Biomedical subjects

P Caiafa

Publications and source records attributed to P Caiafa.

At least 19 recordsLinked to original sources

PubMed1999

Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis.

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.

3T3 Cells↗

PubMed1998

The unmethylated state of CpG islands in mouse fibroblasts depends on the poly(ADP-ribosyl)ation process.

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.

Animals↗

PubMed1998

H1 histone as a trans-acting factor involved in protecting genomic DNA from full methylation.

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.

DNA Methylation↗

PubMed1997

Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?

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.

Animals↗

PubMed1996

H1-H1 cross-linking efficiency depends on genomic DNA methylation.

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.

Circular Dichroism↗

PubMed1996

Different effects of histone H1 on de novo DNA methylation in vitro depend on both the DNA base composition and the DNA methyltransferase.

We have characterized the inhibition exerted by histone H1 on the activity of human placenta DNA (cytosine-5-)-methyltransferase. Our experiments demonstrate that the extent of inhibition depends on the DNA base composition, AT-rich substrates being more severely affected than GC-rich substrates and CpG-rich islands. With bacterial SssI methylase, the effect is completely reversed since its activity on AT-rich substrates undergoes a 4-5-fold stimulation upon the addition of H1. Poly(L-lysine) mimicks H1 effects, suggesting an essential role of lysine residues in both the inhibitory and stimulatory effects of H1. By comparison of the different behaviors of the two enzymes, the inhibitory effect over the eukaryotic enzyme might be accounted for by hypothesizing a competition between minor groove-binding motifs (SPKK-like) present in placenta methylase as well as in histone H1.

Base Composition↗

PubMed1996

Co-operative interactions of oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform.

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.

Animals↗

PubMed1996

Specific inhibitory effect of H1e histone somatic variant on in vitro DNA-methylation process.

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.

Animals↗

PubMed1995

Does hypomethylation of linker DNA play a role in chromatin condensation.

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.

Animals↗

PubMed1995

Specific variants of H1 histone regulate CpG methylation in eukaryotic DNA.

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.

Animals↗

PubMed1995

Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation.

Within the H1 histone family, only some fractions enriched in the H1e-c variants are effective in causing a marked inhibition, in vitro, of enzymic DNA methylation and, in gel retardation and Southwestern blot experiments, in binding double-stranded (ds) CpG-rich oligonucleotides. Both the 6-CpG ds-oligonucleotide and the DNA purified from chromatin fractions enriched in 'CpG islands' are good competitors for the binding of H1e-c to 6-meCpG ds-oligonucleotide. Because of their ability to bind any DNA sequence and to suppress the enzymic methylation in any sequence containing CpG dinucleotides, these particular H1 variants could play some role in maintaining linker DNA at low methylation levels and even in preserving the unmethylated state of the CpG-rich islands which characterize the promoter regions of housekeeping genes.

Animals↗

PubMed1995

Poly(ADP-ribosyl)ation of proteins associated with nuclear matrix in rat testis.

We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.

Animals↗

PubMed1994

Histone-induced condensation of rat testis chromatin: testis-specific H1t versus somatic H1 variants.

Due the likely role of H1 histone variants in inducing the formation of folded DNA filaments with different stabilities, the condensing capacity of the testis-specific H1t versus the somatic variants was tested. Circular dichroism analyses of rat testis H1-depleted oligonucleosomes (5-2kbp) revealed that H1t, which appears in germ cells during the meiotic prophase of mammalian spermatogenesis, exerts the lowest condensing effect as compared to the other variants. The distribution of H1 subtypes among different chromatin fractions was also investigated and gave evidence that H1t is more abundant in chromatin regions which are more sensitive to DNAase I digestion.

Animals↗

PubMed1994

Nuclear matrix-associated poly(ADPribosyl)ation system in rat testis chromatin.

The presence of poly(ADPR)polymerase in the third level of rat testis chromatin, i.e., in stripped chromatin loops and nuclear matrix, was assessed using enzymatic assays, activity blots, and Western blots. The distribution and the size of ADPribose polymers associated to proteins of the same nuclear fractions was analyzed after incubation of intact isolated nuclei with [14C]- or [32P]NAD+. Short ADPribose oligomers, not larger than 3 residues, were found to be associated to tightly bound chromosomal proteins, which resisted extraction by 2 M NaCl, whereas longer oligomers (8-13 residues long) were associated to loosely bound chromosomal proteins. The identity of ADPribose-protein conjugates was determined by autoradiographic analysis of nuclear protein extracts. Tightly bound histone-like proteins appear to be ADPribosylated both in stripped chromatin loops and in nuclear matrix.

Animals↗

PubMed1993

Inhibition of CpG methylation in linker DNA by H1 histone.

H1 exerts a specific in vitro inhibitory effect on enzymic DNA methylation. The experiments reported in this paper were undertaken in order to assess whether the lower methylation level found in internucleosomal DNA compared to core DNA is the in vivo consequence of the well-known localization of this histone in the linker region, as opposed to a possible deficiency of CpG dinucleotides in linker DNA. The methyl-accepting ability of H1-depleted oligonucleosomes from human placenta and of the corresponding core particles were assayed by addition of purified DNA methyltransferase, using S-adenosylmethionine as the methyl group donor. We have found that approx. 80% of newly-incorporated methyl groups are localized in linker DNA, which is indeed a good potential substrate for enzymic DNA methylation. Addition of quasi-physiological amounts of H1 to H1-depleted oligonucleosomes markedly reduced their methyl-accepting ability, while exerting a re-condensing effect on these particles, as revealed by the distortions of their circular dichroism spectra.

Chromatin↗

PubMed1993

Effect of H1 histone isoforms on the methylation of single- or double-stranded DNA.

The loosely and tightly bound H1 histone isoforms were shown to exert, on the in vitro methylation of linker DNA in H1-depleted oligonucleosomes, inhibitory or activating effects respectively similar to those previously shown in the methylation of Micrococcus luteus dsDNA. When assayed on the enzymic methylation of Micrococcus luteus ssDNA, addition of the tightly bound one resulted in a stimulation similar to that exerted on double-stranded bacterial DNA or on linker DNA from mammalian chromatin, while the loosely bound isoform had no effect whatsoever. The transformation of the "typical" loosely bound H1 isoform into its tightly bound counterpart can be visualized as being an essential event in the modulation of DNA methylation process in eukaryotic chromatin.

Cell Nucleus↗

PubMed1991

Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones.

After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.

Chromatin↗

PubMed1991

Histones and DNA methylation in mammalian chromatin. Differential inhibition by histone H1.

Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition. The H1 component was responsible for this inhibition, no effect being exerted by the other histones. H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1. The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.

Animals↗