Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites

Analyse in silico des îlots CpG associés aus régions promotrices des gènes de mammifères

La méthylation de l'ADN est une modification biochimique naturellement présente dans de nombreux organismes procaryotes et eucaryotes sous des formes et des fonctions associées très diffèrentes. Chez les vertébrés, elle est spécifique des cytosines localisées dans les dinucléotides 5'-CG-3' (CpG). Le niveau de méthylation important ainsi que le fort taux de mutation des cytosines méthylées sont supposés être à l'origine de la sous-représentation des CpG dans ces génomes. Singulièrement, certaines régions génomiques sont non-méthylées et relativement riches en CpG : les îlots CpG (CGI). L'origine et le rôle de ces structures sont encore mal connus. Au cours de cette thèse, nous avons étudié in silico les CGI humains et murins afin de déterminer les caractéristiques des gènes possédant un CGI sur leur promoteur (profil d'expression, composition en bases,...) et afin de comprendre les mécanismes présidant la mise en place et le maintien de ces structures. Parallèlement, nous avons développé une méthode d'identification des promoteurs associés aux CGI, CpGProD (http://pbil.univ-lyon1.fr/software/cpgprod.html)LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Determinants of CpG Islands: Expression in Early Embryo and Isochore Structure

In an attempt to understand the origin of CpG islands (CGIs) in mammalian genomes, we have studied their location and structure according to the expression pattern of genes and to the G + C content of isochores in which they are embedded. We show that CGIs located over the transcription start site (named start CGIs) are very different structurally from the others (named no-start CGIs): (1) 61.6% of the no-start CGIs are due to repeated sequences (79 % are due to Alus), whereas only 5.6% of the start CGIs are due to such repeats; (2) start CGIs are longer and display a higher CpGo/e ratio and G + C level than no-start CGIs. The frequency of tissue-specific genes associated to a start CGI varies according to the genomic G + C content, from 25% in G + C-poor isochores to 64% in G + C-rich isochores. Conversely, the frequency of housekeeping genes associated to a start CGI (90%) is independent of the isochore context. Interestingly, the structure of start CGIs is very similar for tissue-specific and housekeeping genes. Moreover, 93% of genes expressed in early embryo are found to exhibit a CpG island over their transcription start point. These observations are consistent with the hypothesis that the occurrence of these CGIs is the consequence of gene expression at this stage, when the methylation pattern is installed

CpG dinucleotides and the mutation rate of non-CpG DNA

The neutral mutation rate is equal to the base substitution rate when the latter is not affected by natural selection. Differences between these rates may reveal that factors such as natural selection, linkage, or a mutator locus are affecting a given sequence. We examined the neutral base substitution rate by measuring the sequence divergence of ∼30,000 pairs of inactive orthologous L1 retrotransposon sequences interspersed throughout the human and chimpanzee genomes. In contrast to other studies, we related ortholog divergence to the time (age) that the L1 sequences resided in the genome prior to the chimpanzee and human speciation. As expected, the younger orthologs contained more hypermutable CpGs than the older ones because of their conversion to TpGs (and CpAs). Consequently, the younger orthologs accumulated more CpG mutations than the older ones during the ∼5 million years since the human and chimpanzee lineages separated. But during this same time, the younger orthologs also accumulated more non-CpG mutations than the older ones. In fact, non-CpG and CpG mutations showed an almost perfect (R2 = 0.98) correlation for ∼97% of the ortholog pairs. The correlation is independent of G + C content, recombination rate, and chromosomal location. Therefore, it likely reflects an intrinsic effect of CpGs, or mutations thereof, on non-CpG DNA rather than the joint manifestation of the chromosomal environment. The CpG effect is not uniform for all regions of non-CpG DNA. Therefore, the mutation rate of non-CpG DNA is contingent to varying extents on local CpG content. Aside from their implications for mutational mechanisms, these results indicate that a precise determination of a uniform genome-wide neutral mutation rate may not be attainable

Diversity and distribution of alpha satellite DNA in the genome of an Old World monkey: Cercopithecus solatus

International audienceBackgroundAlpha satellite is the major repeated DNA element of primate centromeres. Evolution of these tandemly repeated sequences has led to the existence of numerous families of monomers exhibiting specific organizational patterns. The limited amount of information available in non-human primates is a restriction to the understanding of the evolutionary dynamics of alpha satellite DNA.ResultsWe carried out the targeted high-throughput sequencing of alpha satellite monomers and dimers from the Cercopithecus solatus genome, an Old World monkey from the Cercopithecini tribe. Computational approaches were used to infer the existence of sequence families and to study how these families are organized with respect to each other. While previous studies had suggested that alpha satellites in Old World monkeys were poorly diversified, our analysis provides evidence for the existence of at least four distinct families of sequences within the studied species and of higher order organizational patterns. Fluorescence in situ hybridization using oligonucleotide probes that are able to target each family in a specific way showed that the different families had distinct distributions on chromosomes and were not homogeneously distributed between chromosomes.ConclusionsOur new approach provides an unprecedented and comprehensive view of the diversity and organization of alpha satellites in a species outside the hominoid group. We consider these data with respect to previously known alpha satellite families and to potential mechanisms for satellite DNA evolution. Applying this approach to other species will open new perspectives regarding the integration of satellite DNA into comparative genomic and cytogenetic studies

The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys

International audienceAlpha satellite is the major repeated DNA element of primate centromeres. Specific evolutionary mechanisms have led to a great diversity of sequence families with peculiar genomic organization and distribution, which have till now been studied mostly in great apes. Using high throughput sequencing of alpha satellite monomers obtained by enzymatic digestion followed by computational and cytogenetic analysis, we compare here the diversity and genomic distribution of alpha satellite DNA in two related Old World monkey species, Cercopithecus pogonias and Cercopithecus solatus, which are known to have diverged about 7 Ma. Two main families of monomers, called C1 and C2, are found in both species. A detailed analysis of our data sets revealed the existence of numerous subfamilies within the centromeric C1 family. Although the most abundant subfamily is conserved between both species, our fluorescence in situ hybridization (FISH) experiments clearly show that some subfamilies are specific for each species and that their distribution is restricted to a subset of chromosomes, thereby pointing to the existence of recurrent amplification/homogenization events. The pericentromeric C2 family is very abundant on the short arm of all acrocentric chromosomes in both species, pointing to specific mechanisms that lead to this distribution. Results obtained using two different restriction enzymes are fully consistent with a predominant monomeric organization of alpha satellite DNA that coexists with higher order organization patterns in the C. pogonias genome. Our study suggests a high dynamics of alpha satellite DNA in Cercopithecini, with recurrent apparition of new sequence variants and interchromosomal sequence transfer

Additional file 4: Text S2. of Diversity and distribution of alpha satellite DNA in the genome of an Old World monkey: Cercopithecus solatus

Alignment used to generate the phylogenetic tree from Fig. 7b. (TXT 48 kb

Additional file 2: Table S1. of Diversity and distribution of alpha satellite DNA in the genome of an Old World monkey: Cercopithecus solatus

Filtering steps from Cercopithecus solatus raw data to alpha satellite monomer and dimer datasets. Table S2. Alpha satellite family associations in Cercopithecus solatus dimer dataset. (DOCX 40 kb

Additional file 3: Text S1. of Diversity and distribution of alpha satellite DNA in the genome of an Old World monkey: Cercopithecus solatus

Alignment used to generate the phylogenetic tree from Fig. 7a. (TXT 28 kb