Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination.

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

Structures and dynamics of Rpd3S complex bound to nucleosome.

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

A protein network refinement method based on module discovery and biological information.

BACKGROUND: The identification of essential proteins can help in understanding the minimum requirements for cell survival and development to discover drug targets and prevent disease. Nowadays, node ranking methods are a common way to identify essential proteins, but the poor data quality of the underlying PIN has somewhat hindered the identification accuracy of essential proteins for these methods in the PIN. Therefore, researchers constructed refinement networks by considering certain biological properties of interacting protein pairs to improve the performance of node ranking methods in the PIN. Studies show that proteins in a complex are more likely to be essential than proteins not present in the complex. However, the modularity is usually ignored for the refinement methods of the PINs. METHODS: Based on this, we proposed a network refinement method based on module discovery and biological information. The idea is, first, to extract the maximal connected subgraph in the PIN, and to divide it into different modules by using Fast-unfolding algorithm; then, to detect critical modules according to the orthologous information, subcellular localization information and topology information within each module; finally, to construct a more refined network (CM-PIN) by using the identified critical modules. RESULTS: To evaluate the effectiveness of the proposed method, we used 12 typical node ranking methods (LAC, DC, DMNC, NC, TP, LID, CC, BC, PR, LR, PeC, WDC) to compare the overall performance of the CM-PIN with those on the S-PIN, D-PIN and RD-PIN. The experimental results showed that the CM-PIN was optimal in terms of the identification number of essential proteins, precision-recall curve, Jackknifing method and other criteria, and can help to identify essential proteins more accurately.

Two-way feedback between chromatin compaction and histone modification state explains Saccharomyces cerevisiae heterochromatin bistability.

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae, that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins.

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2.

Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Integrative analysis of DNA replication origins and ORC-/MCM-binding sites in human cells reveals a lack of overlap.

Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.

Unveiling the mechanism of efficient ß-phenylethyl alcohol conversion in wild-type Saccharomyces cerevisiae WY319 through multi-omics analysis.

ß-Phenylethanol (2-PE), as an important flavor component in wine, is widely used in the fields of flavor chemistry and food health. 2-PE can be sustainably produced through Saccharomyces cerevisiae. Although significant progress has been made in obtaining high-yield strains, as well as improving the synthesis pathways of 2-PE, there still lies a gap between these two fields to unpin. In this study, the macroscopic metabolic characteristics of high-yield and low-yield 2-PE strains were systematically compared and analyzed. The results indicated that the production potential of the high-yield strain might be contributed to the enhancement of respiratory metabolism and the high tolerance to 2-PE. Furthermore, this hypothesis was confirmed through comparative genomics. Meanwhile, transcriptome analysis at key specific growth rates revealed that the collective upregulation of mitochondrial functional gene clusters plays a more prominent role in the production process of 2-PE. Finally, findings from untargeted metabolomics suggested that by enhancing respiratory metabolism and reducing the Crabtree effect, the accumulation of metabolites resisting high 2-PE stress was observed, such as intracellular amino acids and purines. Hence, this strategy provided a richer supply of precursors and cofactors, effectively promoting the synthesis of 2-PE. In short, this study provides a bridge for studying the metabolic mechanism of high-yield 2-PE strains with the subsequent targeted strengthening of relevant synthetic pathways. It also provides insights for the synthesis of nonalcoholic products in S. cerevisiae.

The GTPase activating protein Gyp7 regulates Rab7/Ypt7 activity on late endosomes.

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.

Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.

The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.

[Enhancing the glycerol utilization of engineered yeast increases its bisabolene production].

Bisabolene is a compound commonly found in essential oils of various plants. It has a broad application in sectors such as chemical, pharmaceutical, and health-care products. This study focuses on modifying the glycerol metabolism pathway to obtain a high bisabolene-producing strain of Saccharomyces cerevisiae. To achieve this, the glycerol transporter gene PtFPS2 from Pachysolen tannophilus and the glycerol dehydrogenase gene Opgdh from Ogataea parapolymorpha were overexpressed in engineered yeast YS036, which was equipped with a GAL promoters-enhanced mevalonic acid pathway. Additionally, the glucose-inhibiting transcription factor MIG1 was knocked out to reduce glucose inhibition. The results showed that the GAL promoter transcription levels of the recombinant yeast strains increased, and the co-utilization of sucrose and glycerol was further improved in MIG1-knockout strain. Moreover, the maximum yield of bisabolene in shaking flask fermentation increased to 866.7 mg/L, an 82.2% increase compared to that of the original strain. By modifying the metabolic pathway of carbon sources, the yield of bisabolene was considerably improved. This study offers an effective strategy for enhancing the yield of terpene compounds in engineered yeast.

Reduced production of Ethyl Carbamate in wine by regulating the accumulation of arginine in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a multisite carcinogenic compound, is naturally produced from urea and ethanol in alcoholic beverages. In order to reduce the content of EC in wine, the accumulation of arginine in Saccharomyces cerevisiae was regulated by genetic modifying genes involved in arginine transport and synthesis pathways to reduce the production of urea. Knockout of genes encoding arginine permease (Can1p) and amino acid permease (Gap1p) on the cell membrane as well as argininosuccinate synthase (Arg1) respectively resulted in a maximum reduction of 66.88% (9.40 µg/L) in EC, while overexpressing the gene encoding amino acid transporter (Vba2) reduced EC by 52.94% (24.13 µg/L). Simultaneously overexpressing Vba2 and deleting Arg1 showed the lowest EC production with a decrease of 68% (7.72 µg/L). The yield of total higher alcohols of the mutants all decreased compared with that of the original strain. Comprehensive consideration of flavor compound contents and sensory evaluation results indicated that mutant YG21 obtained by deleting two allele coding Gap1p performed best in must fermentation of Cabernet Sauvignon with the EC content low to 9.40 µg/L and the contents of total higher alcohols and esters of 245.61 mg/L and 41.71 mg/L respectively. This study has provided an effective strategy for reducing the EC in wine.

Mapping of Prion Structures in the Yeast Rnq1.

The Rnq1 protein is one of the best-studied yeast prions. It has a large potentially prionogenic C-terminal region of about 250 residues. However, a previous study indicated that only 40 C-terminal residues form a prion structure. Here, we mapped the actual and potential prion structures formed by Rnq1 and its variants truncated from the C-terminus in two [RNQ+] strains using partial proteinase K digestion. The location of these structures differed in most cases from previous predictions by several computer algorithms. Some aggregation patterns observed microscopically for the Rnq1 hybrid proteins differed significantly from those previously observed for Sup35 prion aggregates. The transfer of a prion from the full-sized Rnq1 to its truncated versions caused substantial alteration of prion structures. In contrast to the Sup35 and Swi1, the terminal prionogenic region of 72 residues was not able to efficiently co-aggregate with the full-sized Rnq1 prion. GFP fusion to the Rnq1 C-terminus blocked formation of the prion structure at the Rnq1 C-terminus. Thus, the Rnq1-GFP fusion mostly used in previous studies cannot be considered a faithful tool for studying Rnq1 prion properties.

PCNA Unloading Is Crucial for the Bypass of DNA Lesions Using Homologous Recombination.

DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells to progress normally through the cell cycle in the face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex, plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitates an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyze the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that, in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.