Our lab studies novel genetic systems in microbial eukaryotes, combining a mechanistic and comparative approach to understanding genome evolution and diversity. Our research has shown that the surprisingly sophisticated variations on DNA and RNA processing in microbial eukaryotes create an imaginative playground for genome architecture and genetic systems. Some of their pathways erode the notions of a gene (e.g. scrambled genes and RNA editing) and even Mendelian inheritance, reminding us that a genome sequence can be a far cry from knowledge of its products. Genome-wide DNA rearrangements occur in diverse organisms, and contribute to many human diseases, including cancer, but their extreme exaggeration in ciliates, particularly Oxytricha, makes it an ideal model system to study the role of RNA in epigenetic control of genome remodeling. The laboratory is currently focused on understanding the mechanism and evolutionary origin of this remarkable phenomenon in Oxytricha.

Close-up of black and white oxytricha.

By combining molecular, evolutionary, theoretical, and synthetic, experimental biology, we discovered an RNA-guided epigenetic mechanism underlying programmed genome rearrangements (Nowacki et al. 2008 Nature; Fang et al. 2012 Cell), as well as the transitional, or putative intermediate, forms that this complex system passes through during development (Möllenbeck et al., 2008) and evolution (Chang et al. 2005 PNAS) plus its evolutionary origin from a novel mutualistic relationship with thousands of active transposons (Nowacki et al. 2009 Science). Nowacki et al. 2008 also unveiled new roles for RNA, a new class of long noncoding (lnc) RNAs, and a new mechanism for epigenetic wiring (and rewiring) of cellular programs, which research in yeast (Keskin et al. 2014, doi:10.1038/nature13682) suggests may be general to eukaryotes and could influence genome remodeling events that lead to cancer, as well (Fang and Landweber, 2013 and Fang et al. 2012 Biol Direct). We found that the lncRNAs in Oxytricha are also templates for RNA-guided DNA repair (Nowacki et al. 2008) and can regulate gene dosage and chromosome copy number (Nowacki et al. 2010 PNAS). More recently, we discovered an extensive cache of small RNAs (piRNAs) that protect DNA regions against loss or cleavage during genome rearrangement (Fang et al. 2012 Cell). Complementary to this discovery of such tiny guardians of the genome, we found that cytosine methylation or hydroxymethylation of DNA can mark DNA sequences for deletion during Oxytricha genome rearrangement (Bracht et al. 2012).

RNA-Guided Genome Editing

A key advancement in our lab has been a close examination of the molecular basis for programmed genome rearrangement in ciliates. During differentiation of its somatic macronucleus, Oxytricha trifallax destroys roughly 95% of its germline, severely fragmenting its chromosomes, and then descrambles hundreds of thousands of remaining fragments by translocation or inversion. Programmed DNA deletion, inversion, and permutation events provide the main experimental system we are using to explore the mechanism underlying complex genome rearrangements. Massive DNA rearrangements reconstruct a set of ~16,000 gene-sized ‘nanochromosomes’ (Swart et al., 2013; cover of PLoS Biology) for expression in Oxytricha’s somatic macronucleus. These DNA acrobatics include programmed loss of intergenic DNA as well as internal eliminated sequences within genes, accompanied by assembly and reordering of coding segments from precursor germline segments sometimes present in a scrambled, permuted order. Short sequence repeats at the junctions between coding and noncoding sequences facilitate reconstruction of intact chromosomes during development.

We proposed (Angeleska et al. 2007) and experimentally demonstrated (Nowacki et al. 2008) an epigenetic mechanism for DNA unscrambling. Our model requires the existence of long, maternal noncoding RNA templates, which contain complete copies of the previous generation’s reorganized genome, and these molecules guide assembly of cryptic germline fragments in a correct order and orientation. Nowacki et al. 2008 offered several lines of evidence for the transient existence and action of these lncRNAs. Either injection of foreign nucleic acids or feeding the cells bacteria expressing specific double-stranded RNA targeting the templates led to aberrant gene unscrambling in the progeny, blocking the process completely or producing incorrect DNA patterns. This exciting line of experiments provided us with the first opportunity to parse the specific steps in this complex biological process and to develop Oxytricha as a powerful model system. Microinjection of synthetic DNA or RNA templates, or even of small RNAs, can efficiently direct genome editing in Oxytricha, reprogramming DNA rearrangements for multiple sexual generations (Nowacki et al.2008 and Fang et al. 2012). This strongly supports our epigenetic model for genome-wide surveillance. For small RNAs, injection of synthetic piRNAs that correspond to normally-deleted regions leads to their retention across multiple generations (Fang et al. 2012), highlighting the role of small RNAs as powerful transgenerational carriers of epigenetic information for genome remodeling, and creating simpler forward genetic tools for gene knockout. Together, these experiments showcase Oxytricha as a model for RNA-mediated inheritance and revealed a new class of thousands of lncRNAs (Lindblad et al. 2017), as well as new roles for both lncRNAs and piRNAs in controlling somatic genome architecture. The experiments also offered a specific mechanism for the transmission of an acquired genetic state.

The Origin and Evolution of Scrambled Genes and Genome Architectures

A mirror image of oxytrichamirror cells.

In earlier work, we proposed and tested two different step-wise models for how single genes can become extensively fragmented and scrambled over evolutionary time (Chang et al. 2005; Wong and Landweber 2006). The data support a model of frequent germline recombination between coding and noncoding DNA, a trend that leads to exaggerated patterns and increased levels of genome scrambling and complexity over time (Chang et al. 2005). New work in the area of comparative genomics will allow us to ask how whole genomes can rearrange over evolutionary time to produce extremely scrambled germline genome architectures. Two essays in Science (Landweber 2007, 2008) discussed the evolutionary origin of “genomes in pieces” and whether the presence of such a shattered genome architecture might actually confer a selective benefit to some (mostly asexual) organisms. While one might not expect such a Rube Goldberg genetic architecture as Oxytricha’s to confer an adaptive benefit to the host organism, there may be hidden charms. For instance, in Oxytricha’s genetic system, there are two vehicles for inheritance: RNA and DNA. Rather than just the usual storage of genetic information in DNA, Oxytricha can also pass epigenetic information from parent to offspring in the form of RNA (Nowacki et al. 2008; Fang et al. 2012). While the conjecture that this mechanism confers a selective advantage has not been tested in the laboratory, it would be amenable to long-term evolution experiments.

Previous work discovered and characterized new scrambled genes and their orthologs in various species on a case by case basis (e.g. Kuo et al. 2006; Chang et al. 2005, 2006). The availability of a complete PacBio assembly for the O. trifallax germline genome (Chen et al. 2014 Cell) and our bioinformatic pipeline for analysis of precursor–product gene maps allows us to decipher the global patterns and process of rearrangement of its set of over 3,500 newly discovered scrambled genes. Comparative genomics will extend these tools to both closely- and distantly-related species so that we can examine how a lineage’s history allowed it to acquire such a complicated genome architecture. Comparative genomics over the time-course of development, itself, is also underway, to permit us to dissect the cascade of events that produce a new, rearranged genome in real time.

One Cell: Two Complex Genomes

Oxytricha’s macronucleus (Swart et al. 2013) contains over 16,000 tiny chromosomes averaging just 3_kb. Most encode single genes, amplified to ~2000 copies. The smallest chromosome is 469 bp and encodes a 98 aa protein, whereas the largest chromosome is 66 kb and encodes a single, enormous Titin-like protein. Alternative fragmentation of multi-gene chromosomes (containing up to 8 genes) produces isoforms with shared sequence and high levels of variation. Another remarkable feature of Oxytricha’s macronuclear genome is its inordinate fondness for telomeres, with over 60 million chromosome ends per macronucleus!

The sequencing and assembly of Oxytricha’s massively scrambled micronuclear germline genome (Chen et al. 2014) required a tour de force effort of experimental and computational biologists. No other sequenced genome bears the complexity of Oxytricha, with thousands of scrambled genes, many overlapping, and the ability to construct a somatic genome from a set of over 225,000 tiny DNA pieces scattered over long stretches of germline chromosomes. These gene pieces assemble to produce a new somatic genome during every round of sexual reproduction and nuclear development.

Ongoing experiments focus on both comparative genomics and on building and testing a model that combines our observations of the roles of small and long, noncoding RNAs, together with DNA and histone modifications and experimental studies of chromatin, to elucidate the cascade of events that take place during genome rearrangement. The field of epigenetics, particularly RNA-mediated epigenetic inheritance, is presently taking off, and our discoveries have been on the forefront of this discipline, revealing both new classes and functions of small and long, noncoding RNAs, and new roles for RNA in regulating a range of phenomena, from genome rearrangement to RNA-mediated DNA repair and gene dosage.

The genomic tools and functional approaches in our lab will pave the way for future studies of other species with extensive genome rearrangements, including non-ciliate microbial eukaryotes (Landweber, 2007) and disease models (Fang et al. 2013). The lab has worked on a range of non-canonical model systems, including trypanosomes (Landweber & Gilbert 2003, 2004) and slime molds (Horton & Landweber, 2000, 2002; Schaap et al. 2015) and occasionally more conventional model organisms (Liang et al. 2005 Genome Biol; Liang & Landweber, 2006, 2007; Cavalcanti et al. 2006; Fang et al. 2012 Biol Direct). We are also very keen to examine the possible roles of RNA-guided DNA rearrangement and repair (Nowacki et al. 2008) as setting the stage for diseases of the genome such as cancer (Fang et al. 2012 Biol Direct) that may be a direct consequence of genome instability and rogue DNA rearrangements. This is in striking contrast to Oxytricha’s precision control of genome reorganization (Bracht et al. 2013 Cell).

With our move to Columbia University, research in our lab continues to expand in several directions, including single-cell and single nucleus experiments, coupled with genome or proteome analysis. Mass spectrometry facilitates the discovery of proteins present in differentiating nuclei or that interact with key players during development (Fang et al. 2012). It has also confirmed expression of hundreds of germline-limited genes and their post-translational modifications (Chen et al. 2014). These offer candidates for roles in the genome rearrangement process. Furthermore, we continue to develop tools to improve the ease of manipulating Oxytricha cells for genome editing.

The combination of high-resolution genomic analyses and functional molecular experiments in Oxytricha and other species will lead to a broader understanding of the acrobatic processes of genome rearrangement that can accumulate in an organism over both evolutionary time or on the scale of somatic mutation. On a grand scale, these events at the genomic level contribute to the diversification of life on our planet, showcasing the astonishing range of genetic mechanisms available to eukaryotes.