As discussed in a recent post, during lamprey embryogenesis programmed genomic rearrangements lead to deletion of ~20% of the germline genome in the soma. Smith, Amemiya and co-workers have now published a follow-up study in which they further characterise the complement of deleted genes. Their findings have led them to hypothesise that the programmed genomic rearrangements (PGRs) serve to segregate pluripotency functions required in germline that could be deleterious in the soma.
Smith et al used a couple of different genomic techniques to identify somatically deleted sequences. Using microarrays constructed from available germline sequence, they found that ~13 % of the sampled sequence was deleted in the soma (in relative agreement with the ~20% derived from flow cytometry). Within this dataset, they identified 8 new single-copy/low-copy number sequences found only in the germline. RT-PCR showed that 5 of the novel sequences were expressed in germline cells. In situ hybridisation of one of these sequences showed that it was expressed in differentiating primordial germ cells in lamprey embryos.
The main limitation for identifying more genes subject to somatic deletion has been a lack of germline genomic sequence. Smith et al. performed high-throughput shotgun sequencing on lamprey sperm cells, generating short sequence reads covering ~10% of the germline genome. They then compared this dataset with the whole-genome sequence derived from somatic (liver) cells, yielding tens of thousands of putative deletion and recombination sites. A substantial part of the somatically deleted DNA corresponds to single-copy, protein-coding genes; the authors identified 246 instances of homology to individual human genes.
The problem with this comparison however, is that, by necessity, it was generated from 2 different individuals (of different sexes). This meant that apparent cases of deletion or recombination may be due to polymorphisms for insertion or deletion mutations present in lamprey populations. The researchers undertook validation experiments on a subset of the candidate deletion/recombination dataset (using PCR to amplify candidate sequences from testes and blood from 4 different males, blood from 4 different females, as well within an array of somatic tissue types within individuals). Of 48 tested candidate gene deletions or recombination events, they validated 7 sites of programmed deletion, and 3 recombination sites. They also identified 3 insertion/deletion polymorphisms, and 5 gaps in the somatic whole genome sequence. Due to PCR failures, or because of repetitive target sequences, 30 of the candidates were not informative. The validated gene deletions included APOBEC-1 complementation factor, encoding a protein involved in RNA editing, and the secreted developmental signalling molecule encoding WNT7A/B.
In the process of these validation experiments, Smith et al discovered short palindromic sequences at the deletion breakpoints. There was no specific consensus sequence at these positions, but the palindromes may indicate that the mechanism of chromatin diminution utilises site-specific recombination.
Another interesting finding of these experiments is that it appears that the programmed deletions are inherited uniformly throughout all the various somatic lineages. The earlier paper (discussed in the previous post) had suggested that different somatic tissues might have subtly different deleted portions. Microarray experiments, and comparison of the validated gene deletions between different tissues found no evidence of this, although this question may as yet not be answered definitively.
The crux of the paper rests on a computational comparison of ontology terms (in which homology is used to make predictions of cellular function, which are further sorted into broad categories). In the dataset of predicted gene deletions, certain ontologies were overrepresented with respect to the rest of the germline sequence; these included ‘regulation of gene expression’, ‘chromatin organisation’, and ‘development of germ/stem cells’.
Simply put, the paper has shown that a substantial number of protein-coding genes as well as repetitive sequences are deleted from the genomes of lamprey somatic cells. Many of the deleted genes are expressed in the germline, and often appear to have important regulatory functions. The crucial characteristics of the germline are the ability to undergo meiotic recombination, and totipotency. The missexpression of factors involved in these processes in the soma would be seriously detrimental; potentially resulting in aberrant cell fate specification, genome disruption, and hence cancers. The authors postulate that this conflict of interests between the germline and the soma underlies their genomic differentiation.
I find this an attractive and interesting hypothesis. As yet though, I don’t think the data is strong enough to have proved it. Gene ontology terms are relatively crude categorisations, and compounded with the question of what proportion of candidate deletions are bona fide, I’ll withhold judgement on the evolutionary rationale behind the deletions for the moment. Jeramiah Smith and colleagues are currently assembling the entire lamprey germline genome. Complete annotation of the deleted portion of the genome will certainly reveal the function of these fascinating genome rearrangements more clearly. I look forward to new studies investigating the mechanisms underlying the rearrangements, and their developmental progression. The extensive genome remodelling that occurs in ciliates utilises a combination of a small RNA/Argonaute system and domesticated transposase enzymes. I guess that analysis of any transposases encoded in the lamprey genome may be the place to start to unravel the mechanisms of chromatin diminution.
Smith JJ, Baker C, Eichler EE, & Amemiya CT (2012). Genetic consequences of programmed genome rearrangement. Current biology : CB, 22 (16), 1524-9 PMID: 22818913