Analysis of DIE5 and LIA5 reveals the importance of DNA repair in programmed DNA rearrangement of Tetrahymena thermophila

During its somatic nuclear differentiation, the single cell eukaryote Tetrahymena thermophila undergoes genome-wide programmed DNA rearrangement to eliminate transposon-like elements from its future soma. This process involves small RNA-directed heterochromatin formation followed by extensive nuclear reorganization to form subnuclear domains. While more has been known about small RNAs and heterochromatin, the mechanisms and players involved in the process of nuclear reorganization and the subsequent removal of transposon-like elements from the somatic genome are just starting to unravel. My thesis work centers on the study of two novel nuclear proteins Die5p: Chapter 2) and Lia5p: Chapter 3) and their roles in DNA rearrangement. These essential proteins function downstream of small RNA targeted heterochromatin establishment. While Lia5p is essential for nuclear reorganization to form distinct subnuclear structures, Die5p is a protein conserved across ciliate species and appears to be important for the integrity of the differentiating genome. Maintaining genome integrity during somatic nuclear differentiation has proven to be an active process. Similar to V(D)J recombination during mammalian B and T cell maturation, programmed DNA rearrangement in Tetrahymena induces global DNA damage that requires proper response and repair. Through the study of LIA5 and DIE5, we show that nuclear reorganization during Tetrahymena DNA rearrangement is intimately associated with the response to DNA damage. Furthermore, we implicate a chromodomain protein Pdd1 as a component of the DNA damage response system, thus providing evidence to support the link between heterochromatin and DNA repair during the reprogramming of Tetrahymena somatic genome

CpG-creating mutations are costly in many human viruses.

Mutations can occur throughout the virus genome and may be beneficial, neutral or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly than similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are indeed costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses

<i>LIA5</i> Is Required for Nuclear Reorganization and Programmed DNA Rearrangements Occurring during <i>Tetrahymena</i> Macronuclear Differentiation

<div><p>During macronuclear differentiation of the ciliate <i>Tetrahymena thermophila</i>, 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 <i>LIA5</i>, 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 <i>∆LIA5</i> 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 <i>∆LIA5</i> cells contain dephosphorylated Pdd1p. These studies of <i>LIA5</i> 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.</p> </div

Pdd1 dephosphorylation does not occur in

<div><p>Δ<i>LIA5 </i><b>conjugation </b><b>cells</b>.</p> <p>(A) anti-Pdd1 western blot analysis of total protein isolated from conjugating WT, <i>∆LIA5</i> and <i>∆DCL1</i> at indicated time point. (B) Left panel, anti-Pdd1 western blot analysis of two to four fold reduced loading of 12 and 15hrs protein lysates taken from the conjugating Δ<i>LIA5</i> samples shown in (A), right panel, Alkaline phosphatase (AP) treatment of the same Δ<i>LIA5</i> 15hr lysate. Overall protein levels decrease after treatment due to some proteolytic activity in the lysate. (C) Fluorescent images of premature Pdd1-YFP foci in ΔDCL1 conjugants. P, Phosphorylated Pdd1p; O, unphosphorylated Pdd1p.</p></div

Lia5p is a developmentally expressed transposon-like protein.

<p>(A) Representation of Lia5p showing the positions of conserved DDE_Tnp_1_7 and domains. Alignment of Lia5p with (B) the DDD/E and (C) Tnp_zfribbon_2 domains of <i>Trichoplusia ni</i><i>piggyBac</i> transposon and ciliate domesticated transposases, <i>Paramecium</i> PGM and <i>Tetrahymena</i> Tpb2p and Tpb1p: the <i>T. ni</i> catalytic core residues are noted above the alignment. (D) Western blot analyses showing Lia5 mRNA and protein expression from 0 to 12 hrs of conjugation; the hour of conjugation is indicated above each lane. Detection of the abundant histone H3 tri-methylated on K4.</p

<i>LIA5</i> is required for Tetrahymena programmed DNA rearrangement.

<p>(A) The illustration shows the unrearranged M IES (Mic) and the two major alternative rearrangement that excise either 0.6kbp (Δ0.6) or 0.9kbp (Δ0.9). Arrows denote forward and reverse primers used in the second round of nested PCR to amplify across the M IES locus. PCR of single cells to assess M IES excision; M, PstI-digested Lambda DNA size marker; g, genomic DNA from unmated CU428 cells. Each lane (1-8) represents a single exconjugant from WT or <i>∆LIA5</i> crosses. The expected positions for the unrearranged (Mic) and rearranged (Δ0.6 and Δ0.9) products are indicated to the right. (B) Genomic DNA isolated from WT or mutant strains 18 or 30 hours after initiating conjugation (as indicated) was amplified by PCR using primers (designated by arrowheads) flanking five different IESs site. PCR products from IES-containing or IES-eliminated DNA regions are indicated with brackets. The size of relevant DNA standards is indicated at left.</p

<i>LIA5</i> is required for chromosome breakage.

<p>(A) The diagram illustrates the <i>LIA1</i> locus and flanking DNA showing the downstream chromosomal breakage sequence (CBS) (star symbol). <i>Eco</i>RI (RI) restriction sites used for the Southern blot analysis are shown. The probe spans the central <i>Eco</i>RI site and detects: 1) a 7.8 kbp distal fragment common to both micro- and macronuclei; 2) a 10.5kbp micronucleus-specific fragment; 3) a 2.2kbp fragment indicating <i>de </i><i>novo</i> breakage; and 4) variable 2.5kb to 2.6kbp fragments indicative of parental macronuclear chromosomes with fully elongated telomeres (Tel), remaining due to the unmated cells in the population. (B) Southern blot analysis of <i>Eco</i>RI digested genomic DNA from unmated (Veg) or mated populations of WT or mutant strains was used to assess chromosome breakage. The arrow indicates the 2.2 kbp fragment consistent with <i>de </i><i>novo</i> breakage; the approximate sizes of the other fragments are indicated to the right.</p

Lia5 localizes to DNA elimination foci, but not within the Pdd1 central core.

<p>(A) Immunostaining of HALia5 at 9hrs into conjugation. (B, C) Co-localization of Lia5-YFP and Pdd1-CFP or immuno-stained HALia5 and Pdd1-YFP in 14hrs conjugating cells. In (C), Pdd1p and Lia5 localization are shown together in a single developing macronucleus. (D) Immunoprecipitation of HALia5 (anti-HA IP). Untransformed cells (Mock1) and HALia5 transformant lysates immunoprecipitated with rabbit-IgG only (Mock2) are shown as controls. Immunoprecipitated samples (IP) and their respective supernatants (Supe) were analyzed by SDS-PAGE and western blot analysis. HALia5 was visualized with HA epitope antibodies and Pdd1 and Pdd3 were detected specific polyclonal antibodies. HA-Lia5p appears as a doublet after immunoprecipitation, which we have not further investigated.</p

DNA damage rescues foci formation in

<div><p><i>∆LIA5 </i><b>conjugants</b>.</p> <p>(A) Immunostaining of γH2AX in WT and <i>∆LIA5</i> during meiosis (3hrs) and macronuclear differentiation (10hrs) stages of conjugation. (B) Fluorescence images of Pdd1-CFP and LigaseIV-YFP co-localization after UV exposure. (C-D) UV induced DNA damage is sufficient to rescue Pdd1 localization and de-phosphorylation in Δ<i>LIA5</i>. (C) Pdd1-YFP localization in 14hrs conjugating WT, Δ<i>LIA5</i>, and Δ<i>LIA5</i> cells treated with 150mJ of UV. (D) anti-Pdd1 western blot analysis for WT mating at 9hrs and 12hrs. Irradiated (+UV) or control (-UV) <i>∆LIA5</i> mating cells at 12hrs.</p></div

<i>LIA5</i> is essential to complete conjugation.

<p>(A) Illustration of LIA5 gene replacement with the NEO3 selectable marker (MTTNeo), which confers paromomycin resistances upon induction with CdCl2. “X″ s denote homologous recombination directing gene replacement. Arrows indicate restriction enzyme cut sites used to verify gene disruption by Southern blot analysis (B). The region labeled for use as a probe is shown as a black bar. It detects a 6kb (WT) or a 3.9kb (Δ<i>LIA5</i>) EcoRI-PstI fragment indicative of the wt <i>LIA5</i> or knockout allele, respectively. DNA was isolated from wild type (CU427, CU428) and ∆<i>LIA5</i> strains (ms, 4-2). (C) rtPCR for the expression of <i>LIA5</i> in conjugating wild type (WT) and <i>LIA5</i> knockout (Δ<i>LIA5</i>) at indicated hours into conjugation. Primers used span the sixth intron and detect a smaller mRNA product that is easily distinguished from the product amplified from genomic DNA (g) used as control for amplification (or background from possible minor DNA contamination in the PCR). (D) Fluorescent images of representative DAPI stained WT, <i>∆LIA5</i> and <i>∆PDD1</i> strains post conjugation.</p