Setting boundaries for genome-wide heterochromatic DNA deletions through flanking inverted repeats in Tetrahymena thermophila

Abstract

Eukaryotic cells pack their genomic DNA into euchromatin and heterochromatin. Boundaries between these domains have been shown to be set by boundary elements. In Tetrahymena , heterochromatin domains are targeted for deletion from the somatic nuclei through a sophisticated programmed DNA rearrangement mechanism, resulting in the elimination of 34% of the germline genome in ∼10,000 dispersed segments. Here we showed that most of these deletions occur consistently with very limited variations in their boundaries among inbred lines. We identified several potential flanking regulatory sequences, each associated with a subset of deletions, using a genome-wide motif finding approach. These flanking sequences are inverted repeats with the copies located at nearly identical distances from the opposite ends of the deleted regions, suggesting potential roles in boundary determination. By removing and testing two such inverted repeats in vivo , we found that the ability for boundary maintenance of the associated deletion were lost. Furthermore, we analyzed the deletion boundaries in mutants of a known boundary-determining protein, Lia3p and found that the subset of deletions that are affected by LIA3 knockout contained common features of flanking regulatory sequences. This study suggests a common mechanism for setting deletion boundaries by flanking inverted repeats in Tetrahymena thermophila .

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination

“…Different levels of coupling between DNA cleavage and DSB repair may exist in the related ciliate Tetrahymena thermophila. In this species, the vast majority of IESs are excised by Tpb2 [41], a Pgm-related domesticated transposase that introduces DSBs at variable positions around IES boundaries [42], which results in micro-heterogeneous IES excision junctions [43]. T. thermophila encodes a single bona fide Ku80 protein and the closure of its IES excision sites also depends upon the classical NHEJ pathway [44].…”

Programmed genome rearrangements in ciliates

“…In Tetrahymena, flanking sequences are known to have a significant role in the elimination of a number of IESs [ 70 , 71 ]. Together, Lia3p and Lia3-Like 1 (LTL1) regulatory proteins interact with flanking regulatory sequences to determine MDS–IES boundaries for several IESs for excision [ 72 – 74 ]. These data highlight the importance of both IES pointer sequences and their flanking regions in identifying MDS–IES boundaries.…”

Roles of Noncoding RNAs in Ciliate Genome Architecture

“…It was known that cis -acting sequences helped to guide the excision from experiments in which shifting of certain IES-flanking sequences led to shifting of excision boundaries, but these sequences varied from IES to IES and no common consensus could be found [ 73 , [77] , [78] , [79] ]. Recently, a genomewide approach to search for IES-flanking IR sequences was undertaken, and it was found that there are several conserved IR sequences that flank different subsets of IESs at similar distances on both sides of the IES [ 80 ]. The known IES boundary-defining protein LIA3 was found to aid in the excision of a subset of IESs, and these LIA3-dependent IESs share similar polypurine-rich IR sequences at their boundaries [ 80 , 81 ].…”

“…Recently, a genomewide approach to search for IES-flanking IR sequences was undertaken, and it was found that there are several conserved IR sequences that flank different subsets of IESs at similar distances on both sides of the IES [ 80 ]. The known IES boundary-defining protein LIA3 was found to aid in the excision of a subset of IESs, and these LIA3-dependent IESs share similar polypurine-rich IR sequences at their boundaries [ 80 , 81 ]. Meanwhile, a second boundary-defining protein, Ltl1, was found to be required for the excision of another subset of IESs.…”