HpaII Tiny Fragments Research Articles

Profiling the impact of the embryonic microenvironment through global transcriptomic and DNA methylome surveying

Transcriptomicanalyses have been conducted to study how mammalian early embryos cope with their immediate microenvironments especially when stressed from the application of assisted reproductive technologies, maternal nutrition or any other induced external factors. To study potential long term impacts from these stresses, description of early embryo epigenome is necessary. DNA methylation profiling of mammalian early embryo presents important challenges as they are rare biological material. Very recently, a genome-scale, base-resolution timeline of DNA methylation has been characterized for mouse embryo[1], however, such information has not been reported for treated embryos or for other species. We focus on bovine embryonic development since it follows a similar kinetic and developmental success rate as human. Using our transcriptomic platform[2] studies revealed that several key pathways seems to react when embryos are submitted to stress but it was also observed that long non-coding RNAs (IncRNAs) represent the class of RNA that are the most influenced by the embryonic environment further supporting the involvement of epigenetic mechanisms in the embryonic response. Commercial platform specific to the study of the bovine methylome do not exist yet. We therefore set to develop one with the constraints of enabling to survey the general topology of the genomic methylome using the limited amount of starting material offered by early embryos and also to provide a cost effective platform with a complete data analysis pipeline. We report the development of the Bovine Embryonic Global Methylome platform (BEGMP) which allows global DNA methylation from as little as 7-8 ng of gDNA starting material (representing 10 expanded blastocysts). Samples are processed through a modified HpaII Tiny Fragments (HTFs) enrichment by ligation-mediated PCR (HELP) assay. Initial surveying of the methylome found in Day-7 and Day-12 bovine embryos suggests a transition during development of methylation marks in repeat containing elements in a class-specific manner showing the potential of the platform to describe the ontology of the establishment of methylation marks during early embryogenesis. The platform was also used to study the impact of embryonic culture conditions where both the transcriptome and the methylome were surveyed from the same samples. Treatment of in vitro cultured embryos in presence of the methtyl donor S-adenosyl methionin showed that in addition to protein coding genes, IncRNAs were also impacted by the treatment. At the DNA methylation level, it showed global hypermethylation in treated embryos and parallel analysis revealed some similar function and network between early embryo transcriptome and methylome. Overall, BEGMP is the first developed and functional methylome platform for non-model spices which allows to survey the transcriptome and methylome from very restricted samples.

HTF9C gene of 22q11.21 region associates with schizophrenia having deficit-sustained attention

A region at chromosome 22q11.21 has been reported to potentially harbor a candidate gene for schizophrenia, ZDHHC8 (zinc finger, DHHC domain containing 8; also annotated as KIAA1292) in a number of studies. This finding has been replicated in Han Chinese, but not in other ethnicity-specific studies. For further support from within the Han Chinese ethnic group, we selected two single nucleotide polymorphisms (SNP) located at the distal 5'-end (rs1633445; intron 10 of HpaII tiny fragments locus 9C, HTF9C) and the intron 4 (rs175174) of ZDHHC8 gene to test if these were associated with schizophrenia in a study sample of Taiwan. A total of 218 schizophrenia families with at least two affected siblings participated in this study. These two SNPs were genotyped using matrix-assisted laser desorption/localization ionization time of flight (MALDI-TOF) mass spectrometry. Significant associations with schizophrenia were not shown from these two SNPs. After stratifying schizophrenia according to the deficit and the nondeficit of sustained attention assessed by the Continuous Performance Test, the rs1633445 showed significant association with schizophrenia in the presence of a deficit in sustained attention (P<0.04). SNP rs1633445 of the HTF9C gene may be associated with a deficit in sustained attention within schizophrenia, in a Taiwanese cohort. The deficit of sustained attention may be an endophenotype of schizophrenia, and warrants further study.

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.

HpaII library indicates ‘methylation-free islands’ in wheat and barley

A library of wheat genomic DNA HpaII tiny fragments (HTF), sized below 500 bp, has been constructed. Of the clones in the library 80% belong to the single/low-copy category, while 12% of the clones are nuclear repetitive sequences and 8% originate from the chloroplast and mitochondrial DNA. This result shows a substantial enrichment in the single/low-copy sequences of the wheat genome, which contains at least 80% repetitive sequences. Twenty-nine random single/lowcopy clones were analysed further for wheat chromosome location, cross-hybridisation to barley DNA and their association with rare-cutting, C-methylation-sensitive restriction sites. The results show that the HTF clones are associated more frequently than expected with NotI, MluI, NruI and PstI sites in wheat and barley genomic DNA. The 12% repetitive fraction of the clones contain both moderately and highly repetitive sequences, but no tandemly repeated sequences. The level of enrichment for single/low-copy sequences indicates that libraries of this type are a valuable source of probes for RFLP mapping. In addition, the close association of the HTF clones with rare-cutting restriction enzyme sites ensures that HTF clones will have a useful role in the construction of long-range physical maps in wheat.

PCR-mediated cloning of HpaII tiny fragments from microdissected human chromosomes.

Vertebrate DNA contains a small fraction of unmethylated CpG-rich DNA sequences, many of which include the 5' end of a gene. This fraction can be detected by its cleavage to tiny fragments with the methylation-sensitive restriction enzyme HpaII. Thus, the isolation of HpaII tiny fragments (HTFs) from a specific chromosome region may be a useful approach for making an inventory of the genes contained in it. Using microdissection, we have isolated DNA from human chromosome band 8q24.1. The DNA was digested with HpaII, ligated to a ClaI-cut pUC plasmid, and amplified with Taq DNA polymerase and the standard M13/pUC forward and reverse sequencing primers. The amplification products were used to construct an HTF library, which is enriched for CpG-rich single-copy clones.

CpG islands, genes and isochores in the genomes of vertebrates

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [Aïssani and Bemardi, Gene 106 (1991) 173–183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5′-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the ‘extragenic‘ CpG islands located in the 5′-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including ‘extragenic’ CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including ‘extragenic’ CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.

CpG islands: features and distribution in the genomes of vertebrates

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.

Characterization of the human N-CAM promoter

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Methylation of chick UbI and UbII polyubiquitin genes and their differential expression during spermatogenesis

Northern analysis demonstrated that levels of ubiquitin transcript increased during the chicken testis maturation process, in agreement with the previously published increase of ubiquitin during this differentiation process. Specific probes for four different ubiquitin genes (two polyubiquitins, UbI and UbII, and two ubiquitin-fusion genes, UbCep52 and UbCep80) allowed us to analyse the expression of each individual gene. UbI polyubiquitin gene was expressed in all the tissues tested, and its transcript was the most abundant ubiquitin RNA in all of them. Unspliced UbI transcript, already detected in stressed chicken-embryo fibroblast, was also present in immature testis and reticulocytes. UbII, a chicken polyubiquitin gene not previously found expressed and not heat-shock-inducible, was specifically stimulated during the testis maturation process. Two minor ubiquitin fusion transcripts of 0.6 and 0.7 kb, corresponding to UbCep52 and UbCep80 respectively, were also found in chicken testis. Although differentially expressed, it was found that UbI and UbII chicken polyubiquitin genes had an HTF ('HpaII tiny fragments') island (CpG-rich and constitutively unmethylated region) in their 5' proximal non-coding region. In addition, we demonstrated the coexistence of 3' and/or 5' relatively distal methylated sites together with these 5' proximal HTF islands in both chicken polyubiquitin genes. 3' and 5' distal UbI CCGG sites were specifically hypermethylated in mature testis, whereas a 3' distal UbII CCGG site was found to be about 50% methylated in all DNAs tested.

Detection of a rare-cutter RFLP in a CpG-rich island near the cystic fibrosis locus

The probe pCS.7, isolated from an HpaII tiny fragments (HTF) island and tightly linked to the mutation causing cystic fibrosis (CF), detects a polymorphism with the rare-cutter restriction enzyme BssHII. In a single digest, the resulting restriction fragment length polymorphism (RFLP) cannot be detected by either conventional or pulse-field gel electrophoresis. A double digest with BssHII in conjunction with a six-cutter restriction enzyme that does not recognize a site containing a CpG dinucleotide enables the probe to be used routinely for prenatal diagnosis and carrier exclusion. This strategy can be used to identify polymorphisms in HTF islands.

DNA methylation in the developing marsupial embryo

Marsupial development differs from early development of placental mammals in that the blastocyst is unilaminar, so that both embryonic and extraembryonic cells are derived from a single layer of cells (protoderm) which faces the blastocyst cavity. Also, all cells in female marsupial conceptuses so far examined show preferential paternal X-inactivation. To test for a possible correlation between cell position, paternal X-inactivation and DNA hypomethylation, marsupial DNA preparations from three regions, embryo, vascular yolk sac and avascular yolk sac, were digested with methyl-specific restriction endonucleases, separated on agarose gels and end-labelled with 32P-dCTP. The size distribution of the fragments obtained indicated three levels of methylation: high methylation of embryonic DNA, intermediate levels of methylation of vascular yolk sac DNA and hypomethylation of avascular yolk sac DNA. The degree of methylation of repeat sequences, observed as discrete bands in end-labelled HpaII digests, was correlated with the overall methylation of tissue DNA. Thus, the difference in methylation in embryonic and extraembryonic DNA was similar to that described for the mouse conceptus, and the outside cell position of marsupial fetal precursor cells did not correlate with hypomethylation. HpaII tiny fragments, which indicate the presence of CpG-rich islands of DNA, were evident in the marsupial digests. In the mouse DNA, these islands are associated with gene transcription and provide one route to cloning of unique gene sequences.