Genetic consequences of programmed genome rearrangement

Abstract

The lamprey (Petromyzon marinus) undergoes developmentally programmed genome rearrangements that mediate deletion of∼20% of germline DNA from somatic cells during early embryogenesis. This genomic differentiation of germline and soma is intriguing, because the germline plays a unique biological role wherein it must possess the ability to undergo meiotic recombination and the capacity to differentiate into every cell type. These evolutionarily indispensable functions set the germline at odds with somatic tissues, because factors that promote recombination and pluripotency can potentially disrupt genome integrity or specification of cell fate when misexpressed in somatic cell lineages (e.g., in oncogenesis). Here, we describe the development of new genomic and transcriptomic resources for lamprey and use these to identify hundreds of genes that are targeted for programmed deletion from somatic cell lineages. Transcriptome sequencing and targeted validation studies further confirm that somatically deleted genes function both in adult (meiotic) germline and in the development of primordial germ cells during embryogenesis. Inferred functional information from deleted regions indicates that developmentally programmed rearrangement serves as a (perhaps ancient) biological strategy to ensure segregation of pluripotency functions to the germline, effectively eliminating the potential for somatic misexpression.

Figure 1. Summary of germline-specific sequence and gene discovery using arrayCGH

(A) Germline-enriched sequences were identified by comparing observed relative hybridization intensities to a normal distribution with the same number of sampled regions (N) and standard deviation (s.d.). The y-axis is plotted on log₁₀ scale in order to magnify differences at the tails of the distribution. All previously discovered germline-specific sequences [1] (marked by arrows and brackets) and several additional germline-specific sequences were identified in this assay. (B) Examples of PCR validation of single-copy sequences eliminated from soma and their expression in the germline. Sequences are present in testes gDNA (genomic DNA) but absent from blood gDNA. These same fragments can be amplified from testes cDNA, but not from the source RNA (a control for gDNA contamination) or reagent blank. S = Sperm, B = Blood, A = Adult testes, J = Juvenile testes, RB = Reagent Blank, M = 100 bp DNA Ladder.

Figure 2. Expression of a germline-specific marker in embryonic germline

In situ hybridization of an antisense probe of the germline-specific gene 25M04 (putative KRAB domain zinc finger protein) reveals expression in the developing germline cells at day 14 (A, B, I) and day 20 (C, D, J) post-fertilization. Punctate staining reveals specific expression in the presumptive primordial germ cells (PGCs). Staining of PGCs is not observed in embryos that were hybridized with the sense strand probe (E–F), but some background staining is observed due to the presence of non-cellular endogenous alkaline phosphatase activity in the developing gut, pharynx, notochord and otic capsule. Panels B, D, F, and H correspond to the circumscribed regions in A, C, E, G, respectively. Panels I and J are transverse sections of the embryos shown in panels A and C. Sections have been counterstained with eosin in order to enhance contrast; arrows mark the location of PGCs positive for the 25M04 marker. This expression pattern suggests that 25M04 is involved in some aspect of PGC differentiation and/or migration. Nc = notochord, Nt = neural tube, Y = yolk.

Figure 3. Analysis of pilot 454 sequencing data

(A) All 454 reads were categorized on the basis of alignment patterns with the complete lamprey WGS dataset (liver DNA). A majority (82%) of reads appeared as “normal” DNA (multicopy or single-copy). Other alignment patterns were consistent with coverage gaps in the WGS dataset (3.4%), germline-specific DNA (7.6%) or recombination breakpoints (0.66%). Green circles depict the positions of alignment breaks and green arrows depict the generic locations of primer binding sites for validation PCRs. (B) Results of PCR validations of germline-specific/gene-containing (BLAST hit) reads and breakpoint-flanking reads provided positive validation of members of both rearrangement classes and identified segregating (in the population) insertion/deletion (InDel) polymorphisms and WGS coverage gaps, which mimic programmed rearrangement outcomes. Note, the “Germline-Specific” and “Breakpoint” classes result in similar PCR validation patterns because one primer (breakpoint) or both primers (germline-specific) are designed to germline-specific regions. T – template is testes DNA, B – template is blood DNA, M = 100 bp DNA ladder. (C) Overrepresented gene ontologies from 234 predicted germline-specific genes, relative to the entire 454 dataset (p>1e-8, corrected using false discovery rate control, as implemented by Blast2Go [9]).

Figure 4. Sequence of PCR validated breakpoint regions

Breakpoints contain short 5′/3′ palindromes (green) at the junction between somatically retained (blue) and germline-specific (red) sequence. The breakpoint of junction 2 contains an imperfect 5′/3′ palindrome. It is as-yet unclear if these are functionally related to programmed genome rearrangement.