Human RAP1 inhibits non-homologous end joining at telomeres.

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, promote genome stability by distinguishing chromosome termini from DNA double-strand breaks (DSBs). Cells possess two principal pathways for DSB repair: homologous recombination and non-homologous end joining (NHEJ). Several studies have implicated TRF2 in the protection of telomeres from NHEJ, but the underlying mechanism remains poorly understood. Here, we show that TRF2 inhibits NHEJ, in part, by recruiting human RAP1 to telomeres. Heterologous targeting of hRAP1 to telomeric DNA was sufficient to bypass the need for TRF2 in protecting telomeric DNA from NHEJ in vitro. On expanding these studies in cells, we find that recruitment of hRAP1 to telomeres prevents chromosome fusions caused by the loss of TRF2/hRAP1 from chromosome ends despite activation of a DNA damage response. These results provide the first evidence that hRAP1 inhibits NHEJ at mammalian telomeres and identify hRAP1 as a mediator of genome stability.

The human RAP1 and GFAPɛ proteins increase γ-secretase activity in a yeast model system.

Alzheimer's disease (AD) is an age-related disorder that results in progressive cognitive impairment and memory loss. Deposition of amyloid ß (Aß) peptides in senile plaques is a hallmark of AD. γ-secretase produces Aß peptides, mostly as the soluble Aß40 with fewer insoluble Aß42 peptides. Rare, early-onset AD (EOAD) occurs in individuals under 60 years of age. Most EOAD cases are due to unknown genetic causes, but a subset is due to mutations in the genes encoding the amyloid precursor protein that is processed into Aß peptides or the presenilins (PS1 and PS2) that process APP. PS1 interacts with the epsilon isoform of glial fibrillary acidic protein (GFAPɛ), a protein found in the subventricular zone of the brain. We have found that GFAPɛ interacts with the telomere protection factor RAP1 (TERF2IP). RAP1 can also interact with PS1 alone or with GFAPɛ in vitro. Our data show that the nuclear protein RAP1 has an extratelomeric role in the cytoplasm through its interactions with GFAPɛ and PS1. GFAPɛ coprecipitated with RAP1 from human cell extracts. RAP1, GFAPɛ, and PS1 all colocalized in human SH-SY5Y cells. Using a genetic model of the γ-secretase complex in Saccharomyces cerevisiae, RAP1 increased γ-secretase activity, and this was potentiated by GFAPɛ. Our studies are the first to connect RAP1 with an age-related disorder.

Distinct requirements for Pot1 in limiting telomere length and maintaining chromosome stability.

The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.

Identification of Genes in Saccharomyces cerevisiae that Are Haploinsufficient for Overcoming Amino Acid Starvation.

The yeast Saccharomyces cerevisiae responds to amino acid deprivation by activating a pathway conserved in eukaryotes to overcome the starvation stress. We have screened the entire yeast heterozygous deletion collection to identify strains haploinsufficient for growth in the presence of sulfometuron methyl, which causes starvation for isoleucine and valine. We have discovered that cells devoid of MET15 are sensitive to sulfometuron methyl, and loss of heterozygosity at the MET15 locus can complicate screening the heterozygous deletion collection. We identified 138 cases of loss of heterozygosity in this screen. After eliminating the issues of the MET15 loss of heterozygosity, strains isolated from the collection were retested on sulfometuron methyl. To determine the general effect of the mutations for a starvation response, SMM-sensitive strains were tested for the ability to grow in the presence of canavanine, which induces arginine starvation, and strains that were MET15 were also tested for growth in the presence of ethionine, which causes methionine starvation. Many of the genes identified in our study were not previously identified as starvation-responsive genes, including a number of essential genes that are not easily screened in a systematic way. The genes identified span a broad range of biological functions, including many involved in some level of gene expression. Several unnamed proteins have also been identified, giving a clue as to possible functions of the encoded proteins.

Telomere protein RAP1 levels are affected by cellular aging and oxidative stress.

Telomeres are important for maintaining the integrity of the genome through the action of the shelterin complex. Previous studies indicted that the length of the telomere did not have an effect on the amount of the shelterin subunits; however, those experiments were performed using immortalized cells with stable telomere lengths. The interest of the present study was to observe how decreasing telomere lengths over successive generations would affect the shelterin subunits. As neonatal human dermal fibroblasts aged and their telomeres became shorter, the levels of the telomere-binding protein telomeric repeat factor 2 (TRF2) decreased significantly. By contrast, the levels of one of its binding partners, repressor/activator protein 1 (RAP1), decreased to a lesser extent than would be expected from the decrease in TRF2. Other subunits, TERF1-interacting nuclear factor 2 and protection of telomeres protein 1, remained stable. The decrease in RAP1 in the older cells occurred in the nuclear and cytoplasmic fractions. Hydrogen peroxide (H2O2) stress was used as an artificial means of aging in the cells, and this resulted in RAP1 levels decreasing, but the effect was only observed in the nuclear portion. Similar results were obtained using U251 glioblastoma cells treated with H2O2 or grown in serum-depleted medium. The present findings indicate that TRF2 and RAP1 levels decrease as fibroblasts naturally age. RAP1 remains more stable compared to TRF2. RAP1 also responds to oxidative stress, but the response is different to that observed in aging.

Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10.

Histone deacetylases are required for transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox transcription factor, HOXA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.

Synthesis of a FTO inhibitor with anticonvulsant activity.

We describe the rationale for and the synthesis of a new class of compounds utilizing a modular approach that are designed to mimic ascorbic acid and to inhibit 2-oxoglutarate-dependent hydroxylases. Preliminary characterization of one of these compounds indicates in vivo anticonvulsant activity (6 Hz mouse model) at nontoxic doses, inhibition of the 2-oxoglutarate-dependent hydroxylase FTO, and expected increase in cellular N(6)-methyladenosine. This compound is also able to modulate various microRNA, an interesting result in light of the recent view that modulation of microRNAs may be useful for the treatment of CNS disease.

A RAP1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends.

The mechanisms by which telomeres are distinguished from DNA double-strand breaks are poorly understood. Here we have defined the minimal requirements for the protection of telomeric DNA ends from nonhomologous end-joining (NHEJ). Neither long, single-stranded overhangs nor t loop formation is essential to prevent NHEJ-mediated ligation of telomeric ends in vitro. Instead, a tandem array of 12 telomeric repeats is sufficient to impede illegitimate repair in a highly directional manner at nearby DNA ends. The polarity of end protection is consistent with the orientation of naturally occurring telomeres and is well suited to minimize interference between chromosome capping and the repair of DNA double-strand breaks in subtelomeric sequences. Biochemical fractionation and reconstitution revealed that telomere protection is mediated by a RAP1/TRF2 complex, providing evidence for a direct role for human RAP1 in the protection of telomeric DNA from NHEJ.