Foraminifera as a model of eukaryotic genome dynamism.

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Regional mutational signature activities in cancer genomes.

Cancer genomes harbor a catalog of somatic mutations. The type and genomic context of these mutations depend on their causes and allow their attribution to particular mutational signatures. Previous work has shown that mutational signature activities change over the course of tumor development, but investigations of genomic region variability in mutational signatures have been limited. Here, we expand upon this work by constructing regional profiles of mutational signature activities over 2,203 whole genomes across 25 tumor types, using data aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium. We present GenomeTrackSig as an extension to the TrackSig R package to construct regional signature profiles using optimal segmentation and the expectation-maximization (EM) algorithm. We find that 426 genomes from 20 tumor types display at least one change in mutational signature activities (changepoint), and 306 genomes contain at least one of 54 recurrent changepoints shared by seven or more genomes of the same tumor type. Five recurrent changepoint locations are shared by multiple tumor types. Within these regions, the particular signature changes are often consistent across samples of the same type and some, but not all, are characterized by signatures associated with subclonal expansion. The changepoints we found cannot strictly be explained by gene density, mutation density, or cell-of-origin chromatin state. We hypothesize that they reflect a confluence of factors including evolutionary timing of mutational processes, regional differences in somatic mutation rate, large-scale changes in chromatin state that may be tissue type-specific, and changes in chromatin accessibility during subclonal expansion. These results provide insight into the regional effects of DNA damage and repair processes, and may help us localize genomic and epigenomic changes that occur during cancer development.

Epigenetic influences of mobile genetic elements on ciliate genome architecture and evolution.

Mobile genetic elements (MGEs) are transient genetic material that can move either within a single organism's genome or between individuals or species. While historically considered "junk" DNA (i.e., deleterious or at best neutral), more recent studies reveal the potential adaptive advantages MGEs provide in lineages across the tree of life. Ciliates, a group of single-celled microbial eukaryotes characterized by nuclear dimorphism, exemplify how epigenetic influences from MGEs shape genome architecture and patterns of molecular evolution. Ciliate nuclear dimorphism may have evolved as a response to transposon invasion and ciliates have since co-opted transposons to carry out programmed DNA deletion. Another example of the effect of MGEs is in providing mechanisms for lateral gene transfer (LGT) from bacteria, which introduces genetic diversity and, in several cases, may drive ecological specialization in ciliates. As a third example, the integration of viral DNA, likely through transduction, provides new genetic materials and can change the way host cells defend themselves against other viral pathogens. We argue that the acquisition of MGEs through non-Mendelian patterns of inheritance, coupled with their effects on ciliate genome architecture and persistence throughout evolutionary history, exemplify how the transmission of mobile elements should be considered a mechanism of transgenerational epigenetic inheritance.

DAPI staining and DNA content estimation of nuclei in uncultivable microbial eukaryotes (Arcellinida and Ciliates).

Though representing a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of genome structures and changes in genome content over the often-complex life cycles of these lineages remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4',6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates, and the measurements of target organisms were compared to those  of well-known model organisms.The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.