a crucial layer of epigenetic regulation in leukemogenesis. Mutations in DNMT3A
have been identified in over 60% of individuals with clonal hematopoiesis of indeterminate potential (CHIP) 
. Mutations in the DNA methylation machinery have also been found in 44% of patients with AML and many other hematological malignancies, such as myelodysplastic syndromes and myeloproliferative neoplasms 
. This highlights their role in promoting clonal expansion and leukemogenesis.
Traditionally, the role of DNA methylation in the hematopoietic system has been studied through a series of transgenic mouse models, in which the components of the DNA methylation machinery (e.g., Dnmt3a
) are conditionally knocked-out in the hematopoietic system using Mx1-Cre
. Conditional Mx1-Cre-Dnmt3a
knock-out models both displayed increased self-renewal in competitive transplants in vivo, a serial replating capacity, and the aberrant proliferation of the myeloid compartment 
. In Dnmt3a
knock-out mice, Gata3
hypomethylation leads to their overexpression and the concomitant inhibition of hematopoietic differentiation. This provides a mechanistic explanation for the role of DNMT3A in regulating the self-renewal and differentiation of HSCs.
Conditional Mx1-Cre Dnmt1
knock-out leads to functional defects in self-renewal and increased myeloid differentiation upon competitive transplantation 
. This effect can be attributed to the increased cycling of stem and myeloid progenitor cells, leading to the exhaustion of the stem cell pool. Importantly, Dnmt1
knock-out or loss of function delays leukemia onset, indicating that functional DNMT1 is required for the self-renewal of both healthy and leukemic stem cells and, therefore, is also implicated in leukemic progression 
Overall, these studies highlighted the essential roles of histone modifications and DNA methylation in gene expression changes that regulate self-renewal and differentiation in the hematopoietic system and how their disruption leads to clonal expansion and leukemic transformation.