Epigenetic Regulation of Normal and Malignant Hematopoiesis

KL Rice, I Hormaeche and JD Licht

Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

The molecular processes governing hematopoiesis involve the binding of sequence-specific transcription factors to the interplay between lineage-specific transcription factors target gene promoters and enhancers.These factors flag and a series of epigenetic tags, including DNA methyla- thoseregionsofthegenomedestinedtobetranscribedinto tion and covalent histone tail modifications, such as RNA, and work in part by recruitment of basal acetylation, methylation, phosphorylation, SUMOylation transcription factors and RNA polymerase II to target and ubiquitylation. These post-translational modifica- genes.Sequence-specific DNA-binding factors also recruit tions, which collectively constitute the ‘histone code’, are cofactors to gene regulatory regions, many of which are capable of affecting chromatin structure and gene part of multiprotein enzymatic complexes which facilitate transcription and are catalysed by opposing families of or inhibit gene transcription by modification of chromatin, enzymes, allowing the developmental potential of hema- the protein-bound state of DNA present in the cell topoietic stem cells to be dynamically regulated. The (Bottardi et al., 2007). Modulation of gene expression by essential role of these enzymes in regulating normal blood chromatin modification is termed ‘epigenetic’ regulation, development is highlighted by the finding that members and refers to stable and heritable changes in gene from all families of chromatin regulators are targets for expression that do not involve DNA sequence alterations. dysregulation in many hematological malignancies, and Such changes include DNA methylation, nucleosomal that patterns of histone modification are globally affected histone modifications, post-translational modifications in cancer as well as the regulatory regions of specific onco- and antisense miRNA silencing. genes and tumor suppressors. The discovery that these epigenetic marks can be reversed by compounds targeting aberrant transcription factor/co-activator/co-repressor interactions and histone-modifying activities, provides Nucleosomal histones: substrates for epigenetic the basis for an exciting field in which the epigenome of modification cancer cells may be manipulated with potential therapeutic benefits. The organization of DNA into higher order structures, Oncogene (2007) 26, 6697–6714; doi:10.1038/sj.onc.1210755 or nucleosomes, is a central component to epigenetic gene regulation.Each nucleosome, which represents the Keywords: hematopoiesis; epigenetics; histone code; basic repeating unit of chromatin, consists of 147 bp of methylation; leukemia; acetylation DNA wrapped around a core of eight histones including two molecules each of H2A, H2B, H3 and H4 (Luger et al., 1997). Individual nucleosomes are joined to each Overview other by the linker histone H1 and a short length of DNA (B200740 bp) to yield the 10 nm fiber, which Hematopoiesis is a dynamic process in which pluripo- may be further compacted into a helical structure tent, hematopoietic stem cells (HSCs) give rise to all the referred to as a 30 nm fiber via interactions between lineages of the blood, including T and B cells which the more variable, flexible histone tails, which protrude constitute the lymphoid lineage, and neutrophils, from the nucleosomal disk.The concept of a ‘histone eosinophils, basophils, monocytes, macrophages, mega- code’ was proposed following the discovery of specific karyocytes, platelets and erythrocytes which comprise post-translational covalent modifications of these his- the myeloid lineage (Zhu and Emerson, 2002).This tone tails by acetylation, methylation, phosphorylation, process involves the coordination of signal transduction glycosylation, SUMOylation and ubiquitylation.Such pathways, which are responsive to extracellular stimuli, modifications act in a concerted manner to induce and transcriptional networks affecting gene expression, structural changes in the chromatin fiber and to regulate such that the ultimate fate of the active HSC pool is the accessibility of transcription factors to gene regula- linked to the functional needs of the organism.The tory sequences, ADP ribosylate affecting gene expres- regulation of gene transcription is critically mediated by sion (Jenuwein and Allis, 2001).There are a vast number of potential combinations of chromatin modifications that can be displayed by histones but several general- Correspondence: Dr JD Licht, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Lurie 5-123, izations can be made.Transcribed genes may be present 303 East Superior Street, Chicago, IL 60611, USA. in nucleosome-free regions that are highly accessible to E-mail: [email protected] transcription factors, or in regions of chromatin that Epigenetic regulation of hematopoiesis KL Rice et al 6698

Figure 1 Sequence-speciﬁc transcription factors act as docking molecules for the recruitment of DNA and histone-modifying activities to target gene promoters.Active transcription is associated with hyperacetylation and methylation of H3K4, H3K79 and H3K36 residues in promoter regions, whereas gene repression is associated with DNA methylation, hypoacetylation and methylation of H3K9, H3K27 and H4K20 residues.These modiﬁcations are mediated by chromatin-modifying enzymes including DNA methyltransferases (DNMTs), histone acetyltransferases (HATs)/histone deacetylases (HDACs), histone methyltransferases (HMTs) and histone demethylases (HDMs).

tend to be hyperacetylated through the action of histone domains.A number of DNA-binding domains acetyltransferases (HATs).By contrast, heterochromatic have been characterized including the homeodomain, regions, generally silent in terms of gene expression, tend zinc-finger domain, leucine zipper domain, winged helix, to be hypoacetylated through the action of histone erythroblast transformation-specific domain (ETS) and deacetylases (HDACs) and methylated on cytosine- helix–loop–helix domains.In the past, the determination phosphate-guanine (CpG) dinucleotides by DNA methy- of targets of transcription factors was based on a ltransferases (DNMTs).Acetylation of histones can candidate gene approach.With the advent of technol- change the physicochemical properties of these proteins, ogies such as chromatin immunoprecipitation-promoter interfering with the electrostatic attraction between tiling arrays (ChIP–CHIP) and protein-binding micro- positively charged histones and negatively charged arrays, the ability to probe transcription factor-binding DNA.Furthermore, specific histone tail modifications sites on a comprehensive, genome-wide level is now offer binding sites for the recruitment of other chromatin possible (Mukherjee et al., 2004; Oberley et al., 2004). modification machinery.For example, specific histone tail These studies now allow the identification of canonical residue methylation events may be associated with and novel binding motifs within promoter, enhancer and gene activation and others with gene repression transcribed regions of target genes (O’Geen et al., 2007). (Figure 1). ChIP–CHIP experiments also reveal the presence of transcription-priming mechanisms, which involve pre- assembly of transcriptional machinery at promoters in preparation for mitogenic stimulation, and binding site Sequence-specific transcription factors in hematopoiesis selection mechanisms, which reveal that transcription factor binding is not only dependent on sequence During hematopoiesis, the controlled expression of recognition but also on chromatin structure that is lineage-specific genes is crucial for proliferation and associated with specific epigenetic marks found in differentiation cues.Many transcription factors are ‘euchromatic islands’.For example, transcriptional absolutely essential for the development of a hemato- regulation by Myc involves recognition of E-boxes poietic lineage, for example GATA-1 is required for the within the context of trimethylated H3 lysine 4 erythroid and megakaryocytic lineage, while PU.1 is (H3K4) and H3 acetylation and typically correlates required for myeloid development.The retinoic acid with preassembled RNA Pol II (Guccione et al., 2006). receptor (RAR), which is required for neither lineage clearly plays a modulatory role in the production of blood cells.The ability of such factors to influence cell Recruit either co-activators or co-repressors to the fate is related to the ability of transcription factors to: regulatory regions of genes via protein interaction domains Recognize specific DNA sequences via DNA-binding The ability of transcription factors to affect gene domain transcription is dependent on the specific association Transcription factors are modular with generally of activator or repressor regions of transcription factors distinct DNA-binding and transcriptional effector with co-activators or repressors.These cofactors may

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6699 serve to scaffold basal transcriptional machinery to malignancy.In hematological malignancies, recurring target genes, recruit chromatin-modifying enzymes (see chromosomal translocations may lead to the formation below) or to covalently modify transcription factors of novel fusion proteins or overexpression of transcrip- thereby altering activities such as DNA binding, tion factors in inappropriate temporal or developmental protein–protein interactions, nuclear transport and patterns.As a result, factors and enzymes responsible degradation.For example, the p300/CBP co-activator for catalysing these epigenetic modifications, in parti- is recruited by transcription factors to target genes cular DNMTs, HATs, HDACs and histone methyl- frequently via a specific amino acid sequence known as a transferases (HMTs) and nucleosome displacement KIX domain (Kasper et al., 2002), where it catalyses machinery are aberrantly deployed.This may lead to acetylation of core histones, correlating with transcrip- global shifts in gene expression, which frequently lead to tional activation (Blobel, 2002), however p300 can also increased self-renewal in the malignant cells at the enhance transcriptional repression, as in the case of expense of normal differentiation.Elucidating the acetylation of the promyelocytic leukemia zinc-finger mechanisms of aberrant epigenetic deregulation in protein (PLZF; Guidez et al., 2005). Transcriptional specific hematological malignancies including acute repressors may interact with co-repressors via conserved promyelocytic leukemia (APL), lymphoma and myelo- domains, such as the BTB/POZ domain found in the dysplasia has already led to targeted therapies. PLZF-RARa fusion protein present in patients harbor- ing the t(11;17) translocation.Interaction of PLZF- RARa with the nuclear receptor co-repressor (NCoR) DNA methylation: a mark of stable gene silencing and silencing mediator of retinoid and thyroid receptors (SMRT) leads to the recruitment of associated HDACs DNA methylation involves the addition of a methyl and transcriptional repression (Melnick et al., 2002). group at position C5 of the cytidine ring in the context of a CpG dinucleotide, and is catalysed by a family of DNMTs including DNMT1, which preferentially targets Recruit chromatin remodeling machinery hemi-methylated DNA and is required for ‘mainten- Transcriptional regulation frequently requires the move- ance’ methylation during DNA replication; and DNMT3A ment of nucleosomes, which can block access to DNA- and DNMT3B which are required for de novo methy- binding proteins, polymerases and accessory factors lation (Okano et al., 1998). In the mammalian genome, to the gene.Remodeling of chromatin and physical the distribution of CpG dinucleotides, which are displacement of the nucleosome require ATP hydrolysis. predominantly methylated, is statistically underrepresented Chromatin remodeling machinery includes imitation (B1 CpG per 100 bp), however the dinucleotide fre- switch-containing (ISWI) complexes, which mediate quency occurs at near-expected levels in the promoters nucleosome sliding in vitro (Langst and Becker, 2001); of an estimated 60% of human genes (B1 CpG per 10 bp), the SWI/SNF complexes, containing the Brg or Brm where cytosines are typically hypomethylated (Antequera ATPases, which can remove histones from DNA, or and Bird, 1993).This enrichment of CpG dinucleotides in transfer them from one DNA strand to the other (Fan gene promoters is likely the result of spontaneous et al., 2003); and the NuRD multifunctional repression deamination of methylated cytosines to thymidine in complex (Zhang et al., 1999). nonregulatory sequences and these CpG-rich regions are typically referred to as ‘CpG islands’. Respond to hormone through ligand-binding domains The regulation of gene expression by DNA methyla- For example, the RAR and retinoic acid X receptor tion of target gene promoters is crucial for the control of (RXR), which are part of a family of hormone- several developmental processes including X inactiva- responsive nuclear receptors including the estrogen, tion (Goto and Monk, 1998), genomic imprinting vitamin D3, thyroid and steroid hormone receptors, are (Schaefer et al., 2007), embryonic Hox gene patterning typically associated with NcoR and SMRT co-repressor (Terranova et al., 2006) and hematopoiesis. DNA molecules in the absence of ligand.Upon treatment with methylation patterns are perturbed in many human retinoic acid (RA), however, these receptors undergo a cancers and typically involve regional hypermethylation conformational change that results in the release of co- of CpG islands affecting tumor suppressor genes, for repressors and binding of co-activators, thus facilitating example, p15INK4b (CDKN2B) and p16INK4a (CDKN2A), transcription (Mangelsdorf et al., 1995). In some which are silenced in lymphoid and myeloid malignan- instances, the interplay between nuclear hormone cies that occur within an overall setting of genome-wide receptors and lineage-specific transcription factors may DNA hypomethylation, which has been linked to also regulate gene expression.For example, during genomic instability (Galm et al., 2006). The identifica- erythropoiesis, the estrogen receptor has been shown to tion of promoter hypermethylation ‘signatures’ linked to negatively affect GATA-1-dependent transcription in a certain epithelial tumors and leukemia subtypes suggests ligand-dependent manner, suggesting that members of that there is interplay between transcription factors and the steroid receptor family may exert their diverse DNA methylation complexes regulating normal cellular functions by interfering with cell-specific transcription differentiation that are awry in cancer cells (Esteller, factors (Blobel et al., 1995). 2003; van Doorn et al., 2005; Shames et al., 2006). The precise regulation of transcription factors and DNA methylation of CpG islands is associated with associated machinery is frequently deregulated in transcriptionally silent chromatin, however whether

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6700 DNA methylation induces transcriptional silencing silenced, hypermethylated tumor suppressor genes in per se, or functions as a stabilizer of silencing, remains human cancer cell lines.Studies by Cameron et al. enigmatic.Although the exact hierarchy of events is (1999) demonstrated that administration of trichostatin unclear, the net effect of DNA methylation is local A (TSA) following treatment with low doses of histone deacetylation and a closed chromatin config- 5Aza-dC, synergistically reactivated the expression of uration resulting in gene repression via two mechanisms. MLH1, TIMP3, p15INK4b and p16INK4 in tumor cells First, the presence of methyl groups acts to repel the (Cameron et al., 1999). Combinatorial treatment of binding of specific transcription factors, for example, in mice with 5Aza-dC and sodium phenylbutyrate was also the case of the murine H19/Igf2 imprinting control able to significantly reduce lung tumor development region on chromosome 7–69.09cM, which acts as a initiated by a tobacco-specific carcinogen in mice transcriptional insulator.Specifically, methylation of (Belinsky et al., 2003). Since then, clinical trials CpG dinucleotides on the paternal allele blocks binding involving the sequential administration of 5-azacytidine of CTCF and allows a downstream enhancer to activate and sodium phenylbutyrate in patients with myelodys- Igf2 expression via a looping mechanism (Bell and plastic syndrome or acute myeloid leukemia (AML) Felsenfeld, 2000; Yoon et al., 2007). Second, DNA have been conducted.These studies demonstrated methylation leads to the recruitment of methyl-CpG- an enhanced clinical response rate that was associated binding domain (MBD) proteins which include five with demethylation of p15INK4b and acetylation of members: MeCP2, MBD1, MBD2, MBD3 and MBD4 histones H3 and H4 (Gore et al., 2006). Interestingly, (Fatemi and Wade, 2006).These proteins interact with induction of histone acetylation was observed before cytosine methyl groups within the major groove of HDACI was administered, and although the mechanism DNA and through their interactions with DNMTs, by which 5Aza-dC results in histone acetylation is specific transcription factors and chromatin-modifying unknown, it appears that CpG island methylation is the enzymes are capable of repressing gene transcription. dominant epigenetic mark responsible for stable gene For example, MeCP2 has been shown to exist in a silencing. complex with the transcriptional co-repressor Sin3A and Studies of the globin gene locus have also provided HDACs (Nan et al., 1998), and repressed the ability of insights into potentially new mechanisms of epigenetic PU.1 to activate transcription through its cognate- regulation.For example, the initial activation of embryo- binding site (Bird, 2002; Suzuki et al., 2003). nic/fetal genes is thought to be a result of promoter The epigenetic control of the human a-andb-globin demethylation, as opposed to de novo methylation in genes during erythropoiesis is considered a paradigm for adults, since differentiation of HSCs derived from either differentiation-induced methylation changes during baboon fetal liver (FL) and adult bone marrow (ABM) normal hematopoiesis.During erythropoiesis, the ma- into mature erythroblasts is accompanied by a progressive turation of erythrocytes is associated with increased decrease in g-globin promoter methylation and the expression of a-andb-globin genes, which is required to concomitant activation of transcription in both FL and synthesize large amounts of hemoglobin (B3 Â 108 ABM (Singh et al., 2007). These results suggest the molecules per cell).The b-globin locus is located on existence of a DNA demethylase activity to counter- chromosome 11 and consists of five genes e,Gg,Ag, d balance the repression mediated by DNMTs, and would and b which are under the regulation of a locus control also explain the active demethylation of the paternal region, located 6–22 kb upstream of the e-globin gene genome that is observed shortly after fertilization (Levings and Bungert, 2002).In non-erythroid cells, (Kishigami et al., 2006). The identification of ROS1 in these genes exist in a methylated, transcriptionally silent Arabidopsis, which has DNA glycosylase/lyase activity state.During erythroid differentiation, however, indivi- specific for methylated substrates, and whose mutation dual genes within the b-globin locus corresponding to is associated with DNA hypermethylation and gene embryonic (e), fetal (GgAg) and adult (d, b) stages of silencing, provides evidence for such an activity (Kapoor erythropoiesis are expressed in a sequential fashion, et al., 2005). Alternatively, demethylation may occur such that embryonic/fetal genes are ultimately silenced with replication of DNA during differentiation and and adult genes are activated.The reactivation of fetal failure to remethylate daughter strands.The significance g-globin in patients with sickle cell disease using the of a DNA demethylase, however, and other as yet HDAC inhibitor (HDACI) sodium phenylbutyrate undiscovered epigenetic marks/modifying proteins, lies (Dover et al., 1992) and the DNMT inhibitor (DNMTI) in the ability to target the aberrant forms of these 5-aza-20-deoxycytidine (5Aza-dC) (Saunthararajah and activities in a more specific manner. DeSimone, 2004) demonstrated the therapeutic poten- Similar to the globin locus, the expression of specific tial of reversing such epigenetic marks, and set the scene transcription factors required for the differentiation of for its application in hematological malignancies. other hematopoietic lineages is regulated by promoter The mechanism of reactivation using 5Aza-dC is methylation, and as such, these genes also represent related to the depletion of functional DNMTs which potential targets for disruption in hematological malig- become bound in a complex with 5Aza-dC-incorporated nancies.For example, PU.1(SPI1) is highly expressed in DNA, however the ability of 5Aza-dC to selectively HSCs and differentiated B cells, but not in T cells, degrade DNMT1 has also been reported (Ghoshal et al., correlating with the methylation status of the PU.1 2005).The use of DNMTIs in combination with 50UTR, which is hypermethylated in CD4 þ and HDACIs has also been successfully used to reactivate CD8 þ cells (Ivascu et al., 2007). PU.1 overexpression

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6701 has been linked to peripheral T-cell lymphoma (Maha- direct effect of this modification on higher order devan et al., 2005), and mice with deletion of a chromatin structure, which serves to neutralize the regulatory element upstream of PU.1 developed AML charge between histone tails and the DNA backbone, (Rosenbauer et al., 2006; Ivascu et al., 2007). Further- and also by serving as a docking site for bromodomain- more, hypomethylation of PU.1 was observed in containing regulatory factors.In general, hyperacetyla- patients with diffuse large B-cell lymphoma compared tion of histones is associated with structurally ‘open’ to normal lymph nodes, highlighting the requirement for chromatin and gene transcription, whereas histone tight epigenetic control of PU.1 in normal hematopoi- deacetylation is linked to gene repression and/or esis (Ivascu et al., 2007). Differential methylation of heterochromatin formation (Verdone et al., 2006). regulatory elements controlling the expression of other HATs can be divided into three groups on the basis of lineage-determining transcription factors has also been their catalytic domains and comprise GNATs (Gcn5 observed, including GATA3, which displays reduced N-acetyltransferases) which include Gcn5, p300/CBP- methylation in naive and memory CD4 þ cells com- associated factor (PCAF), Elp3, Hat1, Hpa2 and Nut1 pared to CD34 þ , CD8 þ , T and B cells, correlating members; MYSTs, which include MOZ, MORF, Ybf2/ with its known role in maturation of single-positive CD4 Sas3, Sas2, HBO1 and Tip60 members; and p300/CBP cells.As another example, TCF7 and Etv5 display (cAMP response element-binding (CREB) protein) (Lee higher methylation in B and T memory cells compared and Workman, 2007).These enzymes are recruited to to naive counterparts (Ivascu et al., 2007). target promoters by cell-specific transcription factors or The provenance of the aberrant methylation of chromatin-binding subunits such as bromodomain- specific target genes in the cancer cell remains to be containing proteins, or may even directly bind DNA, fully elucidated.One source may be the aberrant as in the case of activating transcription factor-2 expression of DNMTs normally responsible for the (Kawasaki et al., 2000). HDACs can also be divided restricted wave of methylation during blood develop- into categories on the basis of sequence and domain ment.Indeed the overexpression of DNMT1 and 3B, in similarity, and include Class I HDACs (HDAC1–3 and addition to members of the methyl-CpG-binding pro- HDAC8), which possess homology to yeast Rpd3 teins, has been reported in numerous malignancies and are localized to the nucleus; Class II HDACs including ovarian (Ahluwalia et al., 2001), breast (HDAC4–7, HDAC9 and 10), which display similarity (Butcher and Rodenhiser, 2007), prostate (Patra et al., to the deacetylase domain of yeast Hda1 and travel 2002) and lung cancers (Lin et al., 2007). Furthermore, between the nucleus and the cytoplasm; Class III studies by Ostler et al.(2007) reveal that truncated HDACs, which consist of the silent information DNMT3B proteins deficient in the C-terminal catalytic regulator (SIR2) family of nicotinamide adenine dinu- domain are expressed in numerous cancer cell lines and cleotide-dependent deacetylases (SIRT1-8) and Class IV primary acute leukemias.Overexpression of the most HDACs (HDAC11) (Minucci and Pelicci, 2006; Ouaissi frequently expressed aberrant transcript, DNMT3B7 in and Ouaissi, 2006).Like HATs, HDACs function within 293 cells, led to alterations in gene-expression patterns the context of a multiprotein complex that includes which corresponded with DNA methylation at CpG DNA-binding transcriptional factors/unliganded nucle- islands of these promoters, further supporting the role ar receptors and co-repressor proteins such as NcoR, of DNMTs in the abnormal patterns of methylation SMRT, Sin3a and NURD (Cress and Seto, 2000). observed in cancer cells (Ostler et al., 2007). Aberrant In addition to regulating transcription by affecting gene methylation in leukemia may also arise by the chromatin structure, HATs and HDACs are also recruitment of DNMTs and associated chromatin- capable of indirectly affecting gene expression by modifying proteins by cell type-specific transcription modifying non-histone substrates (Minucci and Pelicci, factors, which are commonly dysregulated in hemato- 2006).The acetylation of specific lysine residues of logical malignancies including PML-RARa (Di Croce transcription factors has been shown to affect the et al., 2002) and RUNX1/MTG8 (Liu et al., 2005). The subcellular localization, DNA binding, transcriptional identification of such complexes and their specific target activity, protein–protein interactions and stability of genes is likely to provide important insights into several key transcription factors including p53, STAT3, methylation-induced silencing in leukemic cells. RUNX1 and ETS, and not surprisingly, alterations in HAT/HDAC activity are linked to multiple cancers (Bruserud et al., 2006). During hematopoiesis, lineage-restricted transcription Histone acetyltransferases and histone deacetylases: roles factors regulate specific gene-expression patterns by in normal hematopoiesis and leukemia recruiting HAT or HDAC complexes to the promoters of target genes (Huo and Zhang, 2005).For example, The acetylation of core histone tails in relation to gene during erythropoiesis, erythroid-specific transcription expression has been extensively studied and is regulated factors including GATA-1, which is essential for red by the opposing activities of HATs, which catalyse the blood cell maturation and survival, directly recruit transfer of acetyl groups from acetyl-CoA to lysine HAT-containing complexes to the b-globin locus to residues of target proteins, and HDACs, which catalyse stimulate transcriptional activation.Specifically, the removal of acetyl groups.The ability of histone GATA-1 recruits CBP to the b-globin gene locus, acetylation to regulate gene expression occurs via the resulting in the acetylation of histones H3 and H4,

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6702 and facilitating high-globin gene expression (Letting mechanism, the expression of these fusion proteins is et al., 2003). GATA-1 itself is also acetylated on associated with the loss of monoacetylated H4K16, conserved lysine residues by CBP, and although the which was recently identified as a common mark of effect of this modification remains controversial, the net cancer transformation (Fraga and Esteller, 2005).The result is enhanced transcriptional activity (Boyes et al., mixed lineage leukemia (MLL) gene is also involved in a 1998; Hung et al., 1999). translocation involving CBP, and the resultant MLL- In leukemia, the ectopic expression of wild-type (for CBP fusion has been shown to require both the CBP example, TAL1/stem cell leukemia (SCL), BCL6) or bromodomain and HAT domain for leukemic transfor- chimeric transcription factors (for example, RUNX1- mation (Santillan et al., 2006). These findings highlight MTG8, TEL-AML1, PML-RARa and PLZF-RARa) the importance of HATs and HDACs in regulating results in the aberrant recruitment of histone-modifying genome-wide and loci-specific chromatin structure. activities to target genes that play important roles in cell cycle control and differentiation.TAL1/SCL, first identified by its translocation in T-cell acute lympho- blastic leukemia (T-ALL) (Begley et al., 1989) is a Histone methyltransferases: roles in normal hematopoiesis member of the basic helix–loop–helix (bHLH) transcrip- and leukemia tion factors.TAL1/SCL is essential for the development of erythroid and megakaryocytic lineages, while nega- The methylation of histones on lysine and arginine tively affecting myeloid differentiation.TAL1/SCL residues by HMTs represents another level of gene binds E-box motifs as a heterodimer with other bHLH regulation, and is probably the most complex of the proteins including E12, E47, HEB and E2-2, and is epigenetic modifications: arginines can be monomethy- capable of activating and repressing transcription lated or dimethylated (symmetrically or asymmetri- depending on the specific association with co-activator cally); lysines can be mono-, di- or trimethylated. or co-repressor complexes.For example, acetylation of While arginine methylation is usually associated with TAL1/SCL by the co-activators p300 and the PCAF is gene activation, lysine methylation can be related to linked to increased transcriptional activation and transcriptional activation as well as repression depend- differentiation of murine erythroleukemia (MEL) ing on the residue modified.For example, methylation cells in culture (Huang et al., 1999, 2000), while the of histone H3K4, H3K36 and H3K79 is associated with association with a co-repressor complex including transcriptional activation, whereas H3K9, H3K27 and mSin3A and HDAC1 in MEL and human T-ALL cells H4K20 methylation is usually linked to gene repression was linked to transcriptional repression and inhibition (Shilatifard, 2006).It is becoming increasingly obvious of erythroid differentiation (Huang and Brandt, 2000). that the association between the modification of a The association between TAL1/SCL and mSin3A/ certain residue and its effect on transcription is not so HDAC1 declined upon MEL differentiation, suggesting simple, however, especially in light of recent evidence that mSin3A and HDAC1 may inhibit the ability of demonstrating that the processivity (mono-, di- or tri) TAL1/SCL to potentiate erythropoiesis and highlight- and spatial context of lysine methylation across a given ing a possible mechanism for SCL-induced leukemogen- locus determines the net effect on gene expression.This esis (Huang and Brandt, 2000).Indeed, overexpression can be demonstrated in the case of H3K9, which is of Tal1/Scl in an E2A or HEB heterozygous background typically associated with heterochromatin and gene induced thymocyte differentiation arrest that was linked repression.For example, a recent study of the histone to the depletion of E47/HEB heterodimer and recruit- methylation patterns in a highly active transcribed gene, ment mSin3A/HDAC1 to the CD4 enhancer (O’Neil poly(A)-binding protein C1, revealed increased levels of et al., 2004). These tumors were hypersensitive to H3K9Ac at the expense of H3K9me3 in the transcrip- HDAC inhibitors, consistent with the notion that tional start site ( þ 0.5–5 kb); however, high levels of leukemogenesis by ectopically expressed TAL1/SCL H3K9me3 were identified across the entire transcribed was mediated by aberrant gene repression due to region (Vakoc et al., 2006). The functional significance recruitment of co-repressor complexes. of such coding region methylation may be related to the Translocations affecting HATs have also been im- requirement for recondensation of chromatin following plicated in tumorigenesis.For example, the rare transcription elongation-associated acetylation.For translocations t(8;16)(p11;p13) and t(10;16)(q22;p13) example, Carrozza et al.(2005) demonstrated that the fuse the MOZ (MYST3) and the MORF HATs with deposition of H3K36 methyl marks across the coding CBP in AML (Panagopoulos et al., 2001; Rozman et al., region of active genes, such as STE11, acts as a marker 2004).In the case of the t(10;16)(q22;p13) translocation, for HDAC complexes that restore chromatin configura- the generation of MORF-CBP, which harbors the zinc- tion following RNA polymerase activity, thus suppres- fingers, nuclear localization signals (NLS) and HAT sing erroneous transcription from cryptic promoters. domain of MORF, and the RARa-binding domain, Histone methyltransferases can be classified into three CREB-binding domain, bromodomain and HAT domain groups, and catalyse the transfer of a methyl group main of CBP, is thought to promote aberrant patterns of from the methyl donor S-adenosylmethionine (SAM) to acetylation; however since both reciprocal fusion the e-nitrogen in lysine or the guanidinium nitrogen in proteins are expressed, the leukemogenic potential of arginine.These include (1) lysine-specific SET-contain- these fusion proteins is unclear.Irrespective of the exact ing HMTs involved in the methylation of H3K4, H3K9,

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6703 H3K27, H3K36 and H4K20 residues; (2) non-SET activation.MLL plays a critical role in the proliferation HMTs involved in H3K79 methylation and (3) protein and lineage-determination of hematopoietic progenitors arginine methyltransferases (PRMTs) that specifically during embryonic development, by maintaining the methylate H3R2, H3R17, H3R26 and H4R3.The SET expression of HOX genes, such as Hoxa7 and Hoxa9 domain, which is an acronym for three prototypical (Ernst et al., 2002, 2004). Like many transcriptional chromatin regulators in the fly, Su(var)3-9, enhancer of regulators, MLL is bi-functional, balancing both tran- zeste and trithorax, is a 130–140 amino acid motif found scriptional repression and activation roles: it represses in a number of chromatin-modifying proteins.SET- target genes through the recruitment of PcG proteins, containing proteins from Drosophila (Rea et al., 2000), HDACs and/or SUV39H1 (Xia et al., 2003), and yeast (Nakamura et al., 2002) and human (Rea et al., activates genes through its H3K4 methyltransferase 2000) have HMT activity that maps to the SET domain activity and by recruitment of HATs such as MOF and in some of these proteins, cysteine–rich sequences and CBP (Ernst et al., 2001; Milne et al., 2002; Dou flanking the domain were also required for methyl- et al., 2005). The role of MLL during normal transferase activity.The molecular basis for the speci- hematopoiesis is underscored by the finding that ficity of particular SET proteins for specific histone chromosomal translocations involving the MLL gene residues is not yet understood but is being approached on chromosome 11q23 and more than 60 different by structural biology studies of the SET domain in fusion partners occur in a significant proportion of complex with its substrates (Trievel et al., 2002). The patients with AML and ALL (Daser and Rabbitts, structure of SET domains was solved by a number of 2005).Furthermore, an internal tandem duplication of laboratories, and based upon these structures two MLL is one of the commonest genetic anomalies in critical regions were identified; one that binds the AML with a grossly normal karyotype.Although the methyl donor SAM and the other that binds histones MLL fusion proteins generally lack their own SET and lysine residues.Modification of SET proteins domain, as well as the CBP-binding domain, the DNA- in vitro can lead to changes in the ability of an enzyme binding domain is retained in the MLL fusion protein, to singly or multiply methylate a specific lysine residue and the upregulation of MLL target genes such as (Zhang et al., 2003; Qian and Zhou, 2006). Mutations Hoxa9 and Hoxa7, which have leukemogenic potential, that affect which lysine residue is specifically modified, is thought to account for the oncogenic properties of however, have not yet been identified.The future MLL-X fusions (Kroon et al., 1998; Ayton and Cleary, identification of such a residue that can alter SET 2003). domain specificity could lead to the production of The PcG proteins counterbalance this positive regula- designer HMTs that could be deployed to modify tion of homeotic genes (in addition to silencing genes chromatin in a controlled, artificial manner as well as during processes such as X-inactivation and genomic the design of more specific HMTs inhibitors. imprinting) by acting as negative regulators of tran- These HMTs form large, multiprotein complexes that scription.PcG proteins play essential roles in embryonic typically contain other histone modifier enzymes, such development and stem cell renewal and as such represent as HATs, HDACs, DNMTs and histone demethylases targets for deregulation in leukemia.PcG proteins (HDMs).HMTs may be co-activators or co-repressors function in the context of biochemically distinct, multi- of transcription factors that recruit complex epigenetic protein complexes known as polycomb repressive machinery to specific target promoters involved in complexes (PRC), and in mammals, these complexes critical processes, such as proliferation and differentia- include the PRC1 complex, which contains PcG, RING tion.The important role of these enzymes during domain proteins, and BMI1 (B cell–specific moloney hematopoiesis is underscored by the finding that the murine leukemia virus integration site 1), as well as overexpression or dysregulation of HMTs is found in PRC2, 3 and 4, comprised of enhancer of zeste protein-2 multiple hematopoietic malignancies (Table 1). (EZH2), which specifically methylates H3K27 and H1K26 in a complex-dependent manner, SUZ12, histone-binding proteins RbAp46 and 48 and one of Histone lysine (K) methyltransferases four forms of embryonic ectoderm development (EED). Histone lysine (K) methyltransferases (HKMTs) are Upon recruitment of PRCs to specific loci, transcrip- important regulators of normal hematopoiesis and have tional repression is thought to occur following the been shown to regulate lineage commitment decisions in trimethylation of H3K27 by PRC2 or PRC3, which concert with cell-specific transcription factors.In serves as a marker for PRC1 binding (Squazzo et al., Drosophila, the activation and repression of develop- 2006).PRC1-binding blocks the activating function of mentally regulated loci is maintained by trithorax group the ATP-dependent remodeling enzyme, SWI/SNF, (trxG) and polycomb group (PcG) proteins, respec- thereby facilitating a repressive chromatin environment tively, and since then mammalian homologs of these (de la Serna et al., 2006). In addition, PRC1 also recruits genes have been identified where they have been shown RING domain proteins, which are implicated in the to maintain patterns of Hox gene expression during ubiquitylation of H2A-K119, and BMI1, a protein that embryogenesis.The MLL protein, which resembles trx, activates RING protein activity, to target loci.Although belongs to the SET1 family of HMTs (including SET1A, the exact role of ubiquitylation in relation to gene SET1B and four MLL HMTs) that specifically methy- silencing is unclear, this modification has been shown to lates H3K4, a mark typically associated with gene be required for Hox gene silencing (Cao et al., 2005).

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6704 Table 1 Substrate speciﬁcity of HKMTs and involvement of normal and malignant processes Enzyme Histone residue Methylation Function (effect on transcription) modiﬁcation

SUV39H1 H3K9 (À) me2/me3 Heterochromatin formation Downregulation is associated with genome instability SUV39H2 me2/me3 Heterochromatin formation G9a me1/me2 Euchromatin regulation EuHMT/GLP me1/me2 G9a partner mutated in breast tumors RIZ1/PRDM2 me1/me2 Tumor suppressor mutated in BCL and CML

MLL1 H3K4 (+) me1/me2 Trithorax proteins.Induce cell proliferation and differentiation of MLL2 me1/me2/me3 hematopoietic progenitors.Multiple translocations in leukemia MLL3

G9a H3K27 (À) me1/me2 EEZH2 me1/me2/me3 PRC2 component.Overexpressed in various malignancies such as lymphomas NSD3 me1/me2 t(8;11) associated with AML

HDOT1L H3K79 (+) me1/me2/me3 Co-activator of AF10 and AF4, MLL-AF10, MLL-AF4 (associated with T-ALL and AML)

NSD1 H4K20 (À) me1/me2 t(5;11) associated with AML NSD2/MMSET t(4;14) associated with multiple myeloma SUV420H1 me1/me2 SUV420H2 Overexpressed in breast cancer

The symbols (+) activation or (À) repression refer to the effect of the modiﬁcation on transcription (me1, 2, 3 refer to mono-, di- or trimethylation).

EZH2 overexpression is associated with increased cell demonstrated.For example, during the differentiation growth and is common in prostate cancers and of myeloid and erythroid lineages from common lymphoma.Mice deficient in EZH2 showed impaired progenitors, PU.1 recruits SUV39H1, HP1 and the B-cell development and decreased rearrangement of the retinoblastoma (Rb) proteins and binds to GATA-1 on immunoglobulin heavy chain (Su et al., 2003). Since its target genes, thereby inhibiting erythroid differentia- EZH2 requires the presence of the other PRC2, 3 and 4 tion (Stopka et al., 2005). The interaction of SUV39H1 members for HMT activity, it is not surprising that and HP1 with Rb also implicates these chromatin dysregulation of other PRC components may also be modifiers in cell cycle regulation and cellular senescence linked to tumorigenesis.Indeed, downregulation of the by repressing genes such as Cyclin A, Cyclin E and E2F, polycomb component EED is associated with an and as such, SUV39H1 may function as a tumor increase incidence of carcinogen-induced lymphoma suppressor (Ait-Si-Ali et al., 2004). Indeed, loss of (Richie et al., 2002). Similarly, dysregulation of BMI1 Suv39h1 has been shown to induce dramatic genome also affects the activity of the PRCs, and overexpression instability, associated with loss of H3K9 methylation, and overactivity of BMI1 has been associated with a and increases the development of B-cell lymphomas variety of solid tumors including lung, breast, colon, (Peters et al., 2001) and T-cell lymphomas in mice (Braig prostate and neuroblastoma as well as in malignant et al., 2005). RUNX1, which plays an essential role in hematopoiesis (Breuer et al., 2004; Glinsky et al., 2005; myelopoiesis and is responsible for the silencing of CD4 Steele et al., 2006). Given its critical role in normal stem during T-cell maturation, has also been shown to cell function, it seems likely that BMI1 plays an important interact with SUV39H1.Studies by Reed-Inderbitzin role in the ability of the cancer cell to undergo limitless et al.(2006) demonstrated recruitment of Suv39h1 by self-renewal (Lessard and Sauvageau, 2003). Runx1 to an oligonucleotide containing the band 3 SUV39H1 and SUV39H2 are homologs of the upstream regulatory element, which has been shown to Drosophila Su(var)3-9 (suppressor of position-effect mediate RUNX-1-dependent repression in MEL cells, variegation) HMTase, and were originally shown to be suggesting that RUNX1 may repress a subset of target crucial for the formation of heterochromatin by selective genes by modifying H3K9.RUNX1 function is dis- trimethylation of H3K9 (Aagaard et al., 1999). This rupted through the t(8;21) translocation present in 10– methyl mark is recognized by the chromodomain of 15% of myeloid leukemias, which fuses the N-terminal heterochromatin protein 1 (HP1), which recruits addi- portion of RUNX1 to MTG8, leading to the recruit- tional HP1 molecules and other chromatin-modifying ment of HDACs and aberrant silencing of RUNX1 proteins, thereby spreading and maintaining the target genes.Given that the interaction of RUNX1 and heterochromatin state.Since then, the involvement SUV39H1 involves domains retained in the t(8;21) of SUV39H1 an SUV39H2 in regulating hemato- fusion proteins, this suggests that SUV39H1, in addition poietic-specific gene transcription has been clearly to HDACs and DNMTs may be involved in the

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6705 repression of genes contributing to leukemic transfor- motif, two proline–tryptophan–tryptophan–proline do- mation (Liu et al., 2005; Reed-Inderbitzin et al., 2006). mains which are critical for chromatin targeting (Stec G9a is the major H3K9 mono- and dimethyltransfer- et al., 1998, 2000; Chen et al., 2004; Ge et al., 2004) and ase in euchromatin and can also modify the methylation four plant homeodomain zinc-fingers, often involved in status of H3K27, by functioning as heterodimer with protein–protein interactions (Aasland et al., 1995) G9a-like protein (GLP)/EHMT1 .Similar to SUV39H1, suggest that MMSET is a transcriptional regulator. G9a-mediated repression is initiated by H3K9 methyla- NSD3 is located in a genomic region amplified in breast tion, followed by recruitment of HP1 and DNMT1, and cancer (Angrand et al., 2001) and is also fused to culminating in CpG island methylation.HP1 enhances NUP98 in AML associated with the t(8;11) transloca- DNMT1 activity, while DNMT1 stabilizes the binding tion (Rosati et al., 2002). of HP1 to ensure stable gene silencing (Smallwood et al., hDOT1L is an HMTase that lacks the SET domain 2007).This idea is supported by the finding that siRNA- and specifically methylates H3K79, a residue located mediated knockdown of G9a is associated with DNA within the globular domain of histone H3, rather than hypomethylation (Ikegami et al., 2007). Intriguingly, the histone tail.Intriguingly, hDOT1L has been shown GLP/EHMT1 was recently identified as a gene mutated to interact with AF10, one of several fusion partners in breast cancer, with at least one mutation mapping to of MLL involved in the pathogenesis of leukemia. the active site of the protein, potentially eliminating The direct fusion of MLL and hDOT1L resulted in HMT activity (Cebrian et al., 2006). The functional immortalization of murine myeloid progenitors and was consequences of these mutations on G9a/GLP function associated with the upregulation of several genes are yet to be determined, however this finding and the implicated in leukemogenesis including Hoxa7, Hoxa9 sheer number of SET domain containing HMTs in the and Meis1 (Okada et al., 2005). This activity was genome (B60) suggest that these enzymes might be associated with an increased H3K79 methylation on more commonly affected in malignancy. Hoxa9 genes in cells transduced by MLL-hDOT1L, Consistent with this idea, another HMT that methy- suggesting that the ability of MLL to recruit hDOT1L lates the H3K9 histone tail residue is RIZ1 (PRDM2). to target promoters may be a critical mechanism of This tumor suppressor protein is inactivated in many leukemogenesis.Recently, hDOT1L has also been human cancers by deletion, frameshift mutations, promoter associated with leukemic transformation mediated by hypermethylation and missense mutations (Canote et al., the Clathrin-assembly protein-like lymphoid-myeloid- 2002).Many cancer-associated mutations in RIZ affect AF10 (CALM-AF10) fusion protein identified in its HMT activity, suggesting an important role for this patients with T-ALL and AML (Okada et al., 2006). activity in suppression of tumor formation (Kim et al., The association of hDOT1 with CALM-AF10 results 2003). RIZ1 knockout mice develop B-cell lymphoma in upregulation of Hoxa5 via H3K79 methylation, (Steele-Perkins et al., 2001), and in chronic myelogenous and also contributes to CALM-AF10-mediated leukemia (CML), blastic transformation is associated leukemic transformation by preventing nuclear export with loss of heterozygosity in the vicinity of RIZ1 and a of CALM-AF10. decrease in RIZ1 expression (Pastural et al., 2007). Given the frequent involvement of HMTs in cancer, Overexpression of RIZ1 in a model CML blast crisis cell aberrant histone methylation and HMTs themselves line also reduced cellular proliferation and enhanced represent important potential therapeutic targets.An differentiation, confirming a potential tumor suppressor inhibitor specific for G9a activity, BIX-01294 (diazepin- role mediated by RIZ1. quinazolin-amine derivative) has recently been identi- The NSD family of SET domains includes NSD1, fied, and has been shown to specifically inhibit H3K9 MMSET (NSD2) and NSD3 (Stec et al., 1998). NSD1, dimethylation in the promoters of G9a target genes which methylates H3K36 and H4K20 residues, is (Kubicek et al., 2007). Given that G9a-mediated implicated in AML as a result of the t(5;11) transloca- methylation in euchromatin is a mark for HP1 and tion that fuses NSD1 to NUP98, a subunit of the DNMT1 recruitment, it follows that inhibiting one of nuclear pore complex that is frequently rearranged in the steps in the complicated cascade of event that lead to leukemia (Jaju et al., 2001). In humans, mutation of gene repression could lead to the reactivation of NSD1 is associated with familial gigantism (van Haelst important tumor suppressor genes.Treatment with the et al., 2005), as well as with Sotos syndrome, which is DNMTI 5Aza-dC has also been successful in the characterized by fetal overgrowth, malformations and reactivation of tumor suppressor genes, and recent increased risk of leukemia, suggesting that NSD1 might studies show that this drug also functions by decreasing function as a tumor suppressor (Kurotaki et al., 2002; H3K9 dimethylation in the promoters of breast cancer- Douglas et al., 2005). MMSET (NSD2) was identified at associated TSGs, DSC3 and MASPIN (Wozniak et al., the breakpoint of the t(4;14) translocation present in 2007).This decrease is associated with a post-transcrip- B15% of multiple myelomas, which results in the tional decrease in G9a, and represents a novel inhibitory overexpression of both FGFR3 and MMSET (Chesi mechanism for the DNMTs inhibitors.Recently, the et al., 1998; Stec et al., 1998). Preliminary data from our targeting of PRC2 components EZH2, SUZ12 and EED laboratory suggest that MMSET specifically methylates by the SAM hydrolase inhibitor, DZNep (3-deazane- H4K20 (Licht, unpublished data), and given the planocin A) has also shown promise as a new HMTase- presence of functional domains including NLS, a high targeted therapy.Treatment of breast cancer cells with mobility group box, in some proteins a DNA-binding DZNep resulted in the reactivation of a number of genes

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6706 repressed by PRC2 and induced apoptosis in cancer cells Histone demethylases: the reversibility of stable but not in normal cells (Tan et al., 2007). epigenetic marks Until recently, histone methylation was considered an irreversible epigenetic mark and loss of methylation was Protein arginine methyltransferases explained by histone replacement during replication or The methylation of protein arginine residues has been by the degradation of histone tails (Allis et al., 1980; implicated in numerous biological processes including Ahmad and Henikoff, 2002).The recent discovery of regulation of transcription, cell signaling, RNA proces- two families of HDMs, the amine oxidase enzyme LSD1 sing, subcellular transport and DNA repair (Pahlich and the Jumonji C (JmjC) domain-containing family, et al., 2006). PRMTs can be separated into two classes; however, suggests that regulation of gene expression and Type I enzymes catalyse the formation of asymmetric chromatin structure by histone methylation is more NG,NG-dimethylarginine residues and include PRMT1, dynamic than previously thought. PRMT3, PRMT4/CARM1 and PRMT6, whereas Shi et al.(2004) discovered the first histone lysine Type II enzymes catalyse the formation of symmetric demethylase (LSD1).This enzyme catalyses lysine NG,N0G-dimethylarginine residues and include PRMT5. demethylation in a flavin adenine dinucleotide-depen- Of these, only three PRMTs have been reported to dent manner and permits the specific removal of mono- catalyse histone methylation: PRMT4/CARM1 methy- or dimethylated lysine residues from H3K4 and H3K9 lates H3R2, R17 and R26; PRMT1 methylates H4R3 residues.LSD1 has been purified in several different and PRMT5 methylates H3R8 and H4R3.Formation of complexes and depending on the association with asymmetric dimethylarginine residues in histones by specific factors, may have a dual role in transcriptional PRMT1 and CARM1 is associated with gene activation, regulation.For example, demethylation of H3K4me1 while formation of symmetric dimethylarginine residues and H3K4me2 by LSD1 in promoter regions is by PRMT5 is implicated in gene repression.In addition associated with gene repression and LSD1 has since to affecting chromatin structure by histone methylation been identified as part of a repressive multi-subunit directly, PRMTs can also modify proteins with known complex containing ZNF217, CoREST, HDAC2 and roles in epigenetic regulation.For example, CARM1 has CtBP1, which is capable of repressing the E cadherin been shown to methylate the HAT CBP, which promoter (Cowger et al., 2007). It was proposed that negatively affects the co-activator function of CBP/ following the deacetylation of the histones by HDACs, p300 (Chevillard-Briet et al., 2002), and recent studies LSD1 removes the methyl groups in H3K4, facilitating demonstrate that the methyl-DNA-binding domain gene repression (Forneris et al., 2006). This dependence protein 2 (MBD2), which has been implicated in the of LSD1-mediated demethylation on HDAC activity is formation of colon tumors, is also negatively regulated supported by the negative effect that HDAC inhibitors by arginine methylation (Tan and Nakielny, 2006). have on LSD1 activity (Lee et al., 2006). In contrast, The role of PRMTs in hematopoiesis, specifically in association of LSD1 with the estrogen or androgen myeloid differentiation, was highlighted by Balint et al. receptors leads to demethylation of H3K9me1 and (2005a, b), who demonstrated the methylation of H4R3 H3K9me2, corresponding to an activation function ‘primes’, the regulatory region of specific genes for RA- (Metzger et al., 2005; Garcia-Bassets et al., 2007). In induced myeloid differentiation (Balint et al., 2005b). the context of hematopoiesis, LSD1 has also been Treatment of HL60 myeloid leukemia cells with vitamin identified as a component of a transcription activation D or dimethyl sulfoxide induced a ‘precommitment’ complex containing MLL1, and although the functional state that was linked to a rapid decrease in promoter significance of this interaction is not clear, the dysregu- H3K4 methylation and an increase in enhancer H4R3 lation of MLL in AML suggests that LSD1 may also methylation of the tissue transglutaminase gene, whose have links to leukemogenesis (Nakamura et al., 2002). expression is linked to RA-induced differentiation. The second family of HDMs includes the JmjC These ‘primed’ cells were then treated with RA, resulting enzymes, which catalyse the removal of mono-, di- and in H4 acetylation and H3K4 methylation and transcrip- trimethyl lysines in the presence of Fe(II) and a- tional activation (Balint et al., 2005b). Also implicated ketoglutarate.Several JmjC domain proteins have been in this chain of events is the enzyme peptidyl arginine identified with different specificities including JHDM1 deiminase, PAD4, which removes the methyl mark on (H3K36me1/2), JHDM2 (H3K9me1/2), JMJD2 H4R3, and is also associated with RA-induced gene (H3K9me2/3, H3K36me3) and JARID1 (H3K4me2/3) activation (Wang et al., 2004). Given that methylation of (Table 2).A recent study by Lee et al.(2007) H4R3 has been shown to be a substrate for HATs, these demonstrated the interaction of the JmjC HDM findings fit a model in which PRMT1-specific H4R3 JARID1d with the polycomb-like protein RING6a/ methylation serves as a priming mark for gene activation, MBLR and showed that RING6a/MBLR enhanced which upon exposure to appropriate stimuli (RA) leads to JARD1d-mediated H3K4 demethylation, associated the recruitment of PAD4 and HATs and transcription of with transcriptional repression.Jhdm1b has also been genes required for myeloid differentiation.These studies shown to interact with PcG proteins in a complex open the possibility of treating diseases resulting from the containing Ring1b/Rnf2 and the Bcl6 interacting co- aberrant activity of PRMTs and a number of histone repressor, among other proteins (Sanchez et al., 2007). arginine methyltransferases inhibitors have recently been This co-repressor complex may be implicated in gene synthesized (Spannhoff et al., 2007). repression mediated by Bcl6, a transcription factor

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6707 Table 2 Histone demethylases Enzyme family Enzyme Residue(s) affected Transcriptional activity

Monoamine oxidase family LSD1 H3K4me1/me2 Repression H3K9me1/me2 Activation

JmjC-containing family JHDM1/Fbx11 H3K36me1/me2 Repression JHDM2/JMJD1 H3K9me1/me2 Activation

JmjN subfamily JMJD2A H3K9me3 Activation/repression H3K36me3 JMJD2B H3K9me3 JMJD2C H3K9me3 Activation H3K36me3 JMJD2D H3K9me2/me3 Activation JmjN and Arid subfamily JARID1A-D H3K4me2/me3 Repression

The specific lysine residues targeted and the effect on transcription is outlined. frequently dysregulated in B-cell lymphomas, and could global loss of DNA methylation typically observed in provide the basis for studying the effect of HDM tumor cells (Fraga et al., 2005). Given that trimethyla- inhibitors in this disease.JMJD2A has been also tion of H4K20 is an important marker for repression of implicated in transcriptional repression of E2F-respon- heterochromatin domains, it is postulated that loss of sive promoters via interactions with the retinoblastoma H4K20me3, in conjunction with DNA hypomethyla- protein, HDACs and NCoR, suggesting a role for tion, contributes to genomic instability frequently JMJD2A in cell cycle control (Gray et al., 2005). observed in tumor cells.The finding that H4K20 Although our understanding of HDMs is fragmen- methylation also plays a role in DNA damage response tary, the possibility that these enzymes are deregulated in yeast cells provides another mechanism by which loss in leukemias and other cancers makes them attractive of methylation may aberrantly affect normal cell cycle targets for inhibition.Indeed, LSD1, which has been checkpoints (Sanders et al., 2004). It is likely that shown to be a co-activator for the androgen receptor by specific genes are also affected by the loss of H4K20 removing H3K9 methylation in a ligand-dependent methylation and H4K16 acetylation.For example, manner, is a marker for high-risk relapse prostate acetylation of H4K16 is associated with active tran- cancer (Kahl et al., 2006). Given that LSD1 is similar scription of HOXA9, which is typically dysregulated in in structure to monoamine oxidases (MAOs), it is not myeloid leukemias (Dou et al., 2005). surprising that MAO inhibitors have a negative effect on LSD1 activity.Recent studies revealed that MAO inhibitors including pargyline and the clinically used Epigenetic regulation by noncoding RNAs antidepressant Parnate are capable of inhibiting LSD1, and provide the basis for developing inhibitors with The identification of microRNAs (miRNAs), small greater specificity and potency (Yang et al., 2006, 2007). interfering RNAs (siRNAs) and Piwi-interacting RNAs Polyamine analogs have also been shown to be potent (piRNAs) provides yet another mechanism of gene inhibitors of LSD1 and were capable of inhibiting LSD1 in regulation, and recent studies demonstrate that these human colon carcinoma cells, resulting in the reactivation small regulatory RNAs are capable of regulating gene of aberrantly silenced genes (Huang et al., 2007). expression at the transcriptional, post-transcriptional and epigenetic levels (Ambros and Chen, 2007).Studies in Schizosaccharomyces pombe reveal that an RNAi- Patterns of histone modification may be globally altered induced transcriptional silencing complex (RITS) which in cancer contains the siRNAs from the dg and dh repeats at heterochromatic loci, in addition to Chp1, Ago1 and The analysis of global patterns of histone modifications Tas3 proteins, recruits an RNA-directed RNA poly- using a combination of ChIP and genomic tiling arrays, merase complex (RDRC) that in association with other high-performance capillary electrophoresis and liquid chromatin-modifying proteins is required for hetero- chromatography–electrospray mass spectrometry, has chromatin assembly at target loci (Verdel et al., 2004). It provided intriguing insights into the structure of the is postulated that siRNAs mediate the recruitment of epigenome that characterizes normal and malignant Clr4 (the homolog of Drosophila SU(VAR)3-9) to target cells (Fraga and Esteller, 2005; Bernstein et al., 2007). A loci and that subsequent H3K9 methylation serves as a comprehensive screen of histone H4 modifications in marker for the RITS complex (via the Chp1 chromo- human cancer cell lines and primary tumors compared domain protein) and the recruitment of RDRC.The to normal tissue counterparts revealed an overall loss of generation of additional siRNAs by the RDRC com- both H4K16 monoacetylation and H4K20 trimethyla- plex is thought to generate a feed-forward loop that tion at repetitive DNA sequences, coinciding with the facilitates heterochromatin assembly (Grewal and Elgin,

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6708 2007).Indeed, deletion of several components of this activated by 9-cis RA alone) (Kastner et al., 2001; Zhu pathway in S. pombe resulted in the loss of H3K9 et al., 2001). In the absence of RA, RARa/RXR methylation and expression of integrated transgenes at heterodimers recruit nuclear co-repressors NCoR and centromeric heterochromatin (Volpe et al., 2002). The SMRT, which in turn bind HDACs, leading to the significance of these findings lies in the fact that many of establishment of repressive chromatin structure at the proteins involved in heterochromatin formation in specific target genes and inhibition of granulocytic S. pombe are conserved in mammals, suggesting the differentiation.Physiological concentrations of RA existence of small RNA-induced epigenetic silencing in (10À8 M) trigger the release of repressive HDAC com- mammalian cells.The recent finding that Piwi, an plexes and allow the recruitment of HAT co-activator Argonaute protein that has been shown to associate complexes (including p300, P/CAF and CBP) to with a new class of small RNAs known as piRNAs target genes, resulting in transcriptionally permissive (Aravin et al., 2006), interacts with HP1 suggests that chromatin structure, gene activation and granulocytic these piRNAs may also play a role in heterochromatin differentiation. formation (Ambros and Chen, 2007). The PML-RARa fusion protein acts as a dominant- In animals, small regulatory RNAs in the form of negative RAR by recruiting chromatin-modifying activ- miRNAs are transcribed by RNA polymerase II and are ities to target promoters, leading to the constitutive and subsequently processed in the nucleus by RNase III RA-insensitive repression of RARa target genes.This Drosha and DGCR8 to yield a 70–100 nt stem loop enhanced repression is mediated by PML-RARa oligo- structure (precursor-miRNA; pre-miRNA).These pre- mers, which are tightly bound through the coiled-coil miRNAs are exported to the cytoplasm by Exportin-5, domain of PML and are capable of binding DNA and are digested by a complex including a second RNase independently of RXR (He et al., 1998; Minucci et al., III, Dicer, in addition to TRBP and AGO2, to yield a 2000).PML-RAR a oligomers form high-affinity com- 17–25 nt mature miRNA.The mature miRNA is then plexes with HDACs and DNMTs, leading to hypoace- incorporated in a miRNA-associated RNA-induced tylated histones, DNA methylation and transcriptional silencing complex, where it leads to either the degrada- silencing of target promoters.This functional association of target mRNAs, in the case of perfect target tion between PML-RARa, HDACs, DNMT1 and complementarity, or translational repression via DNMT3A has been well characterized for one such imperfect target binding, typically in the 30UTR of the target gene, RARb2, which encodes a putative tumor mRNA transcript.An evidence that miRNAs can suppressor (Di Croce et al., 2002). Indeed, RARb2is also alter the epigenetic status of the cell comes from not expressed in PML-RARa expressing APL blasts and studies in Arabidopsis, which revealed that mutations cell lines, and this correlates with promoter hypermethy- altering miRNA-binding sites in the PHABULOSA lation, suggesting that silencing of RARb2 may be (PHB) mRNAs affects not only transcript accumula- important for leukemic progression.Following RARb2 tion, but also the methylation status of the PHB gene promoter hypermethylation, the methyl-CpG-binding (Bao et al., 2004). protein, MBD1, is subsequently recruited to the promoter via interactions with HDAC3, where it is required for maintained silencing (Villa et al., 2006). RARa translocations in APL: PML-RARa as a model for Strikingly, however, treatment of patients with pharma- the interplay between genetic and epigenetic events cological doses of all-trans retinoic acid (ATRA, 10À6 M) culminating in leukemia causes the release of PML-RARa HDAC-associated co- repressor complexes and promotes differentiation of The molecular biology of APL has represented the APL blasts, giving rise to the concept that t(15;17) APL leading edge of understanding how transcription is a ‘druggable’ form of leukemia (Grignani et al., 1998). may go awry in hematopoiesis.APL is characterized The dominant-negative effect of PML-RARa on RARa by the accumulation of hematopoietic progenitors transcriptional regulation is unlikely to represent the full blocked at the promyelocyte stage of differentiation spectrum of effects mediated by PML-RARa and the and is associated with chromosomal translocations reciprocal RARa–PML fusion transcripts.Indeed, a that fuse either PML, PLZF, NuMA, NPM or recent study identified a novel PML-RARa transcript STAT5b with the DNA ligand-binding domain of the lacking the functional domains of RARa in a patient RARa (Sirulnik et al., 2003). The PML-RARa fusion who did not respond typically to ATRA treatment, protein generated by the t(15;17) translocation is suggesting that the effect of PML-RARa may be expressed in over 95% of APL cases, and treatment of through interference of PML function (Zayed et al., this AML subtype (FAB M3) is widely accepted as a 2007).The effects of RA may be also be attributed to its paradigm for differentiation therapy based on the ability to alter DNA methylation, since treatment of reversal of epigenetic marks (Goddard et al., 1991; APL blasts with RA led to reduced DNMT1, DNMT3A Vitoux et al., 2007). and DNMT3B expression and activity, associated with In the context of normal hematopoiesis, RARa, demethylation of RARb2 and differentiation of leuke- which is highly expressed in granulocytes, acts as a mic blasts (Fazi et al., 2005). bifunctional modulator of myelopoiesis depending on Acute promyelocytic leukemia fusion proteins also the binding of cognate ligand (RAR can be activated affect histone methylation.A recent study by Carbone by all-trans RA or 9-cis RA, whereas RXRs are et al.(2006) revealed that PML-RAR a interacts with

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6709 SUV39H1, the mammalian ortholog of Drosophila target genes with important roles in cellular prolifera- Su(var)3-9, which belongs to a family of SET-domain, tion and differentiation have been identified including chromatin-modifying proteins.Recruitment of SUV39H1 Hox genes (Kim et al., 2005), C/EBPb (Duprez et al., to the RARb2 promoter in PML-RARa overexpressing 2003), TNFR2 (Park et al., 2003) and RARb2 (Carbone cells was associated with H3K9 trimethylation, a marker et al., 2006), however the identification of additional of heterochromatin and accompanied by strong tran- direct targets which may be used as models for scriptional repression.Furthermore, the association of PML-RARa-induced chromatin modifications is PML-RARa with SUV39H1 significantly inhibited vita- necessary.The importance for such screens is high- min D and transforming growth factor-b (TGFb)-induced lighted by studies demonstrating the unique structure differentiation in the U937 cells, suggesting that the of PML-RARa oligomers that allow the recognition of oncogenic properties of the PML-RARa fusion are novel binding sites, in addition to the canonical RAREs, linked to its ability to stably silence genes required for suggesting that the functions of PML-RARa may also myeloid differentiation by recruitment of specific be related to the acquisition of new target gene chromatin-modifying activities. specificities (Kamashev et al., 2004; Zhou et al., 2006). In addition to recruiting DNMTs, HDACs and In addition, the ability of PML-RARa to interact HMTs, recent studies reveal that PML-RARa is also with other HMTs including G9a, hDOT1L, MMSET capable of recruiting the polycomb repressive complexes as well as the recently discovered HDMs including 2, 3 and 4 (PRC2/3/4) to RARb2 and NFE-2 target LSD1 and the JmjC domain-containing proteins, promoters (Villa et al., 2006). PML-RARa co-purified will also be of interest and may provide insights into with several components of the PRC multiprotein the specificity of PML-RARa and other leukemic complex including SUZ12 and EZH2, a HMTase oncoproteins.Such studies are crucial to lay the specific for H3K27 and H1K26, and transcriptional foundations for the development of specific HMT and repression correlated with an increase in H3K27 HDM inhibitors that may be used to increase the trimethylation.Importantly, knockdown of SUZ12 in efficacy of combinatorial epigenetic-targeted treatments the NB4 APL cell line, which overexpresses the including RA, HDAC inhibitors and DNA demethylating PML-RARa fusion, induced differentiation, suggesting agents. that the oncogenic block induced by PML-RARa may be via PRC-mediated repression of specific target genes.The interaction of PML-RAR a with PRC2/3/4 Epigenetics open questions and therapeutic possibilities was through the PML portion of the fusion, and wild-type PML but not RARa, was capable of During the past decade the understanding of the binding PRC2/3/4.Taken together, these results molecular mechanisms of epigenetic regulation in suggest that the normal function of PML may be the normal and malignant development has greatly in- repression of specific target genes (for example, HOX creased but many questions remain.Some of these genes which are targets of PcG proteins) via interactions include the importance and timing of interplay between with PRC complexes, a function that may be disrupted DNA methylation, histone methylation and histone in t(15;17), and indeed overexpression of HOX genes is a acetylation, the role of small RNAs in epigenetic hallmark of many leukemias.In addition, aberrant regulation of mammalian genes, the full significance of repression of RARa targets by PML-RARa-PRC2/3/4 ubiquitin, SUMO and other chromatin modifications, oligomers is also likely to be a contributory factor in the some of which may yet to be discovered, the relative leukemic transformation induced by this oncogenic importance of epigenetic marks in promoters versus the fusion. body of a gene and the possibility of an active DNA Although the details of histone code are now demethylase. unraveling, the interplay between histone deacetylation, Epigenetic signals represent the final outcome in a DNA methylation and histone tail methylation is not transcriptional hierarchy mediated by transcription completely understood.It is known that certain factors, and the ability to manipulate these marks and epigenetic modifications precede others, for example, potentially reprogram cancerous cells yields exciting H3K9 methylation requires prior deacetylation of the therapeutic possibilities.The use of specific compounds same lysine residue, however the exact sequence of to target aberrant transcription factor/co-repressor events culminating in PML-RARa-mediated repression interactions or histone-modifying activities represents a at the RARb2 promoter remains elusive.Indeed, logical extension of the past decade’s research in demethylating agents were not sufficient to completely elucidating aberrant gene regulation mechanisms.For rescue the PML-RARa/SUV39H1-induced differentia- example, the targeted disruption of Bcl6 and its tion block in U937 cells, while a partial release was associated co-repressor SMRT using inhibitor peptides observed in SUV39H1 overexpressing cells, suggesting caused growth arrest and apoptosis of Bcl6-expressing that both modifications are involved in the transcrip- lymphoma cells, without affecting normal cells, paving tional programs blocking myeloid differentiation.It is the way for future studies to identify novel peptides likely that the degree of repression mediated by PML- capable of specifically inhibiting other oncogenic tran- RARa-chromatin-modifying complexes differs for in- scription factors (Polo et al., 2004). HDAC inhibitors dividual target genes and is also subject to regulation by are already approved for cutaneous T-cell lymphoma different stimuli.To date, a number of PML-RAR a and at least under some conditions these agents may

Oncogene Epigenetic regulation of hematopoiesis KL Rice et al 6710 modulate gene expression to yield a clinical effect (Gore Acknowledgements et al., 2006). HMTs, HDMs and ATP-dependent This study was supported by NIH Grant CA59936 and a chromatin remodeling enzymes all remain potential Burroughs Welcome Foundation Clinical Scientist Award in therapeutic targets, but whether inhibition of these Translational Research (JDL), the Leukemia Research Foun- enzymes will speciﬁcally target the cancer cell in vivo dation (KLR) and a postdoctoral fellowship from the Basque remains to be determined. Government (IH).

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