Telomere Regulation in Arabidopsis thaliana by the CST Capping Complex and DNA Damage Response Proteins

The ends of chormosomes are capped by telomeres, which distinguish the termini from damaged DNA. Paradoxically, DNA repair proteins are also required for telomere maintenance. How DNA repair pathways are regulated to maintain telomeres while remaining competent to repair DNA damage throughout the genome is unknown. In this dissertation, I used a genetic approach to investigate how critical components of telomerase and the telomere protein complex interact with the DNA damage response (DDR). In the flowering plant, Arabidopsis thaliana telomeres are bound by the CST (CTC1/STN1/TEN1) heterotrimer. Loss of any CST component results in telomere shortening, telomere fusions, increased G-overhang length and telomere recombination. To understand the phenotypes caused by CST deficiency, I examined telomeres from plants lacking CTC1 or STN1 and TERT or KU. My analysis showed that CST acts in a separate genetic pathway for telomere length regulation from both KU and TERT. Further, I found that KU and CST act in separate genetic pathways for regulation of G-overhang formation. These demonstrate that multiple pathways are used to maintain telomere length and architecture in plants. My study of the interaction of telomere components with the DDR revealed ATR promotes genome stability and telomere length maintenance in the absence of CTC1, probably by activating programmed cell death of stem cells with high amounts of DNA damage. I also found that poly(ADP-ribosylation) is not required for maintenance of Arabidopsis telomeres, in contrast to human telomeres. Finally, I found an unexpected connection between the DDR and telomerase. My research showed that ATR maintains telomerase activity levels. Further, induction of double- stranded DNA breaks in seedlings led to a rapid decrease in telomerase activity, which correlated with increased abundance of TER2, an alternate Arabidopsis telomerase RNA. I hypothesize that TER2 inhibits telomerase to prevent its inappropriate action at internal sites in chromosomes. These data reveal two ways that DDR pathways work in concert with telomerase to promote genome integrity

Analysis of Poly(ADP-Ribose) Polymerases in Arabidopsis Telomere Biology

Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one

Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP

The conserved histone locus body (HLB) assembles prior to zygotic gene activation early during development and concentrates factors into a nuclear domain of coordinated histone gene regulation. Although HLBs form specifically at replication-dependent histone loci, the cis and trans factors that target HLB components to histone genes remained unknown. Here we report that conserved GA repeat cis elements within the bidirectional histone3–histone4 promoter direct HLB formation in Drosophila. In addition, the CLAMP (chromatin-linked adaptor for male-specific lethal [MSL] proteins) zinc finger protein binds these GA repeat motifs, increases chromatin accessibility, enhances histone gene transcription, and promotes HLB formation. We demonstrated previously that CLAMP also promotes the formation of another domain of coordinated gene regulation: the dosage-compensated male X chromosome. Therefore, CLAMP binding to GA repeat motifs promotes the formation of two distinct domains of coordinated gene activation located at different places in the genome

Loss of p24 function in Drosophila melanogaster causes a stress response and increased levels of NF-κB-regulated gene products

BACKGROUND: Secretory and transmembrane proteins traverse the endoplasmic reticulum (ER) and Golgi compartments for final maturation prior to reaching their functional destinations. Members of the p24 protein family, which are transmembrane constituents of ER and Golgi-derived transport vesicles, function in trafficking some secretory proteins in yeast and higher eukaryotes. Yeast p24 mutants have minor secretory defects and induce an ER stress response that likely results from accumulation of proteins in the ER due to disrupted trafficking. We tested the hypothesis that loss of Drosophila melanogaster p24 protein function causes a transcriptional response characteristic of ER stress activation. RESULTS: We performed genome-wide profiling experiments on tissues from Drosophila females with a mutation in the p24 gene logjam (loj) and identified changes in message levels for 641 genes. We found that loj mutants have expression profiles consistent with activation of stress responses. Of particular note is our observation that approximately 20% of the loci up regulated in loj mutants are Drosophila immune-regulated genes (DIRGs), many of which are transcriptional targets of NF-κB or JNK signaling pathways. CONCLUSION: The loj mutant expression profiling data support the hypothesis that loss of p24 function causes a stress response. Genes involved in ameliorating stress, such as those encoding products involved in proteolysis, metabolism and protein folding, are differentially expressed in loj mutants compared to controls. Nearly 20% of the genes with increased message levels in the loj mutant are transcriptional targets of Drosophila NF-κB proteins. Activation of NF-κB transcription factors is the hallmark of an ER stress response called the ER overload response. Therefore, our data are consistent with the hypothesis that Drosophila p24 mutations induce stress, possibly via activation of ER stress response pathways. Because of the molecular and genetic tools available for Drosophila, the fly will be a useful system for investigating the tissue-specific functions of p24 proteins and for determining the how disrupting these molecules causes stress responses in vivo

<i>Arabidopsis</i> PARPs respond to the absence of telomerase.

<p>(A) RT-PCR of <i>PARP1</i> transcript levels in different generations of <i>tert</i> mutants. (B) RT-PCR of <i>PARP2</i> expression in wild type and 3^rd generation (G3) <i>tert</i> mutants at increasing concentrations of MMS.</p

<i>Arabidopsis</i> telomerase is not stimulated by PARPs.

<p>(A) TRAP analysis on seedlings. Seedlings were treated with either 3-AB (WT) or DMSO (WT and G4 <i>tert</i> mutants) (B) Quantitative TRAP results for 7-day-old 3-AB-treated wild type seedlings relative to untreated seedlings. P-value = 0.03 by Student’s two-tailed t-test.</p

PARPs are not required to maintain telomere length in <i>Arabidopsis</i>.

<p>(A) TRF analysis of bulk telomeres in several individual wild type, <i>parp1</i>, <i>parp2,</i> and <i>parp1 parp2</i> plants. (B–D) PETRA analysis of telomeres on specific chromosome arms in wild type and <i>parp</i> mutants. Primer naming convention is the same as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088872#pone-0088872-g006" target="_blank">Figure 6</a> legend. (B) Analysis of the telomeres on chromosome arms 1L and 2R. DNA was from pooled seedlings. (C) Analysis of four different chromosome arms for individual <i>parp1</i>, <i>parp2,</i> and <i>parp1 parp2</i> mutant plants. Chromosome arms tested are listed below the lanes. (D) Analysis of wild type and G4 <i>tert</i> pooled seedlings grown in either 0.6% DMSO or 5 mM 3-AB/0.6% DMSO using the 2R primer. Molecular weight markers are shown to the left of each gel.</p

Characterization of T-DNA mutants and 3-AB treated seedlings.

<p>(A) Schematic of PARP proteins. The triangles indicate the position of the T-DNA insertions. The T-DNA for PARP1 is located in the intron between exons 6 and 7, within the PARP catalytic domain. The T-DNA for PARP2 is located in exon 10, within the WGR domain. SAP: SAF-A/B, Acinus and PIAS (nucleic acid-binding domain); WGR: Named after conserved central motif (Trp, Gly, Arg) (putative DNA-binding domain); PARP: PARP regulatory and catalytic domain; PADR1: Domain of unknown function found in PARPs; BRCT: BRCA1 C-terminus. (B) Semi-quantitative RT-PCR for <i>PARP1</i> and <i>PARP2</i> expression levels in <i>parp1</i> (top), <i>parp2</i> (middle) and <i>parp1 parp2</i> double mutants (bottom). <i>Actin-2</i> served as a loading control. (C) Wild type (top) and G4 <i>tert</i> mutant seedlings (bottom) grown in 5 mM 3-AB/0.6% DMSO (left) or in 0.6% DMSO (right). (D) qRT-PCR for <i>XRCC2</i> expression in 3-AB-treated wild type seedlings relative to untreated seedlings. * = p-value <0.05 by Student’s two-tailed t-test.</p

PARPs are not required to prevent end-to-end chromosome fusions.

<p>Telomere fusion PCR for (A) 3-AB treated wild type seedlings, and (B) 3-AB treated <i>tert</i> mutants. The pairs of subtelomeric primers used for TF- PCR are indicated below each blot, where 1L refers to the left arm of chromosome 1 and 2R to the right arm of chromosome 2, etc. <i>ctc1</i> mutants were used as a positive control.</p