Polycomb Group Repressive Complex Research Articles

Polycomb group ring finger protein 6 suppresses Myc-induced lymphomagenesis.

Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4, and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6-but not Mga-accelerates Myc-induced lymphomagenesis in Eµ-myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.

Super determinant1A, a RAWULdomain-containing protein, modulates axillary meristem formation and compound leaf development in tomato.

Shoot branching and complex leaf development relies on the establishment of boundaries that precedes the formation of axillary meristems (AMs) and leaflets. The tomato (Solanum lycopersicum) super determinant mutant is compromised in both processes, due to a mutation in Sde1A. Sde1A encodes a protein with a RAWUL domain, which is also present in Polycomb Group Repressive Complex 1 (PRC1) RING finger proteins and WD Repeat Domain 48 proteins. Genetic analysis revealed that Sde1A and Bmi1A cooperate, whereas Bmi1C antagonizes both activities, indicating the existence of functionally opposing PRC1 complexes that interact with Sde1A. Sde1A is expressed at early stages of boundary development in a small group of cells in the center of the leaf-axil boundary, but its activity is required for meristem formation at later stages. This suggests that Sde1A and Bmi1A promote AM formation and complex leaf development by safeguarding a pool of cells in the developing boundary zones. Genetic and protein interaction analyses showed that Sde1A and Lateral suppressor (Ls) are components of the same genetic pathway. In contrast to ls, sde1a mutants are not compromised in inflorescence branching, suggesting that Sde1A is a potential target for breeding tomato cultivars with reduced side-shoot formation during vegetative development.

A novel cardiomyogenic role for Isl1+ neural crest cells in the inflow tract.

The degree to which populations of cardiac progenitors (CPCs) persist in the postnatal heart remains a controversial issue in cardiobiology. To address this question, we conducted a spatiotemporally resolved analysis of CPC deployment dynamics, tracking cells expressing the pan-CPC gene Isl1 Most CPCs undergo programmed silencing during early cardiogenesis through proteasome-mediated and PRC2 (Polycomb group repressive complex 2)-mediated Isl1 repression, selectively in the outflow tract. A notable exception is a domain of cardiac neural crest cells (CNCs) in the inflow tract. These "dorsal CNCs" are regulated through a Wnt/β-catenin/Isl1 feedback loop and generate a limited number of trabecular cardiomyocytes that undergo multiple clonal divisions during compaction, to eventually produce ~10% of the biventricular myocardium. After birth, CNCs continue to generate cardiomyocytes that, however, exhibit diminished clonal amplification dynamics. Thus, although the postnatal heart sustains cardiomyocyte-producing CNCs, their regenerative potential is likely diminished by the loss of trabeculation-like proliferative properties.

OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9–10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Molecular Mechanisms of Facultative Heterochromatin Formation: An X-Chromosome Perspective.

Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.

Genome-wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome‐wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP‐tagged CLF/SWN under their respective native promoters and used them for ChIP‐seq analyses to profile the genome‐wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild‐type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA‐like and Telo‐box‐like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

BCOR regulates myeloid cell proliferation and differentiation.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

PRC1 is taking the lead in PcG repression.

Polycomb group (PcG) proteins constitute a major epigenetic mechanism for gene repression throughout the plant life. For a long time, the PcG mechanism has been proposed to follow a hierarchical recruitment of PcG repressive complexes (PRCs) to target genes in which the binding of PRC2 and the incorporation of H3 lysine 27 trimethyl marks led to recruitment of PRC1, which in turn mediated H2A monoubiquitination. However, recent studies have turned this model upside-down by showing that PRC1 activity can be required for PRC2 recruitment and H3K27me3 marking. Here, we review the current knowledge on plant PRC1 composition and mechanisms of repression, as well as its role during plant development.

Role of PRC2-associated factors in stem cells and disease.

The Polycomb group (PcG) of proteins form chromatin-binding complexes with histone-modifying activity. The two main PcG repressive complexes studied (PRC1 and PRC2) are generally associated with chromatin in its repressed state. PRC2 is responsible for methylation of histone H3 at lysine 27 (H3K27me3), an epigenetic mark that is linked with numerous biological processes, including development, adult homeostasis and cancer. The core canonical complex PRC2, which contains the EZH1/2, SUZ12 and EED proteins, may be extended and functionally manipulated through interactions with several other proteins. In this review, we focus on these PRC2-associated proteins. As PRC2 functions are diverse, the variability conferred by these sub-stoichiometrically associated members may help to understand specific changes in PRC2 activity, chromatin recruitment and distribution required for gene repression.

Expression Profiling Of Selected Polycomb Complex Genes In Childhood Acute Myeloid Leukemia Revealed An Overexpression Of EZH2 In Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) in children and adolescents accounts for about 20% of cases of acute myeloid leukemia (AML) in Brazil. The reasons for the relatively high incidence of APL among children and adolescents in Brazil and other Latin countries remain elusive. Epigenetic constitutional and/or environmental factors might be implicated in the mechanism of APL. The Polycomb group (PcG) genes are critical for differentiation and cell-cycle regulation and maintenance of epigenetic memory of living organisms. Aberrant expression of PcG genes has been observed in human tumors, including AML. In this study, we sought to determine the expression levels of 4 genes from the PcG repressive complexes EZH2, YY1, BMI1 and SUZ12 in acohort of 52 children with AML or APL (male, 32; female, 20; median age 7.8 years, range 4 months-18 years).Cells from healthy children (male, 2; female, 2; median age, 10.7 years, range 6-15 years) were used as the control group. Quantitative determination of mRNA levels was performed using Power SYBR Green PCR Master Mix® (Applied Biosystems, Foster City, CA, USA) in a Rotor Gene® thermocycler (QIAGEN). Expression levels were estimated in triplicate, and ß-actin was used as an internal control. All statistical analyses were performed using the GraphPad Prim 5.0 System. Multiple pairwise comparisons were made using a one-way analysis of variance (ANOVA) test; P<0.05 was considered statistically significant.Despite showing broad variation among patients and controls, the expression levels of YY1 (controls, 1.16 ±SE 0.73; M1/M2, 0.63±1.07; M4/M5 0.217±0.71; APL, 0.62±3.24; p=0.19); SUZ12 (control, 1.11 ±0.93; M1/M2, 0.12±1.65; APL 2.4±3.42; M4/M5, 0.34 ± 10.30; p=0.18), and BMI1 (control, 0.041 ± 0.77; M1/M2: 0.043 ±0.10; M4/M5, 0.016± 0.23; APL, 0.12 ± 0.59; p=0.37)did not differsignificantly between controls and patients grouped according to the major AML subtypes (M1/M2, 15 cases, median age 10.8 years, range 4-18 years; M4/M5, 21 cases,median 4.5 years, range 5 months-18 years; APL 16 cases, median 9.8 years, range, 1-17 years). However, the EZH2 expression levels (control, 0.0008818±0.03675; M1/M2, 0.001495±0.03296; M4/M5 0.008215±0.09313; APL, 0.07180± 0.3402; p=0.0092) were significantly higher in APL. Among patients with APL, the expression levels of EZH2 did not differ significantly according to age (EZH2 expression, 0.23±0.44, age 0-8 years; 0.05±0.46, age 9-18 years; p=0.112), white blood cell count (EZH2 expression 0.07±0.65, WBC < 10 x 109/L; 0.10±0.38, WBC ≥ 10 x 109/L; p=0.148) or platelet count (EZH2 expression 0.20±0.49, platelet count < 40 x109/L; 0.06 ±0.04, platelet count ≥ 40 x 109/L; p=0.13).Several studies have shown that EZH2 is deregulated in several human cancers. Here we extend this data by informing that EZH2 is highly overexpressed in pediatric APL. APL in children less than 8 years of age tends to be associated with higher EZH2 gene expression levels, suggesting that constitutional epigenetic factors may play a driver role in pediatric APL leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

SAM Domain Polymerization Links Subnuclear Clustering of PRC1 to Gene Silencing

The Polycomb-group (PcG) repressive complex-1 (PRC1) forms microscopically visible clusters in nuclei; however, the impact of this cluster formation on transcriptional regulation and the underlying mechanisms that regulate this process remain obscure. Here, we report that the sterile alpha motif (SAM) domain of a PRC1 core component Phc2 plays an essential role for PRC1 clustering through head-to-tail macromolecular polymerization, which is associated with stable target binding of PRC1/PRC2 and robust gene silencing activity. We propose a role for SAM domain polymerization in this repression by two distinct mechanisms: first, through capturing and/or retaining PRC1 at the PcG targets, and second, by strengthening the interactions between PRC1 and PRC2 to stabilize transcriptional repression. Our findings reveal a regulatory mechanism mediated by SAM domain polymerization for PcG-mediated repression of developmental loci that enables a robust yet reversible gene repression program during development.

VAL- and AtBMI1-Mediated H2Aub Initiate the Switch from Embryonic to Postgerminative Growth in Arabidopsis

Plant B3-domain transcription factors have an important role in regulating seed development, in particular seed maturation and germination. Among the B3 factors, the AFL (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], and LEAFY COTYLEDON2 [LEC2]) proteins activate the seed maturation program in a complex network, while the VAL (VP1/ABI3-LIKE) 1/2/3 proteins suppress AFL action in order to initiate germination and vegetative development through an as yet unknown mechanism. In addition, the AFL genes and LEAFY COTYLEDON1 (LEC1), referred as seed maturation genes, are epigenetically repressed after germination by the Polycomb group (PcG) machinery via its histone-modifying activities: the histone H3 lysine 27 trimethyltransferase activity of the PcG repressive complex 2 (PRC2) and the E3 H2A monoubiquitin ligase activity of the PRC1. Both histone modifications are required for the repression; however, the underlying mechanism is far from clear, because the localization and the role of H2Aub marks are still unknown. In this work, we demonstrate that VAL proteins and AtBMI1-mediated H2Aub initiate repression of seed maturation genes. After the initial off switch, the repression is maintained by PRC2-mediated H3K27me3. Our results indicate that the regulation of seed maturation genes does not follow the classic hierarchical model proposed for animal PcG-mediated repression, since the PRC1 activity is required for the H3K27me3 modification of these genes. Furthermore, we show different mechanisms to achieve PcG repression in plants, as the repression of genes involved in other processes has different requirements for H2Aub and H3K27me3 marking.

240 Epigenetic Regulation of Kit Expression in Murine KitLow Interstitial Cell of Cajal Stem Cells (ICC-SC) and KitLow Gastrointestinal Stromal Tumor (GIST) Cells by Polycomb Group (PcG) Repressive Complexes (PRC)

240 Epigenetic Regulation of Kit Expression in Murine KitLow Interstitial Cell of Cajal Stem Cells (ICC-SC) and KitLow Gastrointestinal Stromal Tumor (GIST) Cells by Polycomb Group (PcG) Repressive Complexes (PRC)

Arabidopsis histone deacetylase HDA9 regulates flowering time through repression of AGL19

Flowering time is tightly controlled by several regulatory pathways including photoperiod, vernalization in which epigenetic processes are involved. In this work, we have found that the Arabidopsis histone deacetylase gene HDA9 is involved in flowering time control. Mutation of the gene led to an early flowering phenotype in short day grown plants while without effect in long days. Analysis of flowering time regulatory gene expression revealed that hda9 mutations highly induced the expression of AGL19, but had no effect on CO, SOC1 or FLC. Chromatin immunoprecipitation assays indicated that the mutations led to a clear increase of histone H3K9 and H3K27 acetylation on the AGL19 gene in short days. AGL19 promotes flowering in a way independent of the CO and FLC pathways and has been shown to be repressed by polycomb group repressive complex2 (PRC2) EMF2 but activated by vernalization. The induced levels of AGL19 expression and histone acetylation by the hda9 mutations were comparable to that of the gene under long day conditions, indicating that AGL19 is regulated also by day length and that HDA9 is involved in short day repression of AGL19 by promoting histone H3 deacetylation, which may be related to the PRC2 EMF2 complex.

Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Polycomb group repressive complex protein EZH2 regulates tight junction protein Claudin-1 in craniopharyngiomas

Polycomb group repressive complex protein EZH2 regulates tight junction protein Claudin-1 in craniopharyngiomas

Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia.

BackgroundThe Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples.MethodsQuantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.ResultsFor 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.ConclusionsThe results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Endosperm cellularization defines an important developmental transition for embryo development

The endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endosperm develops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occurs at a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidy crosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seed development. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) and endosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endosperm cellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryo development, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endosperm cellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is a consequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct target gene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endosperm cellularization.

Synergistic antileukemic action of a combination of inhibitors of DNA methylation and histone methylation

DNA methylation and histone methylation are both involved in epigenetic regulation of gene expression and their dysregulation can play an important role in leukemogenesis. Aberrant DNA methylation has been reported to silence the expression of tumor suppressor genes in leukemia. Overexpression of the histone methyltransferase, EZH2, a subunit of the polycomb group repressive complex 2 (PRC2), was observed to promote oncogenesis. This is due to aberrant gene silencing by the trimethylation of histone H3 lysine 27 (H3K27me3) by EZH2. Since both these epigenetic silencing events are reversible, they are interesting targets for chemotherapeutic intervention by using an inhibitor of DNA methylation, such as 5-aza-2′-deoxcytidine (5-AZA-CdR), and 3-deazaneplanocin-A (DZNep), an inhibitor of the EZH2. Human HL-60 and murine L1210 leukemic cells exposed in vitro to 5-AZA-CdR and DZNep in combination showed a synergistic loss of clonogenicity in a colony assay as compared to each agent alone. This positive chemotherapeutic interaction was also observed in mice with L1210 leukemia. Quantitative PCR showed that the combination also produced a remarkable synergistic activation of the tumor suppressor genes, CDKN1A and FBXO32. Microarray analysis showed that 5-AZA-CdR plus DZNep produced a synergistic activation of >150 genes. Our results indicate that 5-AZA-CdR plus DZNep can reactivate target genes that are silenced by two distinct epigenetic mechanisms leading to a loss of the proliferative potential of leukemic cells.