Control of the establishment of aversive memory by calcineurin and Zif268.

Emotional memory is a rapidly acquired and persistent form of memory, and its robustness is in part determined by the initial strength of the memory. Here, we provide new evidence that the protein phosphatase calcineurin (CaN), a potent negative regulator of neuronal signaling that is known to constrain learning and memory, critically regulates the establishment of emotional memory through mechanisms involving the immediate early gene Zif268 (also known as Egr1). We found that CaN is inhibited in the amygdala during the establishment of aversive memory, but Zif268 is activated. Using inducible transgenesis in mice, we further saw that CaN inhibition and Zif268 overexpression during memory establishment strengthen the memory trace and enhance its resistance to extinction. We found that CaN inhibition correlates with increased Zif268 expression and that a common pool of proteins is regulated in the amygdala after CaN inhibition and Zif268 overexpression. Together, these findings reveal a previously unknown mechanism for the control of emotional memory that depends on CaN and Zif268.

Functionally distinct nucleosome-free regions in yeast require Rad7 and Rad16 for nucleotide excision repair.

In yeast, Rad7 and Rad16 are two proteins required for nucleotide excision repair (NER) of non-transcribed chromatin. They have roles in damage recognition, in the postincision steps of NER, and in ultraviolet-light-dependent histone H3 acetylation. Moreover, Rad16 is an ATP-ase of the SNF2 superfamily and therefore might facilitate chromatin repair by nucleosome remodelling. Here, we used yeast rad7 Delta rad16 Delta mutants and show that Rad7-Rad16 is also required for NER of UV-lesions in three functionally distinct nucleosome-free regions (NFRs), the promoter and 3'-end of the URA3 gene and the ARS1 origin of replication. Moreover, rapid repair of UV-lesions by photolyase confirmed that nucleosomes were absent and that neither UV-damage formation nor rad7 Delta rad16 Delta mutations altered chromatin accessibility in NFRs. The data are consistent with a role of Rad7-Rad16 in damage recognition and processing in absence of nucleosomes. An additional role in nucleosome remodelling is discussed.

Changes in the proteome after neuronal zif268 overexpression.

Long-lasting forms of brain plasticity are a cellular basis for long-term memory, and their disturbance underlies pathological conditions such as dementia and cognitive impairment. Neuronal plasticity is a complex process that utilizes molecular cascades in the cytoplasm and the nucleus and involves numerous transcription factors, in particular, immediate early genes (IEGs). The signaling cascades that control IEGs are fairly well described, but the downstream transcriptional response is poorly understood, especially its late components. Here, we investigated the response induced by the IEG Zif268 in the adult brain in relation to long-term memory. Using a mouse model with increased neuronal expression of Zif268 that leads to improved memory, we identified an ensemble of proteins regulated by Zif268 expression and differentiated between direct and indirect targets based on the presence of a consensus binding motif in their promoter. We show that Zif268 regulates numerous substrates with diverse biological functions including protein modification and degradation (proteasome-core complex), phosphorylation, cell division, sensory perception, metabolism, and metal ion transport. The results provide a comprehensive and quantitative data set characterizing the Zif268-dependent proteome in the adult mouse brain and offers biologically important new insight into activity-dependent pathways downstream of IEGs.

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Repair of active and silenced rDNA in yeast: the contributions of photolyase and transcription-couples nucleotide excision repair.

DNA repair by photolyase (photoreactivation) and nucleotide excision repair (NER) are the major pathways to remove UV-induced cyclobutane-pyrimidine dimers (CPDs). The nucleolus is a nuclear subcompartment containing the ribosomal RNA genes (rDNA) of which a fraction is transcribed by RNA polymerase I (RNAP-I), and the rest is silenced. Here yeast was used to investigate how photoreactivation and NER contribute to repair of active and inactive rDNA. Cells were irradiated with UV light and exposed to different repair conditions. Nuclei were isolated, and the active genes were separated from the inactive genes by restriction endonuclease digestion. CPDs were measured in total rDNA, in both fractions, and in the GAL10 gene. Repair in rDNA was as efficient as in GAL10 indicating that both pathways have unrestricted access to the nucleolus. Photoreactivation was much faster than NER and therefore was the predominant repair pathway. Active genes were faster repaired by photolyase than were silenced genes providing evidence for an open chromatin structure during repair. The transcribed strands of active genes, but not of inactive genes, were slightly faster repaired by NER providing evidence for transcription-coupled repair by RNAP-I. There was no pronounced inhibition of photoreactivation by RNAP-I in the transcribed strand, which is in contrast to genes transcribed by RNAP-II and suggests different stabilities of RNAP-I and RNAP-II stalled at CPDs.

Repair of UV lesions in silenced chromatin provides in vivo evidence for a compact chromatin structure.

Genes positioned close to telomeres in yeast are silenced by a heterochromatin-like structure containing Sir proteins. To investigate whether silencing also affects DNA repair, we studied removal of UV lesions by photolyase and nucleotide excision repair (NER) in strains containing the URA3 gene inserted 2 kilobases from a telomere. URA3 was transcriptionally active in sir3delta mutants, partially silenced in SIR3 cells, or completely silenced by overexpression of SIR3 or deletion of RPD3. The active URA3 showed efficient repair by both pathways. Fast repair of the promoter and 3' end by photolyase reflected a non-nucleosomal structure. Partial silencing had no remarkable effect on photolyase but reduced repair by NER, indicating differential accessibility for the two repair reactions. Complete silencing inhibits NER and photolyase in the coding region as well as in the promoter and the 3'-end. Conventional nuclease footprinting analyses revealed subtle changes in the promoter proximal nucleosome under partially silenced conditions but a pronounced reorganization of chromatin extending over the whole gene in silenced chromatin. Thus, both repair systems are sensitive to chromatin changes associated with silencing and provide direct evidence for a compact structure of heterochromatin.