Sequence context analysis in the mouse genome: single nucleotide polymorphisms and CpG island sequences

A genome-wide view of sequence mutability in mice is still limited, although biologists usually assume the same scenario for mice as for humans. In this study, we examined the sequence context in the local environment of 482,528 mouse single nucleotide polymorphisms (SNPs). We found that CpG-containing short sequences, in general, had more representation in the local sequences of SNPs compared to the genome sequences. The extent of this overrepresentation was stronger in mice than in humans, which is inconsistent with previous observations of the weaker neighboring-nucleotide biases on mouse SNPs. To exclude the CpG effect, we compared the distribution patterns of short sequences among the six categories of SNPs. The results revealed an even stronger pattern in the CpG-containing group for C/G substitution compared to for A/G or C/T substitutions. We next performed the first genome-wide sequence context analysis of SNPs in the mouse CpG islands. SNPs occurring at CpG sites were 3.14-fold less prevalent than expected, suggesting the suppression of methylation-dependent deamination in the CpG islands. The extent of this suppression was less in mice than in humans. Finally, compared with humans, the observations of a greater deficit of CpG dinucleotides, a stronger overrepresentation of CpG-containing n-mers surrounding the polymorphic sites, and a higher SNP/genome ratio of CpG dinucleotides in the mouse genome support the "loss of CpG islands" model in the mouse lineage.     

Sequence context analysis of 8.2 million single nucleotide polymorphisms in the human genome

We analyzed n-mers (n=3-8) in the local environment of 8,249,446 human SNPs and compared their distribution with that in the genome reference sequences. The results revealed that the short sequences, which contained at least one CpG dinucleotide, occurred more frequently in the local SNP sequences than in the genome sequences. To exclude the hypermutability effect of the methylated CpG dinucleotides on the sequence context of SNPs, we examined the distribution patterns for each of the six categories of substitution. We observed the similar pattern (i.e., CpG-containing n-mers vs. non-CpG-containing n-mers) in SNP categories A/G, C/T and C/G but the opposite pattern in category A/T. We next identified 34,928 putative CpG islands in the human genome and located 133,591 SNPs within these islands. In the CpG islands, CpG SNPs were 3.92-fold less prevalent relative to the presence of CpG dinucleotides. Conversely, in the human genome, the frequency of CpG dinucleotides at the polymorphic sites was 6.09 times that in the genome reference sequences. These results support the previous views of mutational suppression at the CpG sites in the CpG islands and hypermutability of the methylated CpG dinucleotides that are prevalent in the non-CpG island sequences in the human genome. Our study represents a comprehensive investigation of the sequence context of SNPs in the human genome and in human CpG islands.     

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.     

Sequence context at human single nucleotide polymorphisms: overrepresentation of CpG dinucleotide at polymorphic sites and suppression of variation in CpG islands

Human polymorphisms originate as mutations, and the influence of context on mutagenesis should be reflected in the distribution of sequences surrounding single nucleotide polymorphisms (SNPs). We have performed a computational survey of nearly two million human SNPs to determine if sequence-dependent hotspots for polymorphism exist in the human genome. Here we show that sequences containing CpG dinucleotides, which occur at low frequencies in the human genome, are 6.7-fold more abundant at polymorphic sites than expected. In contrast, polymorphisms in CpG sequences located within CpG islands, important regulatory regions that modulate gene expression, are 6.8-fold less prevalent than expected. The distribution of polymorphic alleles at CpGs in CpG islands is also significantly different from that in non-island regions. These data strongly support a role for 5-methylcytosine deamination in the generation of human variation, and suggest that variation at CpGs in islands is suppressed.     

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 [A?ssani and Bernardi, 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.     

人类基因组上CpG岛的定位和系统分析

为了研究CpG岛产生和消失机制以及位于基因启动子区域外的CpG岛保守性等问题,我们通过序列比对和进化保守性分析等方法,分析在人类和小鼠中保守的基因上的CpG岛。结果显示已有保守序列的突变以及序列插入删除是CpG岛产生和消失的主要原因,进一步分析发现52%的在小鼠基因组上保守序列完全缺失的CpG岛位于两个转座子之间,提示转座子所介导的序列插入是CpG岛形成和消失的重要原因。人类基因组上在启动子区域外的CpG岛中约有79%为新产生的CpG岛,显著高于启动子区域内新产生的CpG岛比例(41%)。GO分析表明与这些CpG岛相关的部分基因与神经系统发育显著相关,提示新产生的CpG岛参与神经发育过程。     

Features and trend of loss of promoter-associated CpG islands in the human and mouse genomes

CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse orthologous gene pairs and found that the mouse genome has notably fewer CGIs in the promoter regions and less pronounced CGI characteristics than does the human genome. We further inferred CGI's ancestral state using the dog genome as a reference and examined the nucleotide substitution pattern and the mutational direction in the conserved regions of human and mouse CGIs. The results reveal many losses of CGIs in both genomes but the loss rate in the mouse lineage is two to four times the rate in the human lineage. We found an intriguing feature of CGI loss, namely that the loss of a CGI usually starts from erosion at the both edges and gradually moves towards the center. We found functional bias in the genes that have lost promoter-associated CGIs in the human or mouse lineage. Finally, our analysis indicates that the association of CGIs with housekeeping genes is not as strong as previously estimated. Our study provides a detailed view of the evolution of promoter-associated CGIs in the human and mouse genomes and our findings are helpful for understanding the evolution of mammalian genomes and the role of CGIs in gene function.     

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.     

一种快速高效的全基因组甲基化CpG岛富集方法的建立

高甲基化的CpG岛所致基因表观遗传学转录失活已经成为肿瘤表观基因组学研究的重要内容。现在已有很多检测CpG岛甲基化的方法,但由于各自的局限,还没有建立一种能快速在全基因组水平上进行甲基化CpG岛的富集方法。本研究利用甲基化结合蛋白MBD2b具有特异性结合甲基化DNA的特性, 建立了一种基于DNA免疫共沉淀技术的全基因组甲基化CpG岛的富集方法。在大肠杆菌中表达重组的GST-MBD2b蛋白,通过Glutathione Sepharose 4B对重组蛋白进行纯化,制备成亲和层析柱,利用在不同的盐离子强度下甲基化DNA和非甲基化DNA的结合能力不同,对甲基化DNA进行富集。用甲基化酶SssI处理过的DNA片段与非甲基化DNA片段进行富集效率的检测,发现0.5M KCl的浓度是甲基化DNA片段和非甲基化DNA片段得以分开的临界条件。样品的富集效率用Real Time PCR进行检测。结果表明,这种方法能够实现对全基因组甲基化DNA的有效富集且最高的富集倍数可达到100多倍。富集到的甲基化DNA可以进行后续的定量PCR, DNA测序和全基因组芯片的分析等工作,为大规模分析全基因组CpG 岛甲基化的改变奠定了基础。     

Particle swarm optimization with reinforcement learning for the prediction of CpG islands in the human genome

Background

Regions with abundant GC nucleotides, a high CpG number, and a length greater than 200 bp in a genome are often referred to as CpG islands. These islands are usually located in the 5′ end of genes. Recently, several algorithms for the prediction of CpG islands have been proposed.

Methodology/Principal Findings

We propose here a new method called CPSORL to predict CpG islands, which consists of a complement particle swarm optimization algorithm combined with reinforcement learning to predict CpG islands more reliably. Several CpG island prediction tools equipped with the sliding window technique have been developed previously. However, the quality of the results seems to rely too much on the choices that are made for the window sizes, and thus these methods leave room for improvement.

Conclusions/Significance

Experimental results indicate that CPSORL provides results of a higher sensitivity and a higher correlation coefficient in all selected experimental contigs than the other methods it was compared to (CpGIS, CpGcluster, CpGProd and CpGPlot). A higher number of CpG islands were identified in chromosomes 21 and 22 of the human genome than with the other methods from the literature. CPSORL also achieved the highest coverage rate (3.4%). CPSORL is an application for identifying promoter and TSS regions associated with CpG islands in entire human genomic. When compared to CpGcluster, the islands predicted by CPSORL covered a larger region in the TSS (12.2%) and promoter (26.1%) region. If Alu sequences are considered, the islands predicted by CPSORL (Alu) covered a larger TSS (40.5%) and promoter (67.8%) region than CpGIS. Furthermore, CPSORL was used to verify that the average methylation density was 5.33% for CpG islands in the entire human genome.     

Probes for CpG islands on the distal long arm of the human X chromosome are clustered in Xq24 and Xq28

We have isolated and characterized 55 EagI-containing genomic DNA clones from the distal long arm of the human X chromosome. The presence of additional sites for rare-cutter restriction enzymes and the demethylation of the corresponding genomic DNA demonstrate that at least 30 clones correspond to CpG islands of the Xq24-Xqter region. All clones were regionally mapped with a hybrid panel. The majority are in Xq28 and Xq24 (18 and 14 clones, respectively), 15 are in the Xq26-Xq27 interval, and none is in Xq25. This analysis demonstrates a nonuniform distribution of CpG islands that may reflect the distribution of coding regions in this part of the genome.     

A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.     

CpG island density and its correlations with genomic features in mammalian genomes

Background  

CpG islands, which are clusters of CpG dinucleotides in GC-rich regions, are considered gene markers and represent an important feature of mammalian genomes. Previous studies of CpG islands have largely been on specific loci or within one genome. To date, there seems to be no comparative analysis of CpG islands and their density at the DNA sequence level among mammalian genomes and of their correlations with other genome features.     

Tissue specific differentially methylated regions (TDMR): Changes in DNA methylation during development

Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.     

A human CpG island randomly inserted into a plant genome is protected from methylation

In vertebrate genomes the dinucleotide CpG is heavily methylated, except in CpG islands, which are normally unmethylated. It is not clear why the CpG islands are such poor substrates for DNA methyltransferase. Plant genomes display methylation, but otherwise the genomes of plants and animals represent two very divergent evolutionary lines. To gain a further understanding of the resistance of CpG islands to methylation, we introduced a human CpG island from the proteasome-like subunit I gene into the genome of the plant Arabidopsis thaliana. Our results show that prevention of methylation is an intrinsic property of CpG islands, recognized even if a human CpG island is transferred to a plant genome. Two different parts of the human CpG island – the promoter region/ first exon and exon2–4 – both displayed resistance against methylation, but the promoter/ exon1 construct seemed to be most resistant. In contrast, certain sites in a plant CpG-rich region used as a control transgene were always methylated. The frequency of silencing of the adjacent nptII (Km^R) gene in the human CpG constructs was lower than observed for the plant CpG-rich region. These results have implications for understanding DNA methylation, and for construction of vectors that will reduce transgene silencing.     

Alu-PCR Approach to Isolating NotI-Linking Clones from the 3p14-p21 Region Frequently Deleted in Renal Cell Carcinoma

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating Not I sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.