Tetrahymena Pot2 is a developmentally regulated paralog of Pot1 that localizes to chromosome breakage sites but not to telomeres

Abstract

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3' overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.

FIG 1

Cell growth and telomere maintenance are unaffected by POT2 macronuclear gene disruption. (A) Schematic of the macronuclear POT1 and POT2 gene loci and the encoded proteins. Black boxes, exons; arrows, transcription start sites; gray boxes, predicted OB folds. (B) Growth curves for wild-type (WT) cells and two POT2 knockout (KO) clones. PD, population doubling. (C) Southern blot showing rDNA telomere length in wild-type cells and three different POT2 knockout clones. The probe was for the subtelomeric region of the rDNA. M indicates size markers. (D and E) Ligation and primer extension assays to measure G-overhang length. (D, top) Diagram illustrating the assay. (Bottom) Diagram illustrating the products expected for a 14-nt overhang. (E) Polyacrylamide gel showing reaction products obtained with DNA from wild-type (lane 1), POT1 knockout (lane 2), or POT2 knockout (lane 3) cells. Products corresponding to overhangs of 14, 20, 26, and 32 nucleotides are marked at the right; positions of the marker oligonucleotides are shown on the left.

FIG 2

Pot2 expression and localization during macronuclear development. (A) Cartoon depicting stages of Tetrahymena conjugation and macronuclear development. After 2 to 3 h of starvation, cells pair, and the micronuclei undergo two rounds of meiosis. Three of the four nuclei are degraded, and the remaining nucleus undergoes mitosis to form two haploid pronuclei. One of the pronuclei is exchanged with the partner cell, and the pronuclei then fuse to form a diploid zygotic nucleus. After two rounds of mitosis (anlagen I), two of the mitotic products begin to develop into new macronuclei (anlagen II), while two remain as micronuclei. The cells separate and complete macronuclear development before degrading one of the new micronuclei. The cells remain with one micronucleus and two macronuclei until nutrients become available, at which point they resume vegetative growth. (B) POT1 and POT2 mRNA expression profiles. RT-PCR was performed with RNA collected from vegetatively growing cells (V), starved cells (S), or mated cells harvested at the indicated times after the initiation of mating. Cells were refed after 24 h, and RNA was collected 2 h later (26 h). PCR with genomic DNA (G) monitored DNA contamination, and RT-PCR with U1 snRNA controlled for RNA quality and loading. (C) Chromosome breakage analysis using the 11-kb PCR assay. The assay was performed with DNA isolated at the indicated time points from the mated cells used for panel B. PCR of the U1 snRNA gene controlled for DNA quality and loading. (D) Immunolocalization of Pot2. Cells at the indicated stages of conjugation and macronuclear development were stained with Pot2 antibody and counterstained with DAPI. Arrowheads, micronuclei; arrows, developing macronuclei; stars, parental macronuclei.

FIG 3

Pot2 localizes to sites of chromosome breakage. (AI) Representative images of acridine orange-stained cells showing stages of macronuclear development used for scoring. (II) Percentage of cells at the indicated stages of macronuclear development at each time point. Values are averages of data from the three ChIP experiments shown in panels B to D; error bars represent standard errors of the means. (III) Eleven-kilobase PCR assay for chromosome breakage. Samples were from one of the time courses used to generate data in panels II and B to D. (B to D) ChIP analysis of chromatin association throughout macronuclear development. Chromatin was collected at 8, 10, 12, and 14 h after the initiation of mating. Input DNA and precipitated DNA were quantified by real-time PCR (n = 3 independent experiments; error bars represent standard errors of the means). ChIP was performed with Pot2 (B), Pot1 (C), and Pdd1 (D) antibodies.

FIG 4

Pot2 lacks binding specificity for CBS or telomeric DNA. Shown are data for mobility shift assays using purified Pot1 and Pot2 and oligonucleotides corresponding to the telomeric G-strand overhang (A) or the CBS 4L-6 duplex and the two strands of CBS 4L-6 DNA (B). The bracket in panel A marks DNA-protein complexes formed by a contaminant in the Pot2 preparation (see Fig. S4 in the supplemental material).

FIG 5

Pat1 and Tpt1 localize to telomeres but not sites of chromosome breakage. (A) mRNA expression profiles for POT2, POT1, PAT1, and TPT1 during conjugation and macronuclear development. RNA was collected and analyzed by RT-PCR as described in the legend of Fig. 2B. (B) ChIP analysis of Pat1 and Tpt1 association with CBS 4L-6, the IES M-element (M-El), and telomeres throughout macronuclear development. Chromatin was from the mating time courses shown in Fig. 2. ChIP was performed with antibody to Pat1 or Tpt1 (n = 3 experiments; error bars represent standard errors of the means).

FIG 6

H3K27Me3 and H3K9Me3 do not accumulate at sites of chromosome breakage. (A and B) ChIP analysis to detect methylated histone H3 at CBS 4L-6, the IES M-element (M-El), and telomeres during macronuclear development. Chromatin was from the mating time courses shown in Fig. 2. ChIP was performed with antibody to H3K27Me3 (A) and H3K9Me3 (B) (n = 3 experiments; error bars represent standard errors of the means). (C) Model showing possible roles for Pot2 in recruiting the endonuclease responsible for chromosome cleavage and telomerase. MDS, macronucleus-destined sequence; RF, recruitment factor.