Contributions of Histone H3 Nucleosome Core Surface Mutations to Chromatin Structures, Silencing and DNA Repair

Histone H3 mutations in residues that cluster in a discrete region on the nucleosome surface around lysine 79 of H3 affect H3-K79 methylation, impair transcriptional silencing in subtelomeric chromatin, and reveal distinct contributions of histone H3 to various DNA-damage response and repair pathways. These residues might act by recruitment of silencing and DNA-damage response factors. Alternatively, their location on the nucleosome surface suggests a possible involvement in nucleosome positioning, stability and nucleosome interactions. Here, we show that the yeast H3 mutants hht2-T80A, hht2-K79E, hht2-L70S, and hht2-E73D show normal nucleosome positioning and stability in minichromosomes. However, loss of silencing in a subtelomeric URA3 gene correlates with a shift of the promoter nucleosome, while nucleosome positions and stability in the coding region are maintained. Moreover, the H3 mutants show normal repair of UV lesions by photolyase and nucleotide excision repair in minichromosomes and slightly enhanced repair in the subtelomeric region. Thus, these results support a role of those residues in the recruitment of silencing proteins and argue against a general role in nucleosome organization

Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair

Chromatin structure modulates DNA repair by photolyase in vivo.

Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light. To study the role of photoreactivation at the chromatin level in vivo, we used yeast strains which contained minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures. The strains were either proficient (RAD1) or deficient (rad1 delta) in NER. In contrast to NER, photolyase rapidly repairs CPDs in non-nucleosomal regions, including promoters of active genes (URA3, HIS3, DED1) and in linker DNA between nucleosomes. CPDs in nucleosomes are much more resistant to photoreactivation. These results demonstrate a direct role of chromatin in modulation of a DNA repair process and an important role of photolyase in repair of damaged promoters with presumptive effects on gene regulation. In addition, photoreactivation provides an in vivo test for chromatin structure and stability. In active genes (URA3, HIS3), photolyase repairs the non-transcribed strand faster than the transcribed strand and can match fast removal of lesions from the transcribed strand by NER (transcription-coupled repair). Thus, the combination of both repair pathways ensures efficient repair of active genes

RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes.

Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes. To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptionally regulated GAL10 gene in a yeast strain deficient in NER [AMY3 (rad1Delta)]. In the active gene (cells grown in galactose), photoreactivation was fast in the NTS and slow in the TS demonstrating preferential repair of the NTS. In the inactive gene (cells grown in glucose), both strands were repaired at similar rates. This suggests that RNA polymerases II blocked at CPDs inhibit accessibility of CPDs to photolyase. In a strain in which both pathways are operational [W303-1a (RAD1)], no strand bias was observed either in the active or inactive gene, demonstrating that photoreactivation of the NTS compensates preferential repair of the TS by NER. Moreover, repair of the NTS was more quickly in the active gene than in the repressed gene indicating that transcription dependent disruption of chromatin facilitates repair of an active gene

Nucleotide excision repair in a constitutive and inducible gene of a yeast minichromosome in intact cells.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction

Transcription through the yeast origin of replication ARS1 ends at the ABFI binding site and affects extrachromosomal maintenance of minichromosomes.

When the function of origins of replication in yeast was compromised by placing ARS sequences downstream of strong promoters, ARS activity might have been affected either by transcription or by an altered chromatin configuration induced by the construct. To distinguish between these possibilities, derivatives of the yeast TRP1ARS1 minichromosome were constructed that contained either the DED1 or the PET56 promoter firing against ARS1 (DEDARS and PETARS constructs). PETARS constructs transformed yeast at high frequencies and were maintained as minichromosomes consistent with efficient ARS1 function, but DEDARS constructs transformed at low frequencies and had to be rescued as minichromosomes by insertion of a second ARS (H4-ARS). Chromatin analysis revealed that the ARS1 regions in PETARS and H4-DEDARS constructs were indistinguishable from the ARS1 region of the host TRP1ARS1 circle showing a nuclease sensitive region flanked by a nucleosome. However, RNA-analysis in the ARS region showed high and low levels of transcripts in H4-DEDARS and PETARS, respectively. Transcription elongated through the A, B1, and B2 elements and ended in B3, the binding site for ABFI. We conclude that transcription through ARS1 and not an altered chromatin structure affected ARS activity in these constructs

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

Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity