Abstract
During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.
Highlights
The organization of DNA within the nucleus reflects how chromosomes are partitioned into functional domains
To test whether Ltl1 contributes to the accuracy of DNA elimination, we examined the rearrangements of several previously studied internal eliminated sequences (IESs), along with the excision events that occur across two selected ∼150 kb regions of the micronuclear genome
We identified the micronucleus-limited IESs as gaps in the alignments of the micro- and macronuclear sequences, designed oligonucleotide primers to sequences flanking the ∼15 IESs found within each region to use in Polymerase chain reactions (PCR) to monitor DNA elimination efficiency and accuracy
Summary
The organization of DNA within the nucleus reflects how chromosomes are partitioned into functional domains. During development of its somatic genome, Tetrahymena packages ∼12 000 loci (totaling ∼1/3 of the 157 Mb genome) dispersed throughout the germline-derived genome into heterochromatin [4]. These cells use small RNAs to identify these loci and target methylation of the associated chromatin on lysines (K) 9 and 27 of histone H3 [5,6,7]. The advantage Tetrahymena offers over other models is that, because all heterochromatic loci are excised from the somatic genome, we can unambiguously identify all loci that are targets for heterochromatin formation during development. The boundaries of these heterochromatic sequences can be defined by the sites of excision
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