Epigenetic regulation of PRAME in acute myeloid leukemia is different compared to CD34+ cells from healthy donors: effect of 5-AZA treatment.

PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies.

Impaired recruitment of the histone methyltransferase DOT1L contributes to the incomplete reactivation of tumor suppressor genes upon DNA demethylation.

Understanding the mechanisms that link changes in DNA methylation with histone modifications is particularly relevant in the case of tumor suppressor genes that undergo transcriptional silencing in cancer cells in association with promoter CpG island hypermethylation. In this study, we show that two histone lysine methylation marks associated with active transcription, dimethylation of H3K79 (H3K79me2) and trimethylation of H3K4 (H3K4me3), are present in all the unmethylated promoters analysed, and both of them are lost when these promoters become hypermethylated. Most importantly, pharmacological and genetic interventions that cause DNA demethylation and partial recovery of gene transcription, result in the restoration of H3K4me3, but not of H3K79me2. We also show that DOT1L, the major H3K79 histone methyltransferase, is no longer recruited to the promoters that are demethylated after 5-aza-deoxycytidine treatment or genetic deletion of DNA methyltransferases. Knock-down and transfection experiments for DOT1L show that this enzyme has a direct role in maintaining the euchromatic and active status of these genes when unmethylated. These findings suggest that DNA demethylating interventions alone are not able to restore a complete euchromatic status and a full transcriptional reactivation of the epigenetically silenced tumor suppressor genes, and reinforce the necessity of targeting multiple elements of the epigenetics machinery for a successful treatment of malignancies.

Identification of (1H)-pyrroles as histone deacetylase inhibitors with antitumoral activity.

Histone deacetylases (HDACs) play a key role in the regulation of gene expression and chromatin structure, and drugs targeting these enzymes might have an important impact in the treatment of human cancer. Herein, we report the characterization of (1H)-pyrroles as a new subfamily of HDAC inhibitors obtained by computational modeling of class-I human HDACs. From a functional standpoint, (1H)-pyrroles are powerful inductors of acetylation of histones H3 and H4, and restore the expression of growth-inhibitory genes. From a cellular view, these compounds cause a marked decrease in the viability of cancer cells in vitro and in vivo, associated with a cell-cycle arrest at G2/M and an inhibition of angiogenesis. Thus, (1H)-pyrroles emerge as a novel group of HDAC inhibitors with promising antitumoral features.

Transforming pathways unleashed by a HDAC2 mutation in human cancer.

Although disruption of histone modification patterns is a common hallmark of human cancer, our knowledge of the mechanistic role of histone-modifying enzymes in its generation is very limited. We have recently identified an inactivating mutation in the histone deacetylase-2 (HDAC2) in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. Since HDAC2 seems to be a central player in epigenetic gene repression, we wondered whether HDAC2-truncating mutations conferred a particular expression signature on these cancer cells. Using unsupervised clustering analysis in microsatellite-unstable colorectal cancer cell lines, we have found that HDAC2 mutant cells (RKO and Co115) show a characteristically different expression microarray signature from HDAC2 wild-type cells (HCT-116, SW48, HCT-15 and LoVo). HDAC2 mutant cells exhibit upregulation of tumor-promoting genes, such as those of tyrosine kinases, mediators of cell cycle progression and angiogenic factors. The overexpression of these genes is associated with a loss of HDAC2 recruitment and a gain of histone H4 hyperacetylation in their particular 5'-end promoters, as observed by chromatin immunoprecipitation. Transfection of wild-type HDAC2 in mutant cells reverted this epigenetic pattern by repressing the transforming genes in association with HDAC2 promoter occupancy. These results suggest a role for HDAC2 mutations in human tumorigenesis through the derepression of key genes from multiple cellular transformation pathways.

Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of methyl-CpG-binding domain proteins.

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5'-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein gamma-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.

Specific hypermethylation of LINE-1 elements during abnormal overgrowth and differentiation of human placenta.

In human post-natal somatic cells, low global levels of DNA methylation have been associated with the hypomethylation of several repetitive elements, a feature that has been proposed to be a surrogate epigenetic marker. These data, mainly derived from the analysis of cancer cells, suggest a potential association between loss of cell-growth control and altered differentiation with hypomethylation of repetitive sequences. Partial hydatidiform moles (PHMs) can be used as an alternative model for investigating this association in a non-tumorigenic context. This gestational disease is characterized by abnormal overgrowth and differentiation of the placenta and spontaneous abortion. Here, we comprehensively analyse the DNA methylation of these trophoblastic tissues in both PHM and normal placenta at global and sequence-specific levels. Analysis of the global 5-methylcytosine content and immunohistochemistry indicate that PHM and normal placenta have identical global levels of DNA methylation. In contrast, bisulfite genomic sequencing shows that, whereas Alu, NBL2 and satellite 2 repetitive elements are equally methylated, LINE-1 sequences are hypermethylated in PHM tissues ( approximately 2-fold relative to normal placenta). Interestingly, altered demethylation is also found in triploid diandric embryos that originate from dispermic fertilization of an oocyte, a common event responsible for most PHMs. In conclusion, alterations of DNA methylation do not seem to be randomly distributed in PHM, as several repeated elements remain unaltered, whereas LINE-1 sequences are hypermethylated. In addition, our findings suggest that the hypomethylation of repetitive elements in cancer is directly linked to the neoplasic process and not a simple consequence of loss of growth control observed in most of the cancer cells.

Multiple binding of methyl-CpG and polycomb proteins in long-term gene silencing events.

Epigenetic regulation is involved in the maintenance of long-term silencing phenomena, such as X-inactivation and genomic imprinting in mammals. Gene repression is mediated by several mechanisms, such as histone modifications, DNA methylation, and recruitment of Polycomb proteins. To understand the mechanistic relationships between these mechanisms for stable gene silencing, we analyzed the mechanisms of X- and Y-inactivation of the PAR2 gene SYBL1, previously showed to be regulated by concerted epigenetic mechanisms. Maintenance of stable repression occurs via the recruitment of both MBDPs and PRC2 complexes to SYBL1 promoter. Their binding is equally sensitive to defective DNA methylation seen in cells derived from ICF syndrome patients. Multiple occupancy is a feature shared within long-term repressed genes, such as the X-inactivated PGK1 and the imprinted IGF2. MBD2, MBD3, and MeCP2 occupy SYBL1 promoter simultaneously, as revealed by sequential ChIP. We did not find this co-occurring binding when looked for members of PRC2 complex together with any of the methyl-binding proteins. Furthermore, in co-transfection assays, MECP2 can silence methylated SYBL1 promoter, whereas the mutated protein fails. However, RNA interference of endogenous MECP2 does not induce the expression of the inactive SYBL1 alleles, suggesting that its silencing activity can be replaced by the other methyl-binding proteins. Our data suggest that maintenance of long-term silencing involves multiple layers of epigenetic control functionally redundant. PRC2 and MBD proteins could collaborate to different phases of this process, the former possibly recruiting DNMTs to the silenced promoters, the latter dictating the lock of the transcription.

Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer.

Colorectal cancer is a major cause of cancer death worldwide. A number of key oncogenes and tumor suppressor genes have been proposed to drive progression from healthy colonic epithelia to malignant tumors, including members of the Wnt/beta-catenin pathway. Recently, CpG island promoter hypermethylation was shown to cause inactivation of two extracellular Wnt inhibitors in colon cancer: secreted frizzled-related proteins (sFRPs) and Wnt inhibitory factor-1 (WIF-1). Here, we show for the first time that another extracellular Wnt inhibitor, the DICKKOPF-1 (DKK-1) gene, is transcriptionally silenced by CpG island promoter hypermethylation in colon cancer cell lines (n=9), whereas treatment with the DNA-demethylating agent 5-aza-2-deoxycytidine restored DKK-1 expression. Restoration of DKK-1 function in non-expressing cells bearing a truncated APC (Adenomatous Polyposis Coli) gene had no effect on beta-catenin/T-cell factor-dependent transcription, but induced tumor suppressor-like features such as reduced colony formation density and tumor growth inhibition in nude mice. These results suggest additional functions for DKK-1 other than inhibiting canonical Wnt signaling. In primary colorectal tumors, DKK-1 was found hypermethylated in 17% (nine of 54) of cases. Furthermore, while for both SFRP-1 and WIF-1 methylation-associated silencing occurred across the whole spectrum of colorectal tumorigenesis, DKK-1 promoter was selectively hypermethylated in advanced colorectal neoplasms (Duke's C and D tumors).

The dioxin receptor is silenced by promoter hypermethylation in human acute lymphoblastic leukemia through inhibition of Sp1 binding.

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2'-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

A Drosophila MBD family member is a transcriptional corepressor associated with specific genes.

DNA methylation in Drosophila melanogaster is restricted temporally during development and occurs at a significantly lower frequency than in mammals. Thus, the regulatory functions, if any, of this form of DNA modification in Drosophila are unclear. However, the presence of homologs of vertebrate methyl-CpG-binding proteins implies functional consequences for DNA methylation in flies. This work describes the properties of dMBD-like, a Drosophila homolog of vertebrate MBD2 and MBD3. dMBD-like and dMBD-likeDelta (a splice variant) failed to bind model methylated DNA probes, inconsistent with their function as mediators of methyl CpG-directed transcriptional repression. However, the MBD-like proteins exhibit transcriptional and biochemical properties consistent with roles as components of a histone deacetylase-dependent corepressor complex similar to the vertebrate Mi-2 complex. The two proteins are differentially expressed during development, suggesting functional specialization. dMBD-like and/or dMBD-likeDelta is present at the chromocenter on larval polytene chromosomes as well as at discrete bands interspersed along the euchromatic chromosome arms, many of which are coincident with known ecdysone-induced loci. This banding pattern suggests gene-specific regulatory functions for dMBD-like and the Drosophila Mi-2 complex.

Conformational changes in the nucleosome followed by the selective accessibility of histone glutamines in the transglutaminase reaction: effects of ionic strength.

Transglutaminases, the enzymes that catalyze the acyl-transfer reaction between glutamine and primary amines, have been used to introduce probes into proteins in order to perform structural studies using physical techniques. Here we use an original approach in which the increasing accessibility of the glutamines of core histones to TGase is used to monitor the salt-induced conformational changes of the nucleosome. The rationale of this strategy is that the accessibility of a glutamine to transglutaminase depends on the blockage due to the presence of either other histones or DNA. At low ionic strength, only glutamines on the N-terminal tails of H2B and H3 are labeled with monodansylcadaverine when core particles are incubated with transglutaminase. The partial unfolding that occurs when going to higher ionic strength values results in an increase in the number of reactive glutamines up to a maximum value of 16 per nucleosome. Labeling of some residues (e.g., Gln(104) and Gln(112) of H2A) requires the unwinding of DNA and the dissociation of the H2A--H2B dimers. Gln(76) of H3 is labeled in the H3--H4 tetramer only when the H2A--H2B dimers are dissociated. Interestingly, the labeling of Gln(95) of H2B exclusively depends on the unwinding of DNA. The accurate analysis of these results indicates that the ionic-dependent unwinding of the DNA may occur following a two-state model.

Methyl-CpG-binding proteins. Targeting specific gene repression.

CpG methylation, the most common epigenetic modification of vertebrate genomes, is primarily associated with transcriptional repression. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins that share the methyl-CpG-binding domain (MBD). The MBD, consisting of about 70 residues, possesses a unique alpha/beta-sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer. All MBDs except MBD4, an endonuclease that forms a complex with the DNA mismatch-repair protein MLH1, form complexes with histone deacetylase. It has been established that MeCP2, MBD1 and MBD2 are involved in histone deacetylase-dependent repression and it is likely that this is also the case for MBD3. The current model proposes that MBD proteins are involved in recruiting histone deacetylases to methyl CpG-enriched regions in the genome to repress transcription. The lack of selectivity for MBD association with particular DNA sequences indicates that other mechanisms account for their recruitment to particular regions in the genome.

Effects of Rett syndrome mutations of the methyl-CpG binding domain of the transcriptional repressor MeCP2 on selectivity for association with methylated DNA.

We have investigated the properties of mutant forms of the methyl-CpG binding transcriptional repressor MeCP2 associated with Rett syndrome, a childhood neurodevelopmental disorder. We find that four Rett syndrome mutations at known sites within the methyl-CpG binding domain (MBD) impair binding to methylated DNA, but have little effect on nonspecific interactions with unmethylated DNA. Three of these mutations (R106W, R133C, and F155S) have their binding affinities for methylated DNA reduced more than 100-fold; this is consistent with the hypothesis that impaired selectivity for methylated DNA of mutant MeCP2 contributes to Rett syndrome. However, a fourth mutant, T158M, has its binding affinity for methylated DNA reduced only 2-fold, indicative either of additional distinct regulatory functions associated with the MBD or of an exquisite sensitivity of developing neurons to the selective association of MeCP2 with methylated DNA.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase.

Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.

Use of the transglutaminase reaction to study the dissociation of histone N-terminal tails from DNA in nucleosome core particles.

We have recently shown that core histones are glutaminyl substrates for transglutaminase (TGase) and that when native nucleosome cores are incubated with monodansylcadaverine (DNC) as donor amine, this fluorescent probe is incorporated into Gln5 and Gln19 of H3 and in Gln22 of H2B [Ballestar et al. (1996) J. Biol. Chem. 271, 18817-18825]. In the present paper, we report that the cause by which Gln22 of H2B is modified in nucleosomes but not in the free histone is the interaction of the region containing that glutamine with DNA. We have used the specificity of the TGase reaction to study the changes induced by increasing ionic strength in the interaction between the histone N-terminal tails and nucleosome DNA by two different approaches. First, the reactivity of the histone tail glutamines was employed to monitor changes in the interactions between the regions containing these residues and DNA. Second, by using reconstituted nucleosome core particles containing either H2B modified with DNC by the TGase reaction at Gln22 or H3 modified with the same procedure at Gln5 and Gln19, the dissociation of the histone tails was followed by the decrease of the fluorescence anisotropy of the probe. These methods allowed us to describe two ionic strength dependent structural transitions of the histone tails not reported to date. In the case of H2B, the first one occurs at very low ionic strength, and it can be assigned to an increase in the mobility of Gln22. The second one results in the cooperative release of a region of the tail that includes lysine residues next to Gln22, and it is followed by the overall release of the entire tail, described by other workers.

Core histones are glutaminyl substrates for tissue transglutaminase.

Chicken erythrocyte core histones are glutaminyl substrates in the transglutaminase (TGase) reaction with monodansylcadaverine (DNC) as donor amine. The modification is very fast when compared with that of many native substrates of TGase. Out of the 18 glutamines of the four histones, nine (namely glutamine 95 of H2B; glutamines 5, 19, and 125 of H3; glutamines 27 and 93 of H4; and glutamines 24, 104, and 112 of H2A) are the amine acceptors in free histones. The use of Gln112 of H2A requires a temperature-dependent partial unfolding of the histone, showing that structural determinants are decisive for the glutamine specificity. The structures of H2A and H2B do not appreciably change upon modification with DNC as determined by circular dichroism, and core particles reconstituted from these DNC-modified histones are indistinguishable from native nucleosome cores. When the reaction is carried out with native nucleosomes, only glutamines 5 and 19 of H3, which are located in the N-terminal tail, and glutamine 22 of H2B, which is not labeled in free histone, are modified. Methylamine and putrescine also are incorporated into nucleosomes by the TGase reaction. Our results reveal several possibilities for the application of the TGase reaction in the chromatin field, and taking into account that histones are easily cross-linked or modified by polyamines in vitro, the possibility that they may be TGase substrates in vivo is discussed.