The clustering of CpG islands may constitute an important determinant of the 3D organization of interphase chromosomes

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.     

CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation

Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Growing evidence has revealed that the multivalent functional zinc finger protein CCCTC-binding factor (CTCF) is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome. Using circular chromosome conformation capture (4C) methodology, we discovered that CTCF may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. We characterized the cell-type specific spatial organization of the kcnq5 gene locus mediated by CTCF in detail using chromosome conformation capture (3C) and 3C-derived techniques. Cohesion also participated in mediating the organization of this locus. RNAi-mediated knockdown of CTCF sharply diminished the interaction frequencies between the chromatin loops of the kcnq5 gene locus and down-regulated local gene expression. Functional analysis showed that the interacting chromatin loops of the kcnq5 gene locus can repress the gene expression in a luciferase reporter assay. These interacting chromatin fragments were a series of repressing elements whose contacts were mediated by CTCF. Therefore, these findings suggested that the dynamical spatial organization of the kcnq5 locus regulates local gene expression.     

Spatial Organization of the Chicken α-Globin Gene Domain in Cells of Different Origins

Hybridization with an oligonucleotide array was used to map the regions of DNA anchorage to the nuclear matrix. Matrix-associated DNA served as a hybridization probe. To obtain the oligonucleotide array, 60-mer oligonucleotides regularly distributed throughout the genome region of interest at 2-kb intervals were immobilized on a nylon filter. The organization of DNA into loop domains was studied in a 100-kb region of chicken chromosome 16, including the α -globin gene cluster. A 40-kb DNA loop, which was fixed to the nuclear matrix and harbored all α-globin genes, was observed in erythroid cells. One of its anchorage regions colocalized with matrix associated region (MAR) and an insulator found previously in the 5′ region of the chicken α-globin gene domain. The spatial (domain-loop) organization of the α-globin gene cluster in lymphoid cells proved to be strikingly different from that in erythroid cells.     

The Chromosomal Distribution of CpG Islands in the Mouse: Evidence for Genome Scrambling in the Rodent Lineage

We have prepared a library of mouse whole CpG islands using a methyl-CpG binding domain column. The distribution of CpG islands in the mouse genome was determined by FISH, using the library as a probe. Unlike in other vertebrate genomes that have been examined (human and chicken), extreme clustering of CpG islands was not seen in the mouse genome. No individual murine chromosome stood out as being either very gene-rich or very gene-poor. Despite the more even distribution of CpG islands in the mouse at a gross chromosomal level, at finer resolution concentrations of CpG islands are seen to correspond to the R-band early replicating regions of the genome.     

Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6

On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21-q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5' UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K(a)/K(s) ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs.     

Spatial organization of the chicken alpha-globin gene domain in cells of different

We have developed a technique for mapping the sites of DNA attachment to the nuclear matrix by hybridization of nuclear matrix DNA with an oligonucleotide array. The latter was made by immobilization of 60-mer oligonucleotides distributed within the area under study with a 2 Kb step on nylon filter. Using this approach we have analyzed the mode of interaction of a 100 Kb fragment of chicken chromosome 16 including the alpha-globin gene domain with the nuclear matrix. The 40 Kb DNA loop including all alpha-globin genes was detected in erythroid cells. One of the borders of this loop colocalized with the previously mapped MAR element and CTCF-dependent enhancer-blocking element. Also, a long transcribed area was found to be preferentially associated with the nuclear matrix. The spatial organization of the area under study in lymphoid cells was drastically different from this observed in erythroid cells.     

Genomics tools for unraveling chromosome architecture

The spatial organization of chromosomes inside the cell nucleus is still poorly understood. This organization is guided by intra- and interchromosomal contacts and by interactions of specific chromosomal loci with relatively fixed nuclear 'landmarks' such as the nuclear envelope and the nucleolus. Researchers have begun to use new molecular genome-wide mapping techniques to uncover both types of molecular interactions, providing insights into the fundamental principles of interphase chromosome folding.     

DNA motifs associated with aberrant CpG island methylation

Epigenetic silencing involving the aberrant methylation of promoter region CpG islands is widely recognized as a tumor suppressor silencing mechanism in cancer. However, the molecular pathways underlying aberrant DNA methylation remain elusive. Recently we showed that, on a genome-wide level, CpG island loci differ in their intrinsic susceptibility to aberrant methylation and that this susceptibility can be predicted based on underlying sequence context. These data suggest that there are sequence/structural features that contribute to the protection from or susceptibility to aberrant methylation. Here we use motif elicitation coupled with classification techniques to identify DNA sequence motifs that selectively define methylation-prone or methylation-resistant CpG islands. Motifs common to 28 methylation-prone or 47 methylation-resistant CpG island-containing genomic fragments were determined using the MEME and MAST algorithms (). The five most discriminatory motifs derived from methylation-prone sequences were found to be associated with CpG islands in general and were nonrandomly distributed throughout the genome. In contrast, the eight most discriminatory motifs derived from the methylation-resistant CpG islands were randomly distributed throughout the genome. Interestingly, this latter group tended to associate with Alu and other repetitive sequences. Used together, the frequency of occurrence of these motifs successfully discriminated methylation-prone and methylation-resistant CpG island groups with an accuracy of 87% after 10-fold cross-validation. The motifs identified here are candidate methylation-targeting or methylation-protection DNA sequences.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.