Epigenetic Control of Chromosome Breakage at the 5′ End of Paramecium tetraurelia Gene A

Abstract

Macronuclei and micronuclei of ciliates have related genomes, with macronuclei developing from zygotic micronuclei through programmed DNA rearrangements. While Paramecium tetraurelia wild-type strain 51 and mutant strain d48 have the same micronuclear genome, qualitative differences between their macronuclear genomes have been described, demonstrating that programmed DNA rearrangements could be epigenetically controlled in ciliates. Macronuclear chromosomes end downstream of gene A (A51 Mac ends) and at the 5 end of gene A (Ad48 Mac ends) in strains 51 and d48, respectively. To gain further insight into the process of chromosome end formation, we performed an extensive analysis of locus A rearrangement in strains d48 and 51, in strain d12, which harbors a gene A deletion, and in interstrain cross progeny. We show that (i) allele A d12 harbors a deletion of >16 kb, (ii) A51 Mac ends distribute over four rather than three DNA regions, (iii) strains d48 and 51 display only quantitative differences (rare Ad48 and A51 Mac ends do form in strains 51 and d48, respectively), (iv) the level of A51 Mac ends is severalfold enhanced in d12-and d48-derived progeny, and (v) this level inversely correlates with the level of Ad48 Mac ends in the d48 parent. Together, these data lead to a model in which the formation of Ad48 Mac ends is epigenetically controlled by a d48 factor(s). We propose that the d48 factor(s) may be derived from RNA molecules transcribed from the Ad48 Mac ends and encompassing the truncated A gene and telomeric repeats.Ciliate cells harbor two types of nuclei that ensure separate functions. Macronuclei that are transcriptionally active carry out the vegetative functions of these unicellular eukaryotes. Micronuclei that are transcriptionally silent during vegetative growth provide genetic continuity between sexual generations. In the course of conjugation, old maternal macronuclei do not replicate their DNA (they will be ultimately lost), while micronuclei enter meiosis. New macronuclei and micronuclei then develop from mitotic copies of zygotic nuclei. Macronuclear genomes result mainly from micronuclear genome fragmentation into short DNA molecules, at the ends of which telomeric repeats are added, excision of internal eliminated sequences (IESs) that are eventually eliminated, and massive DNA amplification that accounts for the large size (and the name) of macronuclei (20,21). Once developed (the whole process takes just a few hours), macronuclear genomes are clonally stable throughout the cellular life span. Although chromosome breakage occurs in a wide range of genomes, the frequency with which this takes place in ciliates is unique and provides an opportunity to study the underlying regulation and mechanisms.In the two species of Paramecium, P. primaurelia and P. tetraurelia, the micronuclear genome is organized into 60 to 90 chromosomes with an average size of 2 Mb, while macronuclear chromosomes range in size from 50 to 800 kb (note that although macronuclear DNA molecules are called chro-

Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

“…The drawings of the scaffolds with the remapped telomere reads show that at least eight other MAC chromosomes harbor such sites. This is a lower limit, however it is clear that not every fragmentation event can also be resolved by internal deletions, as demonstrated by extensive characterization of MAC chromosomes bearing the A SAg in P. tetraurelia (Epstein and Forney 1987;Forney and Blackburn 1988;Phan et al 1984;Amar and Dubrana 2004). We have no data concerning sites where DNA elimination is resolved only by internal deletions, as they would not have been detected by the present analysis.…”

“…Since a few rounds of endoreplication of the diploid zygotic genome precede DNA elimination, both chromosome fragmentation and variable internal deletions occur at this locus, even within a single homozygous cell. Similarly, the use of four different telomere addition regions, separated from each other by ∼10 kb, generates variability downstream from the A surface antigen gene in P. tetraurelia (Forney and Blackburn 1988;Amar and Dubrana 2004).…”

Programmed Rearrangement in Ciliates: Paramecium

“…The ends of MAC chromosomes are capped by a mixture of G 3 T 3 and G 4 T 2 telomeric repeats, which are added by an error-prone telomerase at heterogeneous positions within a telomereaddition region that extends over ∼1 kb (23,24). An additional level of heterogeneity has been attributed to the presence of several telomere-addition regions, distant by several kilobase pairs from each other, at the ends of MAC chromosomes (25,26,27). Thanks to joint efforts of the international community of Paramecium laboratories (28,29,30), a draft assembly of the MAC genome of P. tetraurelia was released in 2006 (7) and a polished version in 2012 (6).…”

Transposon Invasion of theParameciumGermline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

“…Pulse field electrophoresis analyses indicated that the size of Paramecium MAC chromosomes varies between 50 kb and 1 Mb [ 33 ]. The study of particular MAC chromosome ends in P. primaurelia [ 34 ] and P. tetraurelia [ 35 , 36 ] revealed that they are capped by a mixture of G 3 T 3 or G 4 T 2 telomeric repeats added at heterogeneous positions by a single, error-prone telomerase [ 37 , 38 ]; several telomere-addition regions distant of several kbp have been identified for some MAC chromosomes, each one extending over ~1 kb. The MAC genome sequence of Paramecium tetraurelia was obtained in 2006 by a consortium of European labs [ 39 ].…”