Epigenetic transcriptional repression of tumor suppresor genes and its reversion by drugs.

Genetic alterations and deregulation of the epigenetic mechanisms collaborate in the initiation and progression of cancer. In contrast to the genetic defects, the epigenetic abnormalities are potentially reversible. This fact has driven the search for drugs that induce selectively changes in the epigenetic patterns of the tumor cells and thus lead to differentiation, cell death and/or cell growth arrest. Inhibitors of DNA (cytosine-5)-methyltransferases (DNMTs) and inhibitors of Zn(II)-dependent histone deacetylases (HDACs) have been developed with this purpose. The DNMTs inhibitors allow the reactivation of genes, including tumor-suppressor genes, silenced through hypermethylation of the CpG island at their promoter. The HDACs inhibitors allow the reexpression of genes silenced by ypoacetylation of the histones associated at their promoters. Despite all these chemicals have promising effects on cultured cancer cells, many of them have side-effects that limit their use in anticancer chemotherapy. Because of that, we analyzed the properties of the anesthetic procaine (4-aminobenzoic acid 2-diethylaminoethyl ester) as a new inducer of DNA demethylation and we also compared the effects of seven HDACs inhibitors. In both cases, the breast cancer cell line MCF7 was the model system. 1. Procaine reduces the proportion of 5-methylcytosine into global genomic DNA, achieving its maximum effect within 24-48 h of treatment. Low concentrations of procaine decrease the amount of 5- methylcytosine at RAR¦Â2 promoter, which is hypermethylated in MCF7 cells, and reactivate its expression with only small decrease in global DNA methylation. This fact could be an advantage, since global DNA hypomethylation leads to chromosomal instability. Finally, procaine reduces cell proliferation and arrests cell cycle in mitosis, but does not induce apoptosis in MCF7 cells after treatments 3 days long. 2. The seven inhibitors of Zn(II)-dependent HDACs chosen for comparison were: two carboxylic acids (butanoic and valproic acid); one N-(2¡¯-aminophenyl) benzamide (MS-275); and four hydroxamic acids(trichostatin A, suberoylanilide hydroxamic acid, CX and CY). The results of in vitro HDAC activity assays performed on MCF7 nuclear extracts show the existence of a relationship between the chemical structure of these compounds and their activity: low micromolar concentrations of hydroxamic acids are sufficient for inhibiting almost completely the deacetylase activity, whereas millimolar concentrations of carboxylic acids are required for similar effects. Also the alterations that the drugs cause on cell growth and cell cycle arrest depend on its chemical structure. The IC50 for cell treatments 24 h long is in the range of millimolar concentrations for butanoic and valproic acids and low micromolar for the rest of the chemicals. At the IC50, MS-275 induces cell growth arrest in G1/G0, whereas the hydroxamic acids stop cell cycle mostly at G2/M and the carboxylic acids seems to arrest the cycle at both G1/G0 and G2/M. Despite all these inhibitors induce similar changes in the global acetylation of H4 and H3 when employed at their respective IC50, not all of them are able of reactivate the expression of the same genes. Moreover, it seems that the induced expression levels of CDKN1A and GADD45¦Â determine the alterations induced by the drugs on cell cycle. The changes on histone modifications at the promoters of six genes ( CDKN1A, GADD45¦Â, JunD, IGFBP3, MT1X and MT2A) upon CY treatment were studied. HDACs inhibition induces an increase in histone H4 tetraacetylation and in dimethylation of lysine 4 in H3, as well as a decrease in dimethylation of lysine 9 in H3. Additionally, HDAC2 is released from the promoters upon CY treatment. These changes take place also in the promoters of MT1X and MT2A, the genes whose expression remains unaltered after the treatment with CY. __________________________________________________________________________________________________ RESUMEN Las alteraciones gen¨¦ticas y la desregulaci¨®n de los mecanismos epigen¨¦ticos colaboran en la iniciaci¨®n y progresi¨®n del c¨¢ncer. Los defectos epigen¨¦ticos son potencialmente reversibles, lo que ha suscitado la b¨²squeda de f¨¢rmacos que selectivamente causen cambios en los patrones epigen¨¦ticos de las c¨¦lulas tumorales, con la consiguiente diferenciaci¨®n, muerte y/o parada de crecimiento de las mismas. Se han estudiado especialmente inhibidores de metiltransferasas de DNA (DNMTs) e inhibidores de desacetilasas de histonas (HDACs) dependientes de Zn(II). Los inhibidores de DNMTs posibilitan la reactivaci¨®n de genes silenciados mediante hipermetilaci¨®n de la isla CpG de su promotor y los inhibidores de HDACs, de genes silenciados v¨ªa hipoacetilaci¨®n de las histonas asociadas a su promotor. A pesar de sus prometedores efectos en cultivos celulares, muchas de estas sustancias presentan inconvenientes que limitan su aplicaci¨®n en quimioterapia. Por ello, en esta tesis: 1. Se ha estudiado por primera vez la capacidad del anest¨¦sico proca¨ªna para reducir la metilaci¨®n de DNA gen¨®mico y la proliferaci¨®n de la l¨ªnea celular MCF7. Proca¨ªna reduce la cantidad de 5-metilcitosina en DNA, reactiva genes silenciados por hipermetilaci¨®n ( RAR¦Â2) e induce parada del ciclo celular en mitosis. 2. Se han comparado siete inhibidores de HDACs dependientes de Zn(II): ¨¢cido butanoico, ¨¢cido valproico, MS-275, tricostatina A (TSA), SAHA, CX y CY. Existe una relaci¨®n estructura-actividad para los efectos de estas sustancias en ensayos in vitro y en el crecimiento de MCF7. Adem¨¢s, aunque estos inhibidores inducen cambios similares en acetilaci¨®n global de histonas, no todos reactivan los mismos genes. Los niveles expresi¨®n de CDKN1A y GADD45¦Â parecen determinar los efectos de estos compuestos en el ciclo celular. En los promotores de los seis genes estudiados, la inhibici¨®n de HDACs aumenta la acetilaci¨®n de H4 y la dimetilaci¨®n de la lisina 4 de H3, mientras disminuye la dimetilaci¨®n de lisina 9 de H3

Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure

Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster

The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death

The monophosphorylation at S10 decreases as the embryos maturate.

<p>Histone H1 from 2–3 h, 3–6 h, 6–9 h, 9–12 h and 12–15 h was isolated and digested with trypsin. A) Zoom of two MALDI-TOF spectra acquired in the reflector positive mode are shown as examples. The upper spectrum corresponds to the 12–15 h old embryos, the lower to the 3–6 h ones. Note the different intensity of the signal at 2078. B) To compare the proportion of monophosphorylated H1 between the different stages, the area of the clusters corresponding to unmodified 1–21 (both proton and sodium adducts) and the monophosphorylated form (only proton adduct, sodium adduct not detected) was calculated for H1 from staged embryos. For each sample, the relative area of the monophosphorylated species (A_rel) was calculated by division of the area of the monophosphorylated species through the total area (unmodified+monophosphorylated). The results of two independent biological replicates are shown. The error bars indicate the maximum and the minimum obtained values. C) Scheme showing the peptide 1–21 and the numbering of the b and y ions. The residues susceptible of phosphorylation are highlighted in red. D and E) H1 from 3–6 h and 12–15 h old embryos was purified, digested with trypsin, submitted to chemical modification (β-elimination and Michael addition) and finally, the forms of 1–21 were sequenced by ESI-MS/MS. Zooms of the product ions of the monomodified species are shown. * labels the modified species. D) The relative intensities of y12 and y12* indicate that the 12–15 h old embryos have less proportion of S10 phosphorylation than the younger embryos. E) The relative intensities of the b and b* ions suggest that monophosphorylated H1 from 12–15 h old embryos contains more proportion of S1 and/or S3 phosphorylation than the sample from the younger embryos.</p

Release of hypoacetylated and trimethylated histone H4 is an epigenetic marker of early apoptosis

11 p.-5 fig.-1 fig. supl.Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.This work was supported by Grants SAF 2001-0059, BFU2004-02073/BMC and Ramon y Cajal Programme (MCYT), and GR/SAL/0224/2004 (Government of Madrid).Peer reviewe