DNA Damage-Induced Phosphorylation of TRF2 Is Required for the Fast Pathway of DNA Double-Strand Break Repair

Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.Telomeres act as protective caps to disguise the chromosome end from being recognized as a DNA double-strand break (DSB) and play other important roles in maintaining genomic integrity (2, 21, 26). Telomere capping dysfunction resulting in genomic instability is likely a major pathway leading to human cancers and other age-related diseases (8, 27).An increasing number of proteins known to play important roles in DNA repair have also been found to be critical for telomere maintenance (6). Specifically, phosphatidylinositol (PI) 3-kinase-like kinase family members, such as ataxia telangiectasia mutated protein kinase (ATM) and the DNA-dependent protein kinase catalytic subunit in mammals, originally known to act in maintaining genomic stability via DNA repair pathways, have been shown to be important in telomere maintenance (1, 4, 7, 9, 10, 16, 25). Previous reports indicate that ATM is required for the DNA damage-induced phosphorylation of two major telomere-associated proteins in mammals, human TRF1 and TRF2 (16, 28). The specific molecular roles played by the DNA damage-induced phosphorylation of TRF1 and TRF2 in telomere maintenance and/or DNA repair are unclear and under active investigation. We previously reported that upon DNA damage, human TRF2 was rapidly and transiently phosphorylated at threonine 188 (T188) (28). Here, we report that X-ray-induced phosphorylation of human TRF2 at T188 plays a functional role in the fast pathway of DNA DSB repair.     

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2, 7, 34, 36, 39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11, 13, 15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm and transiently localizes to nucleoli (23). In the absence of other viral proteins, UL24 accumulates in the Golgi apparatus and in the nucleus, where it usually exhibits a diffuse staining pattern, but in a minority of cells it is detected in nucleoli (3).During infection, the formation of the viral replication compartments in the nucleus and the action of several viral proteins result in a remodeling of the nucleus. Chromatin is marginalized (29, 40), promyelocytic leukemia bodies are dispersed (26, 27), and the nuclear lamina is disrupted (33, 37). HSV-1 infection also affects the nucleolus, a prominent nuclear substructure implicated in the synthesis of rRNA, cell cycle regulation, and nucleocytoplasmic shuttling (5). Nucleoli become elongated following infection, and the synthesis of mature rRNA is reduced (4, 38, 42). Several HSV-1 proteins have been shown to localize to, or associate with, the nucleolus (12). The viral protein VP22 associates with the nucleolus and with dispersed nucleolin in HSV-1-infected cells (22), and RL1, US11, and ICP0 have also been shown to localize to nucleoli (24, 30, 35). Previously we showed that nucleolin is dispersed throughout the nucleus upon HSV-1 infection and that UL24 is involved in this nuclear modification (23). We further found that the N-terminal portion of UL24 is sufficient to induce the redistribution of nucleolin in the absence of other viral proteins (3).In this study, we sought to test the hypothesis that the endonuclease motif, which is made up of some of the most highly conserved residues in UL24, is important for the dispersal of nucleolin. A panel of substitution mutations in UL24 was generated, and the impact on the function of UL24 was assessed.     

X-chromosome inactivation patterns are unbalanced and affect the phenotypic outcome in a mouse model of rett syndrome

Rett syndrome (RTT), a neurodevelopmental disorder affecting mostly females, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Although the majority of girls with classic RTT have a random pattern of X-chromosome inactivation (XCI), nonbalanced patterns have been observed in patients carrying mutant MECP2 and, in some cases, account for variability of phenotypic manifestations. We have generated an RTT mouse model that recapitulates all major aspects of the human disease, but we found that females exhibit a high degree of phenotypic variability beyond what is observed in human patients with similar mutations. To evaluate whether XCI influences the phenotypic outcome of Mecp2 mutation in the mouse, we studied the pattern of XCI at the single-cell level in brains of heterozygous females. We found that XCI patterns were unbalanced, favoring expression of the wild-type allele, in most mutant females. It is notable that none of the animals had nonrandom XCI favoring the mutant allele. To explore why the XCI patterns favored expression of the wild-type allele, we studied primary neuronal cultures from Mecp2-mutant mice and found selective survival of neurons in which the wild-type X chromosome was active. Quantitative analysis indicated that fewer phenotypes are observed when a large percentage of neurons have the mutant X chromosome inactivated. The study of neuronal XCI patterns in a large number of female mice carrying a mutant Mecp2 allele highlights the importance of MeCP2 for neuronal viability. These findings also raise the possibility that there are human females who carry mutant MECP2 alleles but are not recognized because their phenotypes are subdued owing to favorable XCI patterns.     

Amphetamine-evoked c-fos mRNA expression in the caudate-putamen: the effects of DA and NMDA receptor antagonists vary as a function of neuronal phenotype and environmental context

Dopamine (DA) and glutamate neurotransmission is thought to be critical for psychostimulant drugs to induce immediate early genes (IEGs) in the caudate-putamen (CPu). We report here, however, that the ability of DA and glutamate NMDA receptor antagonists to attenuate amphetamine-evoked c-fos mRNA expression in the CPu depends on environmental context. When given in the home cage, amphetamine induced c-fos mRNA expression predominately in preprodynorphin and preprotachykinin mRNA-containing neurons (Dyn-SP+ cells) in the CPu. In this condition, all of the D1R, D2R and NMDAR antagonists tested dose-dependently decreased c-fos expression in Dyn-SP+ cells. When given in a novel environment, amphetamine induced c-fos mRNA in both Dyn-SP+ and preproenkephalin mRNA-containing neurons (Enk+ cells). In this condition, D1R and non-selective NMDAR antagonists dose-dependently decreased c-fos expression in Dyn-SP+ cells, but neither D2R nor NR2B-selective NMDAR antagonists had no effect. Furthermore, amphetamine-evoked c-fos expression in Enk+ cells was most sensitive to DAR and NMDAR antagonism; the lowest dose of every antagonist tested significantly decreased c-fos expression only in these cells. Finally, novelty-stress also induced c-fos expression in both Dyn-SP+ and Enk+ cells, and this was relatively resistant to all but D1R antagonists. We suggest that the mechanism(s) by which amphetamine evokes c-fos expression in the CPu varies depending on the stimulus (amphetamine vs. stress), the striatal cell population engaged (Dyn-SP+ vs. Enk+ cells), and environmental context (home vs. novel cage).     

Gene cloning and characterization of VcrM,a Na+-coupled multidrug efflux pump,from Vibrio cholerae non-O1

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.