A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration.

Most neurodegenerative diseases such as Alzheimer's disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p-bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.

The asymmetric distribution of RNA polymerase II and nucleosomes on replicated daughter genomes is caused by differences in replication timing between the lagging and the leading strand.

Chromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or whether chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription, and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We found a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNA polymerase II (RNAPII) bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: If transcription and replication are codirectional, the leading strand replicates first; if they are counterdirectional, the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 that impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. We propose that active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state.

Protocol for tracking the inheritance patterns of proteins in live Saccharomyces cerevisiae cells using a photo-convertible fluorescent protein.

Tracking the inheritance patterns of proteins (TrIPP) is a live-cell imaging technique used for tracking maternal protein segregation patterns between mother and daughter cells during asymmetric divisions of budding yeast. We use the photo-convertible fluorescent protein Dendra2 fused to a protein of interest (POI). Irreversible conversion from green to red fluorescence allows for parallel monitoring of old and new proteins for several generations. Single-cell quantitative image analysis of time-lapse microscopy gives synthesis and decay rates, as well as segregation patterns of the POI. For complete details on the use and execution of this protocol, please refer to Auboiron et al. (2021).

TrIPP-a method for tracking the inheritance patterns of proteins in living cells-reveals retention of Tup1p, Fpr4p, and Rpd3L in the mother cell.

Inheritance of chromatin-bound proteins theoretically plays a role in the epigenetic transmission of cellular phenotypes. Protein segregation during cell division is however poorly understood. We now describe TrIPP (Tracking the Inheritance Patterns of Proteins): a live cell imaging method for tracking maternal proteins during asymmetric cell divisions of budding yeast. Our analysis of the partitioning pattern of a test set of 18 chromatin-associated proteins reveals that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3p, Fpr4p, and Tup1p, which are preferentially retained in the mother. Low abundance proteins also tend to be retained in the mother cell with the exception of Sir2p and the linker histone H1. Our analysis of chromatin protein behavior in single cells reveals potentially general trends such as coupled protein synthesis and decay and a correlation between protein half-lives and cell-cycle duration.

Distinct transcriptional roles for Histone H3-K56 acetylation during the cell cycle in Yeast.

Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events.

Dynamics of Nucleosome Positioning Maturation following Genomic Replication.

Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication-how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2'-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.

Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns.

Methylation of histone H3 lysine 4 by the Set1 subunit of COMPASS correlates with active transcription. Here, we show that Set1 levels are regulated by protein degradation in response to multiple signals. Set1 levels are greatly reduced when COMPASS recruitment to genes, H3K4 methylation, or transcription is blocked. The degradation sequences map to N-terminal regions that overlap a previously identified autoinhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or overexpression produce abnormal H3K4 methylation patterns on transcribed genes. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent genes, reflecting differences in their chromatin dynamics. We propose that careful tuning of Set1 levels by regulated degradation is critical for the establishment and maintenance of proper H3K4 methylation patterns.

A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme.

The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 on lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange in which either H2A.Z or H2A can be exchanged from nucleosomes. This result was confirmed in vivo, where genome-wide analysis demonstrated widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a "lock" that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators.

A key role for Chd1 in histone H3 dynamics at the 3' ends of long genes in yeast.

Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5' and 3' ends of genes, accelerating H3 replacement at the 5' ends of genes while protecting the 3' ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3' ends of genes.

Patterns and mechanisms of ancestral histone protein inheritance in budding yeast.

Replicating chromatin involves disruption of histone-DNA contacts and subsequent reassembly of maternal histones on the new daughter genomes. In bulk, maternal histones are randomly segregated to the two daughters, but little is known about the fine details of this process: do maternal histones re-assemble at preferred locations or close to their original loci? Here, we use a recently developed method for swapping epitope tags to measure the disposition of ancestral histone H3 across the yeast genome over six generations. We find that ancestral H3 is preferentially retained at the 5' ends of most genes, with strongest retention at long, poorly transcribed genes. We recapitulate these observations with a quantitative model in which the majority of maternal histones are reincorporated within 400 bp of their pre-replication locus during replication, with replication-independent replacement and transcription-related retrograde nucleosome movement shaping the resulting distributions of ancestral histones. We find a key role for Topoisomerase I in retrograde histone movement during transcription, and we find that loss of Chromatin Assembly Factor-1 affects replication-independent turnover. Together, these results show that specific loci are enriched for histone proteins first synthesized several generations beforehand, and that maternal histones re-associate close to their original locations on daughter genomes after replication. Our findings further suggest that accumulation of ancestral histones could play a role in shaping histone modification patterns.

Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization.

Chromatin domains are believed to spread via a polymerization-like mechanism in which modification of a given nucleosome recruits a modifying complex, which can then modify the next nucleosome in the polymer. In this study, we carry out genome-wide mapping of the Sir3 component of the Sir silencing complex in budding yeast during a time course of establishment of heterochromatin. Sir3 localization patterns do not support a straightforward model for nucleation and polymerization, instead showing strong but spatially delimited binding to silencers, and weaker and more variable Ume6-dependent binding to novel secondary recruitment sites at the seripauperin (PAU) genes. Genome-wide nucleosome mapping revealed that Sir binding to subtelomeric regions was associated with overpackaging of subtelomeric promoters. Sir3 also bound to a surprising number of euchromatic sites, largely at genes expressed at high levels, and was dynamically recruited to GAL genes upon galactose induction. Together, our results indicate that heterochromatin complex localization cannot simply be explained by nucleation and linear polymerization, and show that heterochromatin complexes associate with highly expressed euchromatic genes in many different organisms.

Replication and active demethylation represent partially overlapping mechanisms for erasure of H3K4me3 in budding yeast.

Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as "long-lived" and that is involved in memory during cell state inheritance in metazoans. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, nearly complete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast.

Nucleosome positioning: how is it established, and why does it matter?

Packaging of eukaryotic genomes into chromatin affects every process that occurs on DNA. The positioning of nucleosomes on underlying DNA plays a key role in the regulation of these processes, as the nucleosome occludes underlying DNA sequences. Here, we review the literature on mapping nucleosome positions in various organisms, and discuss how nucleosome positions are established, what effect nucleosome positioning has on control of gene expression, and touch on the correlations between chromatin packaging, sequence evolution, and the evolution of gene expression programs.

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination.

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.

Receipt of the C-terminal tail from a neighboring lambda Int protomer allosterically stimulates Holliday junction resolution.

Bacteriophage lambda integrase (Int) catalyzes the integration and excision of the phage lambda chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (C-terminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the C-terminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the beta5 strand indicate that the observed stimulation arises from specific contacts with the beta5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by beta5 strand contacts are discussed.

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes.

Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.

Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution.

The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.

Arm sequences contribute to the architecture and catalytic function of a lambda integrase-Holliday junction complex.

lambda integrase (Int) mediates recombination between attachment sites on lambda phage and E. coli DNAs. With the assistance of accessory proteins that induce DNA loops, Int bridges pairs of distinct arm- and core-type DNA binding sites to form synapsed recombination complexes, which then recombine via a Holliday junction (HJ) intermediate. We show that, in addition to promoting the proper positioning of Int protomers, the arm sequences facilitate the catalytic activities of the Int tetramer, independent of accessory proteins or physical continuity between the arm and core sites. We have determined the architecture of ternary complexes containing a HJ, Int, and P'1,2 arm-type DNA. These structures accommodate simultaneous binding of Int to direct-repeat arm sites and indirect-repeat core sites and afford a new view of the higher-order recombinogenic complexes.