Eukaryotes specify a genome to be inherited stably, enabling dynamic rearrangements and amplifications of other genomic elements

Laura Wegener Parfrey and Laura A. Katz

Summary

● The presumed constancy of eukaryotic genomes between generations is at odds with the observations of their being dynamic and varying extensively within a generation in some eukaryotic lineages.
● It appears likely that eukaryotes specify the portion of their genome to be inherited, either placing it in a separate germline nucleus or marking it through epigenetics.
● Both cyclic polyploidy and differential amplification elevate gene copy numbers and likely provide advantages to the organism as more templates become available for transcription, thus increasing metabolic efficiency.
● Microbial eukaryotes display diverse mechanisms for reducing ploidy levels and thus maintaining the fidelity of the inherited genome.


Many biologists assume that eukaryotic genomes are transmitted stably between generations with only minor variations. Yet, this presumed constancy is at odds with data indicating that eukaryotic genomes are dynamic, varying extensively in content among many different lineages. Thus, rather than being constant, genomes vary considerably within individuals during their lifetimes.

Despite the dynamic nature of eukaryotic genomes, it appears likely that eukaryotes specify the portion of their genomes that will be inherited, either by placing it in a separate germline nucleus, by marking it through epigenetics, or both. Epigenetic mechanisms such as methylation, histone acetylation, and genome scanning through RNAi may differentiate between the inherited and somatic genetic material within a single nucleus. Such a distinction between somatic and inherited genomes is key to enabling dynamic variation in genomes within life cycles and among individuals within populations, while also maintaining integrity of the genome between generations.

Much of our traditional view of genomes and their inheritance is based on insights from plants and animals, species whose cells carry a single nucleus and a relatively small number of chromosomes. Ploidy variation- changes in the number of whole genome complements in a nucleus-in these organisms is generally limited to fluctuations between haploid (one copy of each chromosome) and diploid (two copies) within life cycles. According to this view, genome content is maintained at a constant ploidy level during cell propagation by mitosis and cytokinesis, resulting in a population or a multicellular organism that is composed of genetically identical cells.

We focus on two genome modifications in which nuclear DNA content changes during the life cycle: cyclic polyploidy and differential amplification. During cyclic polyploidy, the number of whole genome copies increases, in some lineages by a factor of 1,000, and then decreases prior to reproduction such that each daughter cell inherits either one or two genome complements. In contrast, during differential amplification, the copy number for only portions of the genome increases, and these portions range in size from whole chromosomes to single genes. Both cyclic polyploidy and differential amplification elevate gene copy numbers and likely provide advantages to the organism as more templates become available for transcription, thus increasing metabolic efficiency.

Inherited Genome Concept Could Explain How Integrity Is Maintained

Microbial eukaryotes (Fig. 1) have many means for amplifying their genomes while still maintaining hereditary integrity. We use a phylogenetic framework to highlight examples of genomic features across the eukaryotic tree of life along with diverse mechanisms for resetting the inherited genome (Fig. 2 and 3). The broad distribution of these features suggests that a shared mechanism for distinguishing between inherited and somatic genomes enables the evolution of dynamic genomes among diverse eukaryotes.

To bridge the gulf between the traditional and dynamic views of genomes, we propose the inherited genome concept for eukaryotes. This concept involves organisms marking their inherited genome so that it may be transmitted to the next generation with its integrity intact.

Eukaryotic genomes transmit information both genetically, through DNA via its nucleotide sequence, and epigenetically through chemical changes to that DNA or its associated histone proteins. There is increasing recognition that epigenetic processes play important roles in regulating activities within the nucleus, such as heterochromatin formation and transcription levels. We suspect that epigenetic mechanisms also play a role in marking inherited genomes. Once the inherited genome is marked, the somatic genome becomes free to vary in copy number and composition.

We use somatic here to refer to the genome that is used for body cells as opposed to the germ line or inherited material. This distinction between inherited and somatic genomes is unmistakable in animals where the inherited genome is sequestered within germline cells early during embryonic development, while the somatic genome varies among cell types. Indeed, within humans and other mammals, genomes become polyploid in specialized cells such as megakaryocytes and liver cells, while they are dramatically rearranged in immune cells (Fig. 3B). Further, many other eukaryotes have mechanisms for marking their inherited genome without necessarily sequestering them in specialized germ cells, as described below.

Processed Genomes in Ciliates

Genetic material in ciliates (Fig. 1F) is divided between a silenced germline micronucleus and a transcriptionally active macronucleus, each of which is found within the same cell. The micronucleus behaves like a canonical nucleus. It has a relatively small number of large chromosomes that divide by mitosis during asexual reproduction and by meiosis leading up to sexual reproduction. Ciliates exchange meiotic products of the micronucleus during sexual reproduction, and these fuse to form a zygotic nucleus. The zygotic nucleus then divides to produce a new micronucleus and, after a period of genome rearrangement, a macronucleus. During macronuclear development the large micronuclear chromosomes are fragmented, micronuclear-limited sequences are excised, and the remaining material is amplified to yield as many as 25 million gene-sized chromosomes in some lineages. This processing results in numerous small chromosomes with most noncoding sequences removed (Fig. 3A).

With the inherited genome sequestered in the micronucleus, ciliates dramatically modify their macronuclear genome, which may offer selective benefits. For example, we find that elevated rates of protein evolution correlate with the extent of chromosomal processing. Thus, ciliates with extensively processed chromosomes have more divergent proteins than those lineages that undergo limited processing.

The micronuclear genome is not seen by selection because it is transcriptionally inactive, perhaps allowing ciliates to explore protein space and accumulate potentially beneficial compensatory mutations. Finally, processing of the macronuclear genome confers the advantages of polyploidy without the necessity of returning to the inherited genome.

Foraminifera Undergo Complex Nuclear Events

Foraminifera (Fig. 1C) undergo complex nuclear events, sometimes greatly modifying their genomes during vegetative growth but then passing only their inherited genome to the next generation. The life cycle of Foraminifera alternates between uninucleate and multinucleate adults that biologists traditionally assumed to be haploid and diploid, respectively. However, events that occur before reproduction in the uninucleate phase suggest that both polyploidy and differential amplification occur during development.

Consider the dynamics within the nucleus during the uninucleate stage. The single nucleus, which arises from meiosis, is haploid. According to classic accounts of Foraminifera, this nucleus remains haploid while the cell and nucleus grow tremendously-in some species, the nucleus can expand to 400 μm in diameter. Shortly before reproduction, the nucleus enlarges, while DNA and other material within it condense into granules. These granules are subsequently degraded in a process referred to as Zerfall.

The ability of Foraminifera to degrade portions of their nuclear DNA prior to reproduction indicates that DNA is amplified during the growth of the nucleus and the amplified DNA can be distinguished from the inherited genome. Zerfall, aptly described as nuclear cleansing, appears to facilitate a return to the inherited genome before reproduction.

Following Zerfall, the single nucleus divides rapidly by mitosis into hundreds or sometimes thousands of daughter nuclei, demonstrating that this single nucleus must be polyploid. There is no detectable DNA synthesis during the proliferation of daughter nuclei in the only species in which it was measured. The details of this proliferation vary from one species to another, but all indicate polyploidy of the parental nucleus. Thus, it is likely that multiple whole genome copies are produced by polyploidization of the parental nucleus during growth, and that these genome copies segregate into daughter nuclei during reproduction. Other loci are also amplified extrachromosomally in eukaryotes. Foraminifera extensively modify their genome content and have developed at least one mechanism, Zerfall, to return to the inherited genome.

Phylogenetic Diversity of Dynamic Genomes

Beyond the specific life cycles that are the focus of work in our lab, differentiation between inherited and somatic genomes is apparent in other lineages (Fig. 1). Even though data are limited, these lineages appear to be widespread across the eukaryotic tree of life (Fig. 2). The number of amplified loci ranges from the whole genome in ciliates to ribosomal DNA in many other lineages. This broad distribution indicates that the ability to recognize the inherited genome selectively is widespread among eukaryotes.

Ribosomal DNA is the most commonly amplified locus, including among fungi, animals, Entamoeba, slime molds, ciliates, and Euglena. In many cases, amplification of the chromosomal rDNA locus leads to extrachromosomal copies of rDNA. These may be circular, as in animals and Euglena (Fig. 1E), or linear as in ciliates and the slime mold Dictyostelium (Fig. 1B and 3C).

Extrachromosomal copies are presumably excluded from the inherited genome because they lack chromosome features (such as kinetochores) necessary for making it into daughter cells. In contrast, Entamoeba histolytica (Fig. 1H), the causative agent of amoebiasis (or dysentery), contains no chromosomal copy of rDNA. Instead, rDNA is located on up to 200 circular plasmid-like molecules that are maintained at variable copy numbers during the life cycle of Entamoeba and that are inherited through an unknown mechanism.

Other loci are also amplified in eukaryotes. For instance, Sarit Cohen and colleagues at Tel Aviv University in Israel recently demonstrated that extrachromosomal DNA is generated from multiple loci, including loci for ribosomal DNA, in several animal and plant taxa. The unifying feature of the amplified loci is that they are arranged as tandem repeats on chromosomes (Fig. 3D). Extrachromosomal circles of DNA are generated by DNA repair mechanisms, and can replicate independently of the chromosomes. This mechanism for differential amplification may be universal throughout eukaryotes.

Dynamic processes that resemble Zerfall are also found in Polycystinea (a lineage formerly called radiolaria) and Gromia, both of which are large amoebae closely related to Foraminifera. These lineages discard and degrade DNA before reproduction, again suggesting that a portion of the genome was amplified before that phase. As in Foraminifera, the identity of the degraded DNA is unknown, but ribosomal DNA and other highly expressed loci are good candidates.

Cyclic Polyploidy Arose Several Times

Polyploidy arose numerous times in eukaryotes (Fig. 2). Terminal polyploidy is common in differentiated tissues of multicellular organisms such as plants and animals. In these cases, polyploidy is not reduced and these cells eventually die, while the sequestered germline cells give rise to subsequent generations. In contrast, if polyploid singlecells failed to transmit their genome, they would be evolutionary dead ends. Hence, microbial eukaryotes have numerous mechanisms to reduce ploidy levels and to maintain the fidelity of their inherited genome. Cyclic polyploidy is described in members of the radiolaria, Foraminifera, oxymonads, and Apicomplexa.

Radiolaria are a polyphyletic assemblage of large marine amoebae whose ornate shells were drawn by Haeckel. They fall into three clades on the basis of molecular analysis and differences in shell composition: the Phaeodarea, Polycystina, and Acantharea. All become polyploid during their life cycle and then segregate these genome copies into gametic nuclei. The resulting biflagellate cells are described as spores or swarmers, but are likely gametes that will not complete their life cycle in culture. The details of gametogenesis differ among members of these groups, which diverged at least 550 million years ago. In each case, however, the result is a return to the inherited genome during reproduction.

Phaeodarea (Fig. 1D) have a single polyploid nucleus with roughly 1,000 composite chromosomes (Fig. 3E). Prior to reproduction, these composite chromosomes condense and are segregated along microtubules into secondary nuclei. Subsequently, the composite chromosomes break down into 10-12 smaller chromosomes, presumably the inherited genome complement, and divide mitotically to give rise to gametic nuclei.

In Polycystina (Fig. 1G), gametogenesis begins with nucleoli and other peripheral nuclear material pinching off from the nucleus and being degraded in a Zerfall-like process. Then intranuclear microtubule organizing centers (MTOCs) form at intervals along the nuclear envelope, and chromosomes condense onto these MTOCs. Each MTOC and its associated chromosomes give rise to a secondary nucleus, which divides mitotically to produce gametes.

The oxymonad Pyrsonympha (Fig. 1A) is a symbiont of termites and is very large for a flagellate, up to 200 μm in length. Pyrsonympha becomes polyploid as it grows. Upon reproduction, whole chromosomes sets are segregated into numerous daughter nuclei, thereby returning to the inherited genome. The homologous chromosomes are connected by synaptonemal complexes that aid in distributing them to daughter nuclei. The presence of synaptonemal complexes, which are commonly found during meiosis, is intriguing as they may be evidence of meiosis occurring within the polyploid nucleus. Apicomplexa are a diverse lineage of intracellular parasites and include the causative agents of human diseases such as malaria. Apicomplexa have complex life cycles that often include cyclic polyploidy. For example, during growth the genome and microtubule organizing centers in Sarcocystis neuorna, which infects horses, replicate six times without nuclear division, leading to a nucleus with 32 genome copies and MTOCs. Subsequently, Sarcocystis segregates these copies of the inherited genome into daughter nuclei.

Since dynamic genome processes are widespread, eukaryotes must have evolved a mechanism to mark their inherited genome. Such a distinction between somatic and inherited genomes may be key in enabling the variation observed within life cycles while also maintaining integrity of the genome between generations.

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