John R. Bracht

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

The Architecture of a Scrambled Genome Reveals Massive Levels of Genomic Rearrangement during Development

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

Beyond transcriptional silencing: Is methylcytosine a widely conserved eukaryotic DNA elimination mechanism?

Methylation of cytosine DNA residues is a well‐studied epigenetic modification with important roles in formation of heterochromatic regions of the genome, and also in tissue‐specific repression of transcription. However, we recently found that the ciliate Oxytricha uses methylcytosine in a novel DNA elimination pathway important for programmed genome restructuring. Remarkably, mounting evidence suggests that methylcytosine can play a dual role in ciliates, repressing gene expression during some life‐stages and directing DNA elimination in others. In this essay, I describe these recent advances in the DNA methylation field and discuss whether this unexpected novel role for methylcytosine in DNA elimination might be more widely conserved in eukaryotic biology, particularly in apoptotic pathways.

Chromosome fusions triggered by noncoding RNA

ABSTRACT Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

Programmed Genome Processing in Ciliates

The ciliates are a group of protists distinguished by the hair-like cilia on their cell surfaces. Ciliates also possess two types of nuclei, a germline micronucleus and a somatic macronucleus. The micronuclear genome contains segmented genes divided by spacer sequences of DNA that are removed to generate the macronuclear genome during development. For some species, certain micronuclear gene segments can be reordered and/or inverted with respect to their final gene sequence in the macronucleus. This chapter explores the similarities of and differences between micronuclear genomes and the processes of macronuclear development across different ciliate species.

Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

Biochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryote Oxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-length Oxytricha chromosomes in vitro and use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks from Oxytricha chromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcases Oxytricha chromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates. Highlights De novo synthesis of complete, epigenetically defined Oxytricha chromosomes Epigenomic profiles of chromatin organization in Oxytricha’s miniature chromosomes m6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomes Histone acetylation and chromatin remodelers temper the impact of m6dA on chromatin

How Do Cysts Know When to Hatch? The Role of Ecological Communication in Awakening Latent Life

When environmental conditions deteriorate, many ciliates enter a state of suspended animation known as a resting cyst. The cyst is a survival strategy: protected within a thick cell wall, the organism can survive harsh conditions and emerge once conditions improve. A significant literature describes the formation of cysts in the laboratory but surprisingly little is known about the signals that reawaken the dormant organisms. In this review, we discuss signals that awaken cysts in the context of their environment and ecology, and integrate these findings into a conceptual framework. We also discuss the role of cysts (and their awakening signals) in the pathogenicity of human parasitic protozoan diseases: Giardia, Plasmodium, Toxoplasma, Cryptosporidium, Balantidium, and Entamoeba.

Abstract A1-28: Chromosome fusions triggered by noncoding RNA

Chromosomal fusion is a frequent occurrence in cancer cells and can promote transformation through the generation of oncogenic gene products. There are two non-exclusive models for how these chromosome fusions may occur: (1) the instability of the cancer genome produces chromosomal fusions randomly and those promoting transformation enjoy a selective advantage, or (2) some non-random mechanism drives chromosome fusion. Evidence for (2) comes from recurrent fusion events and correlative human data linking noncoding RNA expression with chromosome fusion. To help address this important fundamental question, we functionally tested the role of noncoding RNA, specifically piRNAs, in promoting chromosomal fusion in the ciliate, Oxytricha trifallax , a microbial eukaryote. We microinjected custom piRNAs into cells and observed chromosome fusion in four cases (of four tested), including a double fusion event and a chromosome circularization event (self-fusion). Furthermore, these RNA-mediated inter-and-intra-chromosomal fusions are heritable over multiple sexual generations, illustrating the ability of noncoding RNAs to program genome architecture, including chromosome fusion and circularization. Citation Format: John Bracht, Xing Wang, Keerthi Shetty, Xiao Chen, Mariusz Nowacki, Sierra McCloud, Derek Clay, Laura Landweber. Chromosome fusions triggered by noncoding RNA. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-28.

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.

Background Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells.