Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
2.
Leukemia ; 22(8): 1503-18, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18548105

ABSTRACT

During embryonic development and adult life, the plasticity and reversibility of modifications that affect the chromatin structure is important in the expression of genes involved in cell fate decisions and the maintenance of cell-differentiated state. Epigenetic changes in DNA and chromatin, which must occur to allow the accessibility of transcriptional factors at specific DNA-binding sites, are regarded as emerging major players for embryonic and hematopoietic stem cell (HSC) development and lineage differentiation. Epigenetic deregulation of gene expression, whether it be in conjunction with chromosomal alterations and gene mutations or not, is a newly recognized mechanism that leads to several diseases, including leukemia. The reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. Here we review some of the epigenetic mechanisms that regulate gene expression in pluripotent embryonic and multipotent HSCs but may be deregulated in leukemia, and the clinical approaches designed to target the chromatin structure in leukemic cells.


Subject(s)
Cell Lineage , Chromatin/metabolism , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Hematopoietic Stem Cells/metabolism , DNA Methylation , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Humans , Polycomb-Group Proteins , Repressor Proteins/metabolism
3.
Int J Oncol ; 19(4): 773-7, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11562754

ABSTRACT

Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.


Subject(s)
Cell Count , Cell Cycle/physiology , Fibroblasts/cytology , Histone Deacetylases/metabolism , Tumor Cells, Cultured/pathology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Blotting, Northern , Butyrates/pharmacology , Cell Division , DNA Probes , Fibroblasts/metabolism , Histone Deacetylase 1 , Histone Deacetylases/genetics , Humans , Male , Mice , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Ribonuclease, Pancreatic/metabolism , Tumor Cells, Cultured/metabolism
4.
Biochemistry ; 39(34): 10413-8, 2000 Aug 29.
Article in English | MEDLINE | ID: mdl-10956031

ABSTRACT

It is well-known that H1-H1 interactions are very important for the induction of 30 nm chromatin fiber and that, among all posttranslational modifications, poly(ADP-ribosyl)ation is one of those capable of modifying chromatin structure, mainly through H1 histone. As this protein can undergo both covalent and noncovalent modifications by poly(ADP-ribosyl)ation, our aim was to investigate whether and how ADP-ribose polymers, by themselves, are able to affect the formation of H1-H1 oligomers, which are normally present in a condensed chromatin structure. The results obtained in our in vitro experimental system indicate that ADP-ribose polymers are involved in chromatin decondensation. This conclusion was reached as the result of two different observations: (a) H1 histone molecules can be hosted in clusters on ADP-ribose polymers, as shown by their ability to be chemically cross-linked, and (b) H1 histone has a higher affinity for ADP-ribose polymers than for DNA; ADP-ribose polymers compete, in fact, with DNA for H1 histone binding.


Subject(s)
Histones/chemistry , Histones/metabolism , Poly Adenosine Diphosphate Ribose/pharmacology , Animals , Binding, Competitive , Cell Line , Cross-Linking Reagents/pharmacology , DNA/metabolism , In Vitro Techniques , Mice , Poly Adenosine Diphosphate Ribose/metabolism , Protein Structure, Quaternary
5.
FASEB J ; 13(12): 1518-22, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10463942

ABSTRACT

The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.


Subject(s)
Plasmids/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism , 3T3 Cells , Animals , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , DNA Methylation , Dinucleoside Phosphates/metabolism , L Cells , Mice , Molecular Sequence Data , Poly(ADP-ribose) Polymerase Inhibitors , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Transfection
6.
FASEB J ; 13(1): 89-93, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9872933

ABSTRACT

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP-ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP-ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP-ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti-5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP-ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the heterochromatic regions colocalized with the extended methyl-rich domains.


Subject(s)
DNA Methylation , Heterochromatin/drug effects , Poly(ADP-ribose) Polymerase Inhibitors , 3T3 Cells , 5-Methylcytosine , Animals , Benzamides/pharmacology , Cytosine/analogs & derivatives , Cytosine/metabolism , Enzyme Inhibitors/pharmacology , Image Processing, Computer-Assisted , Mice
7.
Mol Biol Rep ; 26(4): 261-7, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10634509

ABSTRACT

Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene. The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle. Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle. Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase. The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity. In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.


Subject(s)
Cell Cycle , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Gene Expression , Saccharomyces cerevisiae Proteins , Transfection/methods , Acetyltransferases/metabolism , Animals , Cell Line , Chromatin/chemistry , Chromatin/metabolism , Enzyme Inhibitors/pharmacology , Fibroblasts , Genes, Reporter , Histone Acetyltransferases , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Mice , Plasmids/genetics
8.
Biol Chem ; 379(6): 647-54, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9687013

ABSTRACT

This review aims to explain why H1 histone can be considered as a protein involved in protecting genomic DNA from full methylation. Some of our results indicated that, to explain the multiple roles in which H1 histone seems to be involved, it is important to consider that it is not a unique protein but a family of genetic somatic variants and that every one of them can be dynamically modified by different post-synthetic enzymatic modifications. Our data show that H1 histone plays an inhibitory effect on DNA methylation through its H1e variant and that poly(ADP-ribosyl)ation is a post-synthetic modification involved in this regulatory role. The idea that the poly(ADP-ribosyl)ated isoform of H1e could be present in decondensed chromatin structure, where the housekeeping genes are located, will be discussed.


Subject(s)
DNA Methylation , Histones/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Trans-Activators/metabolism , Humans
9.
J Biol Chem ; 273(26): 16517-20, 1998 Jun 26.
Article in English | MEDLINE | ID: mdl-9632720

ABSTRACT

In vivo and in vitro experiments carried out on L929 mouse fibroblasts suggested that the poly(ADP-ribosyl) ation process acts somehow as a protecting agent against full methylation of CpG dinucleotides in genomic DNA. Since CpG islands, which are found almost exclusively at the 5'-end of housekeeping genes, are rich in CpG dinucleotides, which are the target of mammalian DNA methyltransferase, we examined the possibility that the poly(ADP-ribosyl)ation reaction is involved in maintaining the unmethylated state of these DNA sequences. Experiments were conducted by two different strategies, using either methylation-dependent restriction enzymes on purified genomic DNA or a sequence-dependent restriction enzyme on an aliquot of the same DNA, previously modified by a bisulfite reaction. With the methylation-dependent restriction enzymes, it was observed that the "HpaII tiny fragments" greatly decreased when the cells were preincubated with 3-aminobenzamide, a well known inhibitor of poly(ADP-ribose) polymerase. The other experimental approach allowed us to prove that, as a consequence of the inhibition of the poly(ADP-ribosyl)ation process, an anomalous methylation pattern could be evidenced in the CpG island of the promoter fragment of the Htf9 gene, amplified from DNA obtained from fibroblasts preincubated with 3-aminobenzamide. These data confirm the hypothesis that, at least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects the unmethylated state of the CpG island.


Subject(s)
CpG Islands , Poly Adenosine Diphosphate Ribose/metabolism , ran GTP-Binding Protein , Animals , Fibroblasts/metabolism , GTP-Binding Proteins/genetics , Leucine Zippers/genetics , Mice , Nuclear Proteins/genetics , Promoter Regions, Genetic , Proteins/genetics , RNA-Binding Proteins , Restriction Mapping
10.
Biochemistry ; 36(26): 7937-43, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9201939

ABSTRACT

The existence of a possible correlation between poly(ADP-ribosyl)ation and DNA methylation processes was investigated. In vivo and in vitro experiments were carried out on L929 mouse fibroblasts preincubated for 24 h with or without 3-aminobenzamide, a well-known inhibitor of poly(ADP-ribose) polymerase. Both experimental approaches evidenced a close relationship between these two important nuclear enzymatic mechanisms, suggesting that the poly(ADP-ribosyl)ated isoform of H1 histone and/or long and branched protein-free ADP-ribose polymers could act as protecting agents against full methylation of the CpG dinucleotides in genomic DNA.


Subject(s)
DNA Methylation , DNA/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , Fibroblasts/metabolism , Histones/metabolism , Mice , Poly(ADP-ribose) Polymerases/metabolism
11.
Biochem Biophys Res Commun ; 227(3): 768-74, 1996 Oct 23.
Article in English | MEDLINE | ID: mdl-8886008

ABSTRACT

Oligonucleosomal DNA preparations from condensed-inactive chromatin were examined, before and after artificial methylation by bacterial SssI methylase, for their ability to allow cooperative H1-H1 interactions under conditions of different ionic strength. Our results support the conclusion that, within the highly methylated genomic DNA, there are some CpG's whose unmethylated state is critical for chromatin folding. Circular dichroism spectra indicate that artificial overmethylation of native oligonucleosomal DNA reduces its efficiency in inducing an ordered conformation of H1 histone. Temperature melting profiles confirm on the other hand that the native and the artificially overmethylated forms of oligonucleosomal DNA are both able to bind H1 histone.


Subject(s)
DNA Methylation , Histones/chemistry , Circular Dichroism , Cross-Linking Reagents , Hot Temperature , Humans , Nucleic Acid Denaturation , Succinimides
12.
Biochem J ; 316 ( Pt 2): 475-80, 1996 Jun 01.
Article in English | MEDLINE | ID: mdl-8687390

ABSTRACT

H1 histone somatic variants from L929 mouse fibroblasts were purified by reverse-phase HPLC. We analysed the ability of each H1 histone variant to allow the H1-H1 interactions that are essential for the formation of the higher levels of chromatin structure, and we investigated the role played by the poly(ADP-ribosyl)ation process. Cross-linking analysis showed that H1e is the only somatic variant which, when bound to DNA, is able to produce H1-H1 polymers; the size of polymers was decreased when H1e was enriched in its poly(ADP-ribosyl)ated isoform. Measurement of the methyl-accepting ability in native nuclei compared with nuclei in which poly(ADP-ribosyl)ation was induced showed that the poly(ADP-ribosyl)ated H1 histone had not been removed from linker regions, in spite of its different interaction with DNA.


Subject(s)
DNA/metabolism , Histones/metabolism , Nucleosomes/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Chromatin/chemistry , Chromatin/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Histones/genetics , Histones/isolation & purification , Methylation , Methylnitronitrosoguanidine/pharmacology , Mice , Mutagens/pharmacology
13.
Biochem Biophys Res Commun ; 220(1): 102-7, 1996 Mar 07.
Article in English | MEDLINE | ID: mdl-8602826

ABSTRACT

H1e and H1c histone variants were purified from mouse L929 fibroblasts using a reverse phase HPLC, and their effect on in vitro DNA methylation was investigated, together with their ability to bind unmethylated or methylated CpG-rich 44bp oligonucleotides. In a "physiological" range of H1:DNA ratios only H1e, at variance from H1c, was found to cause a marked inhibition of in vitro enzymic DNA methylation. It was also shown that both variants have a similar affinity in binding a methylated CpG-rich oligonucleotide, but that the binding to the same oligonucleotide in the unmethylated form occurs preferentially with H1e rather than with H1c. H1e is therefore likely to be directly involved in maintaining CpG-rich sequences in the unmethylated state.


Subject(s)
DNA/metabolism , Histones/genetics , Histones/metabolism , Animals , Base Sequence , Cell Line , CpG Islands , DNA/genetics , Genetic Variation , Histones/pharmacology , Methylation , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Protein Binding
14.
Gene ; 157(1-2): 247-51, 1995 May 19.
Article in English | MEDLINE | ID: mdl-7607500

ABSTRACT

The inhibitory effect that H1 histone exerts on the in vitro DNA methylation process, catalysed by mammalian DNA methyltransferase, together with the relative hypomethylation of linker DNA in eukaryotic cells chromatin, suggest that this hypomethylated state of linker DNA can be of importance in allowing or regulating H1-dependent chromatin condensation. In native oligonucleosomes (olnu), i.e., in chromatin fragments consisting of 5-20 nucleosomes each, there was a correlation between the effects of H1 on the DNA ellipticity at 280 nm and the in vitro assayed methyl-accepting ability. The same was true in H1-depleted or in H1-reconstituted preparations. Artificial methylation caused olnu DNA to lose its ability to allow cooperative H1-H1 interactions under ionic strength conditions similar to those known to affect the transition of the 10-nm filament to the 30-nm chromatin fiber. These results suggest that hypomethylation of linker DNA plays a role in the H1-H1 interactions that are needed for solenoid condensation.


Subject(s)
Chromatin/physiology , DNA/chemistry , DNA/metabolism , Mutagenesis, Insertional , Animals , Chromatin/ultrastructure , Circular Dichroism , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel , Female , Histones/metabolism , Humans , Mammals , Methylation , Nucleosomes/physiology , Nucleosomes/ultrastructure , Placenta/metabolism , Pregnancy
15.
Gene ; 157(1-2): 253-6, 1995 May 19.
Article in English | MEDLINE | ID: mdl-7607502

ABSTRACT

Upon HPLC fractionation of human placenta or calf thymus H1 histone preparations, only some fractions enriched in the H1e-c variants were able to exert a severe inhibition on in vitro enzymatic DNA methylation. These fractions, though similar to the other variants in interacting with genomic DNA, were also the only ones which could bind CpG-rich ds-oligodeoxyribonucleotides (oligos). Both the 6-CpG ds-oligo and the DNA purified from chromatin fractions enriched in 'CpG islands' were good competitors for the binding of H1e-c to the 6meCpG ds-oligo. This ability to bind any DNA sequence and to suppress the enzymatic methylation in any sequence containing CpG dinucleotides suggests, for these particular H1 variants, a possible role in maintaining CpG island DNA and linker DNA at low methylation levels.


Subject(s)
DNA/metabolism , Genetic Variation , Histones/genetics , Histones/metabolism , Oligodeoxyribonucleotides/chemistry , Animals , Base Sequence , Cattle , Chromatin/chemistry , Chromatin/metabolism , DNA/chemistry , DNA/isolation & purification , Dinucleoside Phosphates , Female , Histones/isolation & purification , Humans , Methylation , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Placenta/metabolism , Pregnancy , Thymus Gland/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...