Evolution of CpG Islands within the myc Gene Family

CpG islands are discrete regions of DNA with significantly greater frequencies of CpG doublets than bulk genomic DNA. They are most frequently associated with the 5′-ends of housekeeping genes and are involved in the regulation of their expression. In this study, the structure and evolution of CpG islands within genes of the myc family were evaluated with the protein-coding sequences of animals and their transducing viruses. These evaluations relied on a gene tree for the entire myc family to test the origins of CpG islands within their two protein-coding exons. Overall, CG-very rich and CG-rich islands are associated with exon 2 of the different myc genes of warm-blooded vertebrates and with exon 3 of the N-myc and s-myc sequences of mammals, but not birds. These overall distributions of well-developed islands can be related to the major transitions of the CG-rich genomes of warm-blooded vertebrates from the CG-poor ones of other animals. In turn, the greater variability of well-developed islands within exon 3 of the N-myc gene and among the different retrogenes of the myc family can be attributed to their reduced functional constraints, as evidenced by their limited and very restricted patterns of expression, respectively.     

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.     

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.     

Non-methylated islands in fish genomes are GC-poor.

In the vertebrate genomes studied to date the 5' end of many genes are associated with distinctive sequences known as CpG islands. CpG islands have three properties: they are non-methylated; the dinucleotide CpG occurs at the frequency predicted by base composition; and they are GC-rich. Unexpectedly we have found that CpG islands in certain fish only have the first two properties; that is, their GC-content is not elevated compared to bulk genomic DNA. Based on this finding, we speculate that the GC-richness of CpG islands in vertebrates other than fish is a passive consequence of a higher mutation rate in regions of open chromatin under conditions where the nucleotide precursor pools are biased.     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.     

Structure and expression of B-myc, a new member of the myc gene family.

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.     

Gene-associated CpG islands in plants as revealed by analyses of genomic sequences

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG-rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG-rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG-rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5'-end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5'-end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue-specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5'-end of the genes.     

A species-generalized probabilistic model-based definition of CpG islands

The DNA of most vertebrates is depleted in CpG dinucleotides, the target for DNA methylation. The remaining CpGs tend to cluster in regions referred to as CpG islands (CGI). CGI have been useful as marking functionally relevant epigenetic loci for genome studies. For example, CGI are enriched in the promoters of vertebrate genes and thought to play an important role in regulation. Currently, CGI are defined algorithmically as an observed-to-expected ratio (O/E) of CpG greater than 0.6, G+C content greater than 0.5, and usually but not necessarily greater than a certain length. Here we find that the current definition leaves out important CpG clusters associated with epigenetic marks, relevant to development and disease, and does not apply at all to nonvertabrate genomes. We propose an alternative Hidden Markov model-based approach that solves these problems. We fit our model to genomes from 30 species, and the results support a new epigenomic view toward the development of DNA methylation in species diversity and evolution. The O/E of CpG in islands and nonislands segregated closely phylogenetically and showed substantial loss in both groups in animals of greater complexity, while maintaining a nearly constant difference in CpG O/E between islands and nonisland compartments. Lists of CGI for some species are available at http://www.rafalab.org.     

CpG islands in genes showing tissue-specific expression

Patterns of DNA methylation at CpG dinucleotides and their relations with gene expression are complex. Methylation-free CpG clusters, so-called HTF islands, are most often associated with the promoter regions of housekeeping genes, whereas genes expressed in a single-cell type are usually deficient in these sequences. However, in the human carbonic anhydrase (CA) gene family, both the ubiquitously expressed CAII and the muscle specific CAIII appear to have such CpG islands although erythrocyte-specific CAI does not. The CAII island is quantitatively more CpG rich than that of CAIII, with a CpG:GpC ratio of 0.94 compared with 0.82 for CAIII. Estimation of CpG:GpC ratios in the proximal-promoter regions of 44 vertebrate genes suggest that 40% of genes with tissue-specific or limited tissue distribution may show methylation-free CpG clusters in their promoter regions. In many cases the CpG:GpC ratio is less than that found in housekeeping genes and this may reflect variation in the interaction of CpG clusters with regulatory factors that define different patterns of tissue expression.     

Compositional compartmentalization and gene composition in the genome of vertebrates

Summary The compositional distribution of coding sequences from five vertebrates (Xenopus, chicken, mouse, rat, and human) is shifted toward higher GC values compared to that of the DNA molecules (in the 35–85-kb size range) isolated from the corresponding genomes. This shift is due to the lower GC levels of intergenic sequences compared to coding sequences. In the cold-blooded vertebrate, the two distributions are similar in that GC-poor genes and GC-poor DNA molecules are largely predominant. In contrast, in the warm-blooded vertebrates, GC-rich genes are largely predominant over GC-poor genes, whereas GC-poor DNA molecules are largely predominant over GC-rich DNA molecules. As a consequence, the genomes of warm-blooded vertebrates show a compositional gradient of gene concentration. The compositional distributions of coding sequences (as well as of DNA molecules) showed remarkable differences between chicken and mammals, and between mouse (or rat) and human. Differences were also detected in the compositional distribution of housekeeping and tissue-specific genes, the former being more abundant among GC-rich genes.     

Directional fixation of mutations in vertebrate evolution

Summary We have made pairwise comparisons between the coding sequences of 21 genes from coldblooded vertebrates and 41 homologous sequences from warm-blooded vertebrates. In the case of 12 genes, GC levels were higher, especially in third codon positions, in warm-blooded vertebrates compared to cold-blooded vertebrates. Six genes showed no remarkable difference in GC level and three showed a lower level. In the first case, higher GC levels appear to be due to a directional fixation of mutations, presumably under the influence of body temperature (see Bernardi and Bernardi 1986b). These GC-richer genes of warm-blooded vertebrates were located, in all cases studied, in isochores higher in GC than those comprising the homologous genes of cold-blooded vertebrates. In the third case, increases appear to be due to a limited formation of GC-rich isochores which took place in some cold-blooded vertebrates after the divergence of warm-blooded vertebrates. The directional changes in the GC content of coding sequences and the evolutionary conservation of both increased and unchanged GC levels are in keeping with the existence of compositional constraints on the genome.