Douglas L. Chalker

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

ABSTRACT Abundant ∼28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (ΔDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These ΔDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation ΔDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, ΔDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA.

Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila. These deletion elements are usually not detected in macronuclear chromosomes. We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation. Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei. Little or no interference with the deletions of adjacent elements or of unlinked elements was observed. The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion. This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner. Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements. The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.

DNA Elimination in Ciliates: Transposon Domestication and Genome Surveillance

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.

Nucleus-Specific Importin Alpha Proteins and Nucleoporins Regulate Protein Import and Nuclear Division in the Binucleate Tetrahymena thermophila

ABSTRACT The ciliate Tetrahymena thermophila, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how Tetrahymena can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro- and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) α- and 11 imp β-like proteins revealed that imp α-like proteins are nucleus specific—nine localized to the germ line micronucleus—but that most imp β-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp α adapters. The different imp α proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the IMA10 gene encoding an imp α-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in Tetrahymena are regulated by diverse, specialized karyopherins.

Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila.

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3′ exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena.

Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement.

Dynamic nuclear reorganization during genome remodeling of Tetrahymena.

The single-celled ciliate Tetrahymena thermophila possesses two versions of its genome, one germline, one somatic, contained within functionally distinct nuclei (called the micronucleus and macronucleus, respectively). These two genomes differentiate from identical zygotic copies. The development of the somatic nucleus involves large-scale DNA rearrangements that eliminate 15 to 20 Mbp of their germline-derived DNA. The genomic regions excised are dispersed throughout the genome and are largely composed of repetitive sequences. These germline-limited sequences are targeted for removal from the genome by a RNA interference (RNAi)-related machinery that directs histone H3 lysine 9 and 27 methylation to their associated chromatin. The targeting small RNAs are generated in the micronucleus during meiosis and then compared against the parental macronucleus to further enrich for germline-limited sequences and ensure that only non-genic DNA segments are eliminated. Once the small RNAs direct these chromatin modifications, the DNA rearrangement machinery, including the chromodomain proteins Pdd1p and Pdd3p, assembles on these dispersed chromosomal sequences, which are then partitioned into nuclear foci where the excision events occur. This DNA rearrangement mechanism is Tetrahymenas equivalent to the silencing of repetitive sequences by the formation of heterochromatin. The dynamic nuclear reorganization that occurs offers an intriguing glimpse into mechanisms that shape nuclear architecture during eukaryotic development.

FLANKING REGULATORY SEQUENCES OF THE TETRAHYMENA R DELETION ELEMENT DETERMINE THE BOUNDARIES OF DNA REARRANGEMENT

ABSTRACT In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the leftcis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymenarearrangement.

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

ABSTRACT Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena ΔDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

LIA5 is required for nuclear reorganization and programmed DNA rearrangements occurring during tetrahymena macronuclear differentiation.

During macronuclear differentiation of the ciliate Tetrahymena thermophila, genome-wide DNA rearrangements eliminate nearly 50 Mbp of germline derived DNA, creating a streamlined somatic genome. The transposon-like and other repetitive sequences to be eliminated are identified using a piRNA pathway and packaged as heterochromatin prior to their removal. In this study, we show that LIA5, which encodes a zinc-finger protein likely of transposon origin, is required for both chromosome fragmentation and DNA elimination events. Lia5p acts after the establishment of RNAi-directed heterochromatin modifications, but prior to the excision of the modified sequences. In ∆LIA5 cells, DNA elimination foci, large nuclear sub-structures containing the sequences to be eliminated and the essential chromodomain protein Pdd1p, do not form. Lia5p, unlike Pdd1p, is not stably associated with these structures, but is required for their formation. In the absence of Lia5p, we could recover foci formation by ectopically inducing DNA damage by UV treatment. Foci in both wild-type or UV-treated ∆LIA5 cells contain dephosphorylated Pdd1p. These studies of LIA5 reveal that DNA elimination foci form after the excision of germ-line limited sequences occurs and indicate that Pdd1p reorganization is likely mediated through a DNA damage response.