Differentiated tissues are elaborated during mammalian ontogeny by the coordinated sequential execution of cell type-specific gene expression programs. A common supposition is that basic inherited genomic structure remains constant during this process. This supposition is critically important for the recently developed procedures for obtaining induced pluripotent stem cells from somatic tissues, with great potential for clinical applications.
Is this supposition comprehensively true? Would it not make sense, in terms of energy conservation, to program the necessary changes permanently through alterations in genome structure? Several special-case examples of such purposeful, site-specific manipulation of primary genome structure have long been known to exist Citation[1,2] and have obvious functional consequences during a specific life-cycle stage. These are most common in unicellular organisms but also occur in higher organisms, for example, amplification of specific genes for rapid production of proteins, such as those of the chorion of insect eggs, and rearrangements to produce diversification of variant surface glycoprotein in trypanosomes. Vertebrates also utilize genomic rearrangement to enhance diversity of antigen receptors, through the action of the DNA sequence-specific V(D)J recombinases.
Evidence is emerging that programmatic modification of the primary structure of the inherited genome may in fact be common, during development and in adult tissues. Structural genetic differences are known to occur between cell populations within an individual, including individual humans, constituting somatic mosaicism Citation[3]. These differences, though frequent, have been interpreted as abnormalities, resulting from uncorrected somatic mutations, happening spontaneously or from environmental insult.
Mammalian brains are somatic mosaics, due to aneuploidy and genomic copy number variations. Recently, it also has been found that active transposable elements, long interspersed nuclear elements 1 (LINE-1) in neuronal tissue, contribute to the mosaicism, with tissue specificity Citation[4,5]. LINE-1 elements influence chromosome integrity and gene expression. Retrotransposition activity for the LINE-1 elements was strong during neuronal differentiation and occurred with high frequency. Activity was also found in the adult brain. Regulatory events leading to LINE-1 activation were elucidated Citation[6]. These observations led to the proposition that LINE-1 element transposition has a purpose: adaptive increase in neuronal variation and plasticity and in brain-controlled phenotypes Citation[7]. However, specific sites of insertion will need to be located to confirm this idea.
Another line of evidence, implicating the occurrence of developmentally-programmed genomic structural changes, comes from a recent study of the gene for ribosomal DNA (rDNA) during ontogeny in the mouse Citation[8]. There are hundreds of copies of rDNA in each cell on several chromosomes. The rDNA of the mice was found to present several SNPs in promoter regions of the gene. The relative percentages of these variant SNPs, indicative of rDNA structural status, were determined in sperm, embryos and two differentiated tissues, lung and liver, using a high-throughput quantitative pyrosequencing technique. The percentage of the variants changed in the differentiated tissues: in the males they differed significantly in lung and liver compared with sperm, and in the females they differed in lung compared with liver. Second, within-litter variances were calculated. These variances should be constant throughout development, if the genomic ratio of rDNA SNPs is established at fertilization. By contrast, for each of the five rDNA SNP variants, within-litter variances were significantly (p < 0.0001) greater for lung, liver and sperm than for embryos Citation[8]. Thus, there was tissue-specific developmental adjustment in the structure of the rDNA. This phenomenon is uniquely different from previously reported specific structural genomic changes noted above. Rather than pertaining only to a particular stage of development or class of proteins, or a noncoding repetitive element, rDNA is an ubiquitously expressed gene that is of central importance to the growth and energy economy of all cells Citation[9]; rRNA biogenesis is rate-limiting for ribosome production and protein synthesis.
Are the nonrandom shifting ratios of rDNA variants during differentiation of mouse tissues of functional significance? Information on this possibility was limited but suggestive. The rDNA SNP sites in the main promoter all have potential involvement in control of expression of the gene. The -218 and -178 sites are located within the repetitive enhancer elements. The -178 site is only a few bases downstream from the termination of the RNA transcript initiated at the spacer promoter, and a few bases upstream from the binding site for transcription termination factor-1, a critical regulator of rDNA silencing and expression. The -104 SNP is within the sequence for the regulatory promoter RNA (pRNA) Citation[10].
Methylation of specific CpG sites is a critical aspect of rDNA expression and regulation Citation[11]. In the mouse tissues, the degree of methyation of a concentration of CpG sites in one of the rDNA promoters showed ontogenetic regulation, with the highest methylation in sperm, and was higher in lung than in liver tissues Citation[8]. There was a significant negative or positive correlation between the percentage of CpG methylation and the percentage of one of the rDNA variants in the majority of tissues. Interestingly, the percentage of the variant, which showed the most prominent ontogeny-related shift in relative amounts, was strongly correlated with the percentage of CpG methylation in all differentiated tissues, but less so in embryos. In an in vitro transcription assay Citation[8], the mouse rDNA ontogenetic variants were differentially methylated, and differentially transcribed. Thus, genomic and epigenetic modulations may work hand-in-hand during tissue differentiation.
These results suggest that the structural shifting of the rDNA SNP copies during ontogeny is functionally purposeful; more research will be required to confirm this. Published reports indicate that functional differences among rDNA variants are plausible. Recently, polymorphisms in the size of the hypervariable region, just upstream from the main promoter region of rDNA, have revealed that the intergenic rRNA, transcribed from the spacer promoter, is derived from a subclass of variants Citation[12]. Several mouse rDNA variants with SNPs in the transcribed region had tissue specific expression Citation[13], and SNP profiles in the rDNA promoter region were associated with the binding of specific regulatory factors Citation[14].
The mechanism of the variant- and tissue-specific genomic shifts in rDNA is an intriguing question. Expression of the V(D)J recombinase, RAG, has recently been described in the brain, leading to speculation that site-specific recombination could have functional significance beyond the immune system Citation[15]. There is one perfect CACAGTG and four CACTGTG sites in murine rDNA (GenBank BK000964) for potential recognition by the RAG recombinase. Nonconsensus substitutions at positions four or five of the RAG recognition sequence destabilized but did not prevent RAG binding, with retention of potential for recombination Citation[16]. The RAG complex can target DNA without the canonical heptamer nearby, by engaging HMGB1/2 activity Citation[17], such as might be provided by upstream binding factor, the master regulator of rDNA chromatin conformation. In addition, sequence-specific recombination in rDNA may occur as a result of p53 binding Citation[18], and sequence-specific cleavage of rDNA by topoisomerases has been noted Citation[19,20].
The ontogenetic shifts in ratios of rDNA variants and the putative functional consequences of these shifts obviously may be a component of the required close regulation of rRNA production, in the context of cell economy and growth. In addition, there are hints that rDNA and rRNA may have wider roles. It has been proposed that rDNA, due to its high intrinsic instability, plays a global role in maintenance of overall genome stability Citation[21]. In Drosophila, rDNA copy number influenced degree of heterochromatin for unlinked genes Citation[22]. Biogenesis of rRNA has been implicated in some diverse human outcomes, including suicide Citation[23] and Down‘s syndrome Citation[24]. In humans, sizes of rDNA clusters showed a very high degree of variability and essentially complete heterozygosity in white blood cells Citation[25]. There was a high rate of meiotic recombination and evidence for somatic recombination in approximately a third of individuals. Furthermore, human rDNA gene clusters have been discovered to be recombinational hotspots in cancer: half of lung or colorectal cancers had rDNA rearrangements relative to surrounding tissues or blood Citation[26]. rRNA is strongly implicated in cancer Citation[27] and is currently a target in anticancer therapeutic design Citation[28]. In human lung cancer cell lines, there was a negative correlation between a SNP in the 5´ leader of rRNA and levels of a ncRNA Citation[29]. Might there be disease-related variant – and/or cell-type specificity – in these rDNA polymorphisms and recombination phenomena?
A final question is whether these observations of rDNA are specific to this particular gene, or might other ubiquitously expressed genes also undergo specific, functionally relevant genomic structural evolutions during ontogeny? This could especially be likely for those present in multiple copies with a high likelihood for recombination, such as repetitive elements that have acquired regulatory roles as promoters, enhancers, splice sites and insulators. But the special case of V(D)J recombinase in the immune system illustrates that any gene could, in theory, be subject to ontogenetic structural processing. This possible new dimension for developmental genetics deserves further exploration, particularly in view of its implications for stem cell usage and for cancer development.
Financial & competing interests disclosure
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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References
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