Isolation and enrichment of human genomic CpG islands by methylation-sensitive mirror orientation selection

CpG islands (CGIs) in human genomic DNA are GC-rich fragments whose aberrant methylation is associated with human disease development. In the current study, methylation-sensitive mirror orientation selection (MS-MOS) was developed to efficiently isolate and enrich unmethylated CGIs from human genomic DNA. The unmethylated CGIs prepared by the MS-MOS procedure subsequently were used to construct a CGI library. Then the sequence characteristics of cloned inserts of the library were analyzed by bioinformatics tools, and the methylation status of CGI clones was analyzed by HpaII PCR. The results showed that the MS-MOS method could be used to isolate up to 0.001% of differentially existed unmethylated DNA fragments in two complex genomic DNA. In the CGI library, 34.1% of clones had insert sequences satisfying the minimal criteria for CGIs. Excluding duplicates, 22.0% of the 80,000 clones were unique CGI clones, representing 60% of all the predicted CGIs (about 29,000) in human genomic DNA, and most or all of the CGI clones were unmethylated in human normal cell DNA based on the HpaII PCR analysis results of randomly selected CGI clones. In conclusion, MS-MOS was an efficient way to isolate and enrich human genomic CGIs. The method has powerful potential application in the comprehensive identification of aberrantly methylated CGIs associated with human tumorigenesis to improve understanding of the epigenetic mechanisms involved.     

Human-specific CpG “beacons” identify loci associated with human-specific traits and disease

Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10^?3) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10^?3). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.     

Inter-population Differences in Retrogene Loss and Expression in Humans

Gene retroposition leads to considerable genetic variation between individuals. Recent studies revealed the presence of at least 208 retroduplication variations (RDVs), a class of polymorphisms, in which a retrocopy is present or absent from individual genomes. Most of these RDVs resulted from recent retroduplications. In this study, we used the results of Phase 1 from the 1000 Genomes Project to investigate the variation in loss of ancestral (i.e. shared with other primates) retrocopies among different human populations. In addition, we examined retrocopy expression levels using RNA-Seq data derived from the Ilumina BodyMap project, as well as data from lymphoblastoid cell lines provided by the Geuvadis Consortium. We also developed a new approach to detect novel retrocopies absent from the reference human genome. We experimentally confirmed the existence of the detected retrocopies and determined their presence or absence in the human genomes of 17 different populations. Altogether, we were able to detect 193 RDVs; the majority resulted from retrocopy deletion. Most of these RDVs had not been previously reported. We experimentally confirmed the expression of 11 ancestral retrogenes that underwent deletion in certain individuals. The frequency of their deletion, with the exception of one retrogene, is very low. The expression, conservation and low rate of deletion of the remaining 10 retrocopies may suggest some functionality. Aside from the presence or absence of expressed retrocopies, we also searched for differences in retrocopy expression levels between populations, finding 9 retrogenes that undergo statistically significant differential expression.     

Modeling genomic data with type attributes, balancing stability and maintainability

Background

The computational prediction of DNA methylation has become an important topic in the recent years due to its role in the epigenetic control of normal and cancer-related processes. While previous prediction approaches focused merely on differences between methylated and unmethylated DNA sequences, recent experimental results have shown the presence of much more complex patterns of methylation across tissues and time in the human genome. These patterns are only partially described by a binary model of DNA methylation. In this work we propose a novel approach, based on profile analysis of tissue-specific methylation that uncovers significant differences in the sequences of CpG islands (CGIs) that predispose them to a tissue- specific methylation pattern.

Results

We defined CGI methylation profiles that separate not only between constitutively methylated and unmethylated CGIs, but also identify CGIs showing a differential degree of methylation across tissues and cell-types or a lack of methylation exclusively in sperm. These profiles are clearly distinguished by a number of CGI attributes including their evolutionary conservation, their significance, as well as the evolutionary evidence of prior methylation. Additionally, we assess profile functionality with respect to the different compartments of protein coding genes and their possible use in the prediction of DNA methylation.

Conclusion

Our approach provides new insights into the biological features that determine if a CGI has a functional role in the epigenetic control of gene expression and the features associated with CGI methylation susceptibility. Moreover, we show that the ability to predict CGI methylation is based primarily on the quality of the biological information used and the relationships uncovered between different sources of knowledge. The strategy presented here is able to predict, besides the constitutively methylated and unmethylated classes, two more tissue specific methylation classes conserving the accuracy provided by leading binary methylation classification methods.     

Human-specific CpG “beacons” identify loci associated with human-specific traits and disease

Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10⁻³) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10⁻³). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.     

Identifying differentially methylated genes using mixed effect and generalized least square models

Background  

DNA methylation plays an important role in the process of tumorigenesis. Identifying differentially methylated genes or CpG islands (CGIs) associated with genes between two tumor subtypes is thus an important biological question. The methylation status of all CGIs in the whole genome can be assayed with differential methylation hybridization (DMH) microarrays. However, patient samples or cell lines are heterogeneous, so their methylation pattern may be very different. In addition, neighboring probes at each CGI are correlated. How these factors affect the analysis of DMH data is unknown.     

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.     

Unmethylated state of 5′ upstream CpG islands may be necessary but not sufficient for the testis-enriched expression of ZNF230/Znf230

The testis-enriched genes ZNF230/Znf230 are located on human chromosome 11p15/mouse chromosome 7 near conserved imprinting control regions. Typical CpG islands (CGIs) extend from the promoter to the first exon in each of these genes. To investigate the correlation between the methylation status of the above CGIs and the expression patterns of the two genes, we performed bisulfite genomic sequencing of genomic DNA from human and mouse tissues and cells. The results showed that the CGIs of ZNF230/Znf230 were completely unmethylated in all selected tissues and cells, regardless of the expression levels of the two genes. Further experiments using Znf230-second-exon-knockout mice to investigate the imprinting status of Znf230 showed that its expression was not affected by genomic imprinting. However, an in vitro methylation assay illustrated that the methylation of these CpG sites could repress the expression of the luciferase reporter gene. Furthermore, chromatin immunoprecipitation with anti-Specificity protein 1 (Sp1) antibody showed that Sp1 could bind to the CGIs in the ZNF230/Znf230 gene promoter. Thus, we propose that the unmethylated state of ZNF230/Znf230 CGIs may be a prerequisite for their expression but not sufficient for their abundant expression in the testis, and that Sp1 binding may be one factor involved in preserving the methylation-free state of ZNF230/Znf230 CGIs.