Jaime F. Angulo

UV-Induced Stabilization of c-fos and Other Short-Lived mRNAs

ABSTRACT Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IκB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

The mouse Kin-17 gene codes for a new protein involved in DNA transactions and is akin to the bacterial RecA protein

We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.

Expression of Kin17 and 8-OxoG DNA glycosylase in cells of rodent and quail central nervous system

Kin17 and 8-Oxoguanine DNA glycosylase (Ogg1) are proteins, respectively, involved in illegitimate recombination and DNA repair in eukaryotic cells. To characterize the expression of these proteins in cell types of rodent and avian brains, we combined immunocytochemistry for either Kin17 or Ogg1 proteins with glial fibrillary acidic protein (GFAP, an astrocyte marker) immunodetection on the same tissue section. Both Kin17 and Ogg1 proteins were localized in cell nuclei and were extensively distributed in neuronal populations of quail and rodent brains. However, GFAP-immunoreactive cells were never labeled by Kin17 protein. This was observed in nerve fiber tracts, in the cerebral cortex, the hippocampal formation, the hypothalamic region, and the periventricular regions of the brain of both species studied. These results were confirmed by combining in situ hybridization of kin17 mRNA and GFAP immunodetection. On the contrary, GFAP-immunoreactive cells were often labeled by the Ogg1 protein in brain structures such as fiber tracts, the cortical surface, the cerebellum, and the ependymal surface of both quail and mouse brains. Our results suggest that the expression of the Kin17 protein (observed in neurons) and that of the Ogg1 protein (observed in neurons and glial cells) is conserved in brain phylogeny.

Overexpression of kin17 protein forms intranuclear foci in mammalian cells

We used antibodies against E coli RecA protein to identify in mouse cells a 45-kDa DNA-binding protein called kin17, which has an active zinc finger and a nuclear localisation signal. Kin17 protein produced in E coli binds preferentially to the curved DNA of a bacterial promoter in vivo and in vitro, suggesting a transcriptional regulation activity. The fact that in rodent cells kin17 protein levels increase after gamma-irradiation suggests its participation in a cellular response to ionising radiation. We raised polyclonal antibodies against the whole kin17 protein and against its derived synthetic peptides. We report the detection of kin17 protein and of truncated forms of the protein by Western blot or by immunocytochemistry after transient overexpression in cultured human cells. Our results indicate that the cross-reactivity with the anti-RecA antibodies is due to an antigenic determinant located in the core of kin17 protein, between residues 129 and 228. The kin17 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm. The RecA homologous region seems to play an essential role in the localisation of kin17 protein since the deletion of this particular region dramatically changes the form and the distribution of the intranuclear foci. We hypothesise that these dot-like structures reflect nuclear metabolism compartmentalization.

Preferential expression of kin, a nuclear protein binding to curved DNA, in the neurons of the adult rat

The KIN17 gene product has been identified by cross immunoreactivity with anti-RecA antibodies and by DNA recombination techniques, and is probably part of the DNA recombination-repair machinery. Following Western blotting and immunocytochemistry using anti-RecA antibodies, and in situ hybridization with specific KIN17 cDNA probes, we here report the detection of high levels of KIN protein and KIN17 mRNA in the CNS of adult rats. The RecA cross-reacting protein has an apparent molecular weight of 41 kDa and is located in the nucleus of brain cells. Both the KIN17 transcript and the protein were found to be widespread, but they were present in different proportions, depending on the type of brain cells. High levels of KIN protein were seen in neurons of the motor nuclei of the brainstem, the locus coeruleus, hippocampal formation, entorhinal cortex, Purkinje cells, pyramidal cells of the cortex and mitral cells. In contrast, using a combination of KIN17 mRNA in situ hybridization and GFAP immunocytochemistry (a marker of glial cells) showed that the KIN17 messenger is preferentially transcribed in neurons, the post-mitotic and long lived brain cells. We postulate that KIN17 play a role in the illegitimate recombination of DNA sequences and/or the repair of alterations of the genome in neurons.

Kin protein expression : laminar specificity during rat cerebral cortex development

Kin is a mammalian nuclear protein involved in DNA recombination-repair and the regulation of gene expression. The present study explored the expression of the Kin nuclear protein during postnatal development of the rat cerebral cortex, using immunocytochemistry with anti-RecA antibodies. The immunostaining of the Kin protein preferentially occurs within layers IV-V and VIb of the cortex in early postnatal developing brain, whereas in the adult rat this expression is observed unequivocally in all cortical layers. 35S-isotopic in situ hybridization for Kin-17 mRNA confirmed this Kin protein expression pattern and demonstrated its transcription in cortical neurons. This gradual age-related expression during development may have functional implications in the maturation processes of the cortex.

Expression of Kin, a nuclear protein binding to curved DNA, in mammal and avian brains

Kin is a nuclear protein which presents cross-immunoreactivity with the bacterial RecA protein and which efficiently binds to curved DNA. This genomic interaction could be implied in DNA repair and illegitimate recombination in eukaryotic cells. Using immunocytochemistry with anti-RecA antibodies, we report the ubiquitous presence of the Kin protein in the CNS of mice and quails. However, some brain structures such as the hippocampal area, the locus coeruleus and Purkinje cells are preferentially immunolabelled and show some homologies between the two species. In conclusion, the expression of the Kin protein is preserved in the phylogeny of the brain of higher vertebrates.

Putative Roles of kin17, a Mammalian Protein Binding Curved DNA, in Transcription

In bacteria, RecA protein is indispensable for recombination, mutagenesis and for the induction of SOS genes. Curiously, anti-RecA antibodies recognize kin 17, a human nuclear Zn-finger protein of 45 kDa that preferentially binds to curved DNA and participates in a general response to diverse genotoxics. KIN17 gene is conserved from yeast to man and codes for a protein involved in DNA replication. Recent observations suggest that kin 17 protein may also participate in RNA metabolism. Taken together all these data indicate the participation of kin 17 protein in a pathway that harmonizes transcription, replication and repair in order to circumvent the topological constraints caused by unusually complex lesions like multiply damaged sites.

Expression of Kin, a nuclear protein binding to curved DNA, in the brain of the frog (Rana esculenta), turtle (Trachemys scripta), quail (Coturnix coturnix) and mouse (Mus musculus)

The distribution of Kin protein, the vertebrate homologue of the bacterial recA nuclear protein involved in illegitimate recombinant DNA repair and gene regulation, was analysed in the brain of the mouse, quail, turtle and frog by immunocytochemical methods. The protein was expressed in all brains, but not in a uniform manner. Immunoreactivity was absent from major fibre tracts. In the cerebral nuclei, immunolabelling in the various species showed an important variation. A comparative analysis, based on the homologies between different brain structures in these species, showed that this variation was not due to interspecific variation but that of an ancestral pattern of distribution of Kin protein. It is also shown that whatever the species examined, Kin protein is consistently more highly expressed in those regions of the brain with a conservative evolutionary history (e.g. the olfactory and limbic systems, the hypothalamus, the monoaminergic system, the cerebellum, and the nuclei of sensory and motor cranial nerves). The protein is markedly less heavily expressed in the dorsal striatum and the sensory nuclei of the thalamus.