New & Noteworthy
Acetylation Regulates the Nuclear Pore Complex
July 15, 2022
Several recent studies have done an excellent job characterizing the architecture of the yeast nuclear pore complex (NPC). With so much new information, researchers are now able to ask probing questions about how NPCs mediate communication between the nucleus and the rest of the cell. Considering that signals perceived from the environment need to reach the transcriptional machinery in the nucleus, and that mRNA transcripts made in response to these signals need to get back out to get translated, the NPC has a lot of communicating to do. A study in a recent issue of the EMBO Journal by Gomar-Alba et al. makes strong strides toward understanding how this communication is accomplished.
On the nuclear side of the NPC resides a substructure called the nuclear basket that has previously been shown to play roles in regulating gene expression and mRNA export. The nuclear basket also interacts with lysine acetyltransferases (KATs) and deacetylases (KDACs) that are best known for modulating transcription via reversible acetylation of histones in chromatin. These enzymes, however, can also act on non-histone proteins and have been linked to numerous cell processes, including DNA damage repair, cell division, and signal transduction.
Promotion of mRNA export is another function linked to acetylation, specifically by the NuA4 histone acetyltransferase complex, for which the catalytic subunit is Esa1p. Gomar-Alba et al. show in this recent study that Esa1p is the primary lysine acetyltransferase that promotes cell cycle entry—and also that it acetylates the nuclear pore protein Nup60p.
Acetylation of Nup60p promotes mRNA export, which in turn triggers fast entry into the Start phase of the cell cycle, thereby promoting cell division. Nup60p accomplishes this increased export by recruiting the TREX-2 transcription-export complex to the nuclear basket once Nup60p becomes acetylated. The deacetylated form of Nup60p has lower affinity for TREX-2 and thus mRNA export decreases. Deacetylation of Nup60p is performed by Hos3p, which acts in opposition to Esa1p in removing Esa1p-transferred acetyl residues.
Perhaps the most intriguing finding in this study is that Hos3p localizes primarily to daughter cells after cell division, causing displacement of the mRNA export complex and thus slowing G1/S phase transition. This action prevents premature division in the smaller daughter cells, as they require additional growth to meet the size control threshold for entry into a new cell cycle. Accomplishing this level of control with a single enzyme acting on a single nuclear pore protein is a simple, elegant solution.
As usual, studies in yeast make enormous impact on understanding cell division in other organisms.
Categories: Research Spotlight
Tags: acetylation, cell cycle control, mRNA export, nuclear export, nuclear pore, Saccharomyces cerevisiae
Pathway Between Copper and Disease Revealed in Yeast
July 08, 2022
Several lethal genetic disorders in humans are caused by mutations that cause symptoms of copper (Cu) deficiency, even in the presence of copper. These copper-deficiency disorders are fatal and include Menkes disease, Friedreich’s ataxia, and neurological and cardiac defects in infants due to lack of copper supply to cytochrome c oxidase in mitochondria. Given that no treatments are currently available for these terrible disorders, researchers have been interested in drugs that might improve copper bioavailability. The copper-binding oncological drug elesclomol (ES) has been identified as a candidate.
In a recent report by Garza et al. in the Journal of Biological Chemistry, the authors use the facility of yeast genetics to ask detailed questions about how ES affects metal homeostasis in yeast cells. It was previously established that perturbation in the levels of one metal tends to cause perturbations in supply of other metals, and thus they asked directed questions about both copper and iron (Fe). Deficiency in copper can cause linked deficiencies in bioavailable iron, and both are critically important for metal-dependent enzymes in mitochondria.
The crux of the team’s findings was that supplementing copper-deficient yeast cells with Cu-bound ES (ES-Cu) not only increased Cu levels, but nearly doubled Fe levels in mitochondria. They were able to show that ES transports copper by an alternate route that bypasses the major yeast copper importer (Ctr1p). While this is an intriguing and perhaps encouraging result, perturbations in metals have so much potential to be toxic that it remains critically important to understand the relationships between the components.
The authors found that application of preformed ES-Cu complex is more efficient than ES at transporting Cu, and that transport of Cu across the plasma membrane by the drug occurs by passive transfusion, not active transport. Further, they made the critical discovery that copper delivered by ES goes first to the Golgi lumen, not directly to mitochondria as the authors had expected based on studies in other models. At the Golgi, the Cu is made available to the copper-transporting ATPase Ccc2p, which in turn assists in metalating Fet3p, a multicopper oxidase that oxidizes ferrous (Fe2+) to ferric iron (Fe3+). Once activated with copper in the Golgi, Fet3p-Cu is transported to the plasma membrane, where it oxidizes iron to Fe3+, the form that can be taken up by the iron transporter Ftr1p. This increased transport of iron leads to increased bioavailability in mitochondria. Thus, the link between copper and iron by means of an alternative copper transporter becomes more clear.
Interestingly, copper and iron metabolism are linked in both humans and yeast. The yeast protein Fet3p has two homologs in humans (ceruloplasmin and hephaestin), both of which are multicopper oxidase proteins critical for normal iron metabolism. From these powerful studies in yeast, it appears that disorders of Cu metabolism cause defects in Fe metabolism due to disrupted metalation of these cuproenzymes within the Golgi. Indeed, this is an intriguing finding that opens avenues for possible therapies, and it would be hard to imagine making this connection without the use of the yeast model.
Categories: Research Spotlight
Tags: copper, copper transport, cytochrome c oxidase, iron, iron transport, mitochondria, Saccharomyces cerevisiae, yeast model for human disease
Snf5p Senses pH to Combat Starvation
July 01, 2022
Yeast are keenly sensitive to internal pH. Several membrane proteins pump H+ ions out of the cell to keep the internal pH near neutral. When carbon becomes scarce, however, it is essential for survival that these pumps get inactivated so the internal space is rapidly acidified. This acidification is postulated to conserve energy and trigger a number of subsequent pathways to combat starvation. Key among these adaptive responses is the derepression of the glucose-repressed genes. The well-studied SWI/SNF complex has been established as a key mediator for this, but the details of how the transcriptional boost is effected have not been known.
A recent study in eLife by Gutierrez et al. has shown the pivotal subunit of the SWI/SNF complex to be Snf5p, which performs a regulatory role by sensing pH. But how would a protein sense pH?
In studying the sequence and structure of the eleven subunits of the SWI/SNF complex, the authors noted that ten of the eleven subunits had large intrinsically disordered regions and that four of the eleven contained glutamine-rich low-complexity sequences (QLCs) that contain multiple histidine residues. QLCs were previously identified as important for binding transcription factors.
In looking for a link between pH and activation, the authors postulated that the histidine residues might be important because the histidine sidechain has an intrinsic pKa of 6.9, and thus might change conformation when pH drops.
Detailed comparative analysis of QLCs from yeast and other organisms led the authors to conclude that, in yeast, the histidines are salient features of QLCs that have been evolutionarily conserved. Given this, they noted that the N terminus of Snf5p has one of the largest QLCs in the whole yeast proteome and is in the top three for number of histidines.
Naturally, given the tools of the yeast model, the next step was to mutate the protein, for which they compared a full deletion against an N-terminal deletion of the QLC and a targeted allele with four histidines within the QLC mutated to alanine.
They found that total loss of the gene was phenotypically distinct from either of the QLC-targeted mutants. Total absence of the protein caused disruption of the SWI/SNF architecture, while QLC-mutants maintained an intact complex but showed disruptions in transcriptional reprogramming in response to starvation, as specifically measured by derepression of the ADH2 gene.
By a subsequent series of elegant biochemical experiments—conducted both in vivo and in vitro—the authors show with great precision how the Snf5p QLC specifically senses pH to trigger widespread reprogramming of genes that will help yeast metabolize non-preferred carbon sources. Even more specifically, they show how acidification leads to protonation of the histidines in the QLC, causing that region of the protein to expand and change conformation, thereby affecting the binding properties of the whole SWI/SNF complex.
The ability to do these experiments and develop a model of how the cell accomplishes delicate regulation once more astounds us with the awesome power of yeast genetics.
Categories: Research Spotlight
Tags: glucose metabolism, glucose repression, glucose starvation, glucose-repressed genes, pH sensing, Saccharomyces cerevisiae, signal transduction
The Remarkable Nuclear Pore Complex
June 24, 2022
The nuclear pore complex (NPC) is a complicated assembly embedded in the nuclear envelope that has the ability not only to assemble and disassemble quickly, but to adapt to changing needs for transport of macromolecules. The critical function of this elaborate complex has led researchers to invest intensive study, which has recently yielded remarkable new understanding.
In a January study in Cell, Akey et al. describe resolving the yeast nuclear pore complex to astounding detail. Using both in situ and isolated complexes, they dissect the layered organization of the pore to characterize the flexible inner ring, the adaptin-like central layer, and then the membrane-interacting layer that anchors the complex.
Each of these layers employs complex protein connections that together form “spokes” in the pore. The authors speculate that the multiple layers and flexible connectors provide the means for NPCs to assemble and disassemble as quickly as they do, giving the ability to react to cell cycle stages and environmental conditions.
Interestingly, upon close examination of crystalized structures, the authors observed that yeast has both single and double outer rings. The double outer ring has been observed in two other fungi to date and appears to represent a functional variant.
Upon further examination, the authors identified a third variant in yeast and were able to show that the variants co-exist in cells. One variant has two single outer rings that frame the inner ring, a second form has a single ring on the cytoplasmic surface and a double ring on the nuclear surface (both with nuclear baskets); while a third variant has two single rings, no baskets, and is specifically enriched over the nucleolus.
These variants provide further clues as to how the NPC might assemble as modular structures with multiple forms that adapt to different conditions. Further, the inner ring appears to have the ability to dilate and contract to allow smaller versus larger macromolecules to pass through, thereby adding another means of adaptability.
Understanding the yeast nuclear pore complex provides a foundation for understanding the eukaryotic NPC in general. A paper released this past week by Petrovic et al. in Science looked closely at just this set of relationships, comparing the human NPC to both the S. cerevisiae and Chaetomium thermophilum fungal NPCs. They show how, despite low conservation of sequence among nucleoporins and the other components of the pore complex, there is strong evolutionary conservation of the linker-scaffold architecture between humans and fungi. Once more, studies on model organisms throw bright light on the inner workings of our own cells.
Categories: Research Spotlight
Tags: nuclear pore, nucleocytoplasmic transport, Saccharomyces cerevisiae, yeast model for human cells
Mpe1p essential for mRNA processing
June 17, 2022
Messenger RNA (mRNA) 3′ end processing is an evolutionarily conserved and highly controlled process which requires several components from translation/transcription machinery. This processing involves monitoring nascent mRNAs for specific sequences, endonucleolytic cleavage, adding poly(A) tails, and triggering transcription termination. In budding yeast, the 3′ end processing machinery involves the cleavage and polyadenylation factor (CPF) complex and RNA-binding cleavage factors CF IA and CF IB. Based on the different enzymatic roles, the CPF complex has three distinct modules: polymerase, phosphatase, and nuclease.
A new study by Rodriguez-Molina et al. in Molecular Cell provides exciting insights into the role of the CPF complex in the 3′ end processing. The study provides strong evidence that Mpe1p, a nuclease module subunit of CPF, is involved in polyadenylation, cleavage, and transcription termination. The data show that in the presence of RNA, Mpe1p directly interacts with the polymerase module subunit Pfs2p through residues 207-268. The authors designate this region as a pre-mRNA-sensing region (PSR) of Mpe1p. However, another nuclease module subunit, Cft2p, hinders this Mpe1-polymerase module interaction.
It is known that the polymerase module subunit Yth1p, interacts with RNA via a polyadenylation signal (PAS), a conserved sequence of A1A2U3A4A5A6. The authors show that in addition to Yth1p, Mpe1 interacts with PAS through the P215 residue. The authors hypothesize that Mpe1-PSR interacts with the polymerase module only after the Yth1p recognizes the PAS RNA, suggesting that Mpe1p may be able to ‘sense’ the RNA-polymerase module binding.
Further, to understand the importance of the PSR region in Mpe1p, the authors analyzed mutants where its interaction with Pfs2p (mpe1-W257A,Y260A) or PAS (mpe1-P215G) is disrupted. Both mutants show reduced endonuclease and polyadenylation activities. Thus, suggesting that the same residues in Mpe1p involved in PSR-RNA/Pfs2 binding are also responsible in activating cleavage and regulating polyadenylation. A similar effect on endonuclease and polyadenylation activities is observed in a mutant where the CPF complex lacks the Mpe1p subunit, further corroborating the role of Mpe1p in mRNA 3′ end processing.
In the CPF complex, Mpe1p interacts with Ysh1p, another nuclease module subunit, via its N-terminal ubiquitin-like domain (UBL). This interaction stabilizes Mpe1p with the nuclease module (and CPF complex) even in the absence of RNA. To evaluate the importance of this interaction, a variant where the UBL region of Mpe1p is disrupted (mpe1-F9A,D45K,R76E,P78G) was generated. This variant is unable to form a stable CPF complex and show deficiencies in activating cleavage and polyadenylation activities. Thus the authors conclude that Mpe1p-Ysh1p interaction is essential for proper processing of 3′ end mRNA.
Another important aspect of mRNA 3′ end processing is the timely termination of transcription. The data show that the CPF complex lacking Mpe1p is unable to successfully terminate the transcription in time. Furthermore, the authors show that the PSR region specifically influences the role of Mpe1p in timely transcription termination.
Thus, the study highlights the role of Mpe1p as an essential subunit of the CPF complex in mRNA 3′ end processing, specifically in cleavage, polyadenylation, and transcription termination.
Categories: Research Spotlight
Tags: CPF complex, Mpe1p, mRNA 3' end processing, Saccharomyces cerevisiae, transcription
Chromatin Modulators Act in Fungal Drug Resistance
June 10, 2022
Candida glabrata and Saccharomyces cerevisiae are closely related yeasts of which the former is pathogenic to mammals and the latter is used to make bread, wine, and beer. At present, the ~400,000 annual cases of life-threatening Candida infection are at risk of increase due to multidrug-resistant strains. Fungal drug resistance relies on the fungal-specific pleiotropic drug resistance (PDR) pathway, in which ATP is consumed to pump drugs out of cells. The PDR pathway is similar among related fungi and a recent study by Nikolov et al. in Nature Communications leveraged this similarity to delve further into genetic regulation of PDR drug resistance in the tractable and benign S. cerevisiae. The team then applied these findings to the virulent C. glabrata.
In S. cerevisiae, the genes involved in PDR share a common promoter element called the PDR responsive element (PDRE). Nikolov et al. discovered that the histone chaperone Rtt106p has an unexpectedly strong binding affinity for PDREs, suggesting a role in transcriptional activation separate from histone modulation. They further show that Rtt106p works through the PDR gene PDR3, a transcriptional activator, to regulate basal expression of PDR-related genes. They found that basal expression of PDR5, the main multidrug transporter of the PDR pathway, requires Rtt106p binding the PDR5 promoter in concert with Pdr3p.
In response to drugs, however, a different set of regulators come into play. The authors show that neither RTT106 nor PDR3 is responsible for drug induction of resistance genes, but rather that PDR1 is critical for drug-induced upregulation of PDR5. By means of the minichromosome isolation technique, they further show how a different histone-modulating mechanism—the SWI/SNF ATP-dependent chromatin-remodeling complex—both binds to and activates the PDR5 promoter in response to an azole-type antifungal drug.
As the goal of this work was to better understand PDR in pathogenic yeasts, the team next looked at the orthologs of the histone modulation genes in C. glabrata. The summary of their findings is that neither CgRtt106 nor CgSWI/SNF acts in maintenance of basal expression of PDR network genes, but each is clearly required for drug-induced expression. From this work, the authors conclude that these chromatin modulators studied in budding yeast constitute potential therapeutic targets in the pathogenic yeast. The use of a tractable model yeast has once more proven its value in combat of fungal multidrug resistance.
Categories: Research Spotlight
Tags: fungal drug resistance, fungal multidrug resistance, pleiotropic drug resistance, Saccharomyces cerevisiae, yeast model for pathogenic yeasts
New Twist on INO80 Chromatin Remodeling
June 03, 2022
The INO80 chromatin remodeling complex has long been the subject of intense study. Despite this, a recent report by Hsieh et al. in Molecular Cell reveals a new and unexpected biological activity: the INO80 complex (as compared to the other classes of chromatin remodelers) has a unique ability to act not only on nucleosomes but to enable transient detachment of an H2A–H2B histone dimer to form smaller hexasomes, which are slid and repositioned differently from nucleosomes.
Intriguingly, the authors demonstrate that the INO80 complex not only has the ability to act on hexasomes, but prefers to remodel hexasomes. Using in vitro biochemistry, they show that hexasomes are better substrates for the enzyme complex, better stimulate the enzyme’s ATPase activity, and are remodeled faster than full-size nucleosomes.
To explore the mechanisms underlying these observations, the authors asked about the acidic patches on H2A-H2B dimers. Given how previous studies had shown the importance of these patches for remodeling activity, the loss of one dimer of the two might be expected to hamper remodeling—not improve it. Instead, the team used a clever experiment with asymmetric nucleosomes containing mixtures of wild-type versus acidic patch mutant (APM) dimers to show how INO80 requires only a single acidic patch to maintain remodeling rates.
Arp5p is the protein within the INO80 complex that interacts most directly with acidic patches on histone H2 dimers. Using another series of in vitro experiments on reconstituted chromatin with a restriction enzyme accessibility assay and INO80(Δarp) (i.e. the complex lacking Arp5p), the authors show how the acidic patch specifically promotes formation of a key intermediate that primes the nucleosome for sliding along DNA.
That these complex experiments are so informative relies on the long history of studying yeast genes and proteins. These newer studies build on the breadth of earlier examinations to look at the complex abilities of protein assemblies to perform both overlapping and unique biochemical actions. The study of how chromatin is opened to allow transcription in a regulated fashion remains a critical area of study, for which yeast is an ideal model.
Categories: Research Spotlight
Tags: chromatin, chromatin remodeling, hexasomes, INO80 complex, nucleosomes, Saccharomyces cerevisiae, transcription
HIR Complex Influences Transcription Interference
May 27, 2022
Eukaryotic transcription is most often initiated by RNA polymerase II (RNAPII) and regulated by several factors, including epigenetic factors, histone modification, DNA methylation, and non-coding antisense transcripts. Non-coding antisense transcripts, produced from the strand opposite to the sense strand, use transcription interference (TI) to regulate gene expression. TI is a phenomenon where one transcription activity negatively impacts the other in cis. One such example is when transcription of long non-coding RNAs (lncRNAs) overlaps with coding gene promoters, causing repression of that gene. Antisense transcripts are involved in several biological processes and show dysregulation in different diseases; however, the mechanisms underlying the antisense mediated transcription interference (AMTI) are not well understood.
An exciting study by Soudet J et al. in Nucleic Acid Res has identified new components in budding yeast to be involved in AMTI. The authors highlight a strong connection between HIR histone chaperone complex binding and antisense transcription in context with SAGA or TFIID-dependent gene regulation. Likewise, the study shows that induction of antisense transcription influences HIR binding, nucleosome repositioning, and (de novo) histone deposition.
Furthermore, data show that antisense transcription into promoters at SAGA-dependent genes restricts the access of the transcription factor binding sites (TBSs) and transcription factors (TFs) by closing the promoter nucleosome depleted regions (NDRs) with nucleosomes, thus repressing the genes. When antisense elongation begins, nucleosomes are randomly lost from the promoters, and chromatin has a chance to reopen again before new nucleosomes are incorporated. In the absence of HIR complex, the promoters stay open and upregulate the SAGA-dependent genes.
The study confirms that SAGA-dependent genes are associated with higher HIR binding and antisense transcription into promoters. Interestingly, TFIID-dependent genes do not show the same effect even in the presence of high antisense transcription levels. Although SAGA and TFIID-dependent gene classes share a high level of antisense transcription as a common feature, the authors propose that genes can interchange these classes when the balance between TF binding and nucleosome incorporation is changed.
Thus, the study sheds light on the mechanism of the antisense mediated transcription interference and emphasizes the role of the HIR histone chaperone complex in gene regulation.
Categories: Research Spotlight
Tags: Antisense transcription, gene regulation, HIR Complex, Saccharomyces cerevisiae, SAGA, Transcription Interference
A Revelation in Loosening Chromatin to Allow Replication
May 20, 2022
The orderly replication of chromosomes is a daily miracle, driving growth of all higher organisms. Yet chromosomes are packaged into tight little nucleosome balls that must be systematically unpacked before replication can commence. Much is yet to be learned about this process, but a recent study by Chacin et al. in Nature Communications has provided a new piece of the puzzle.
The ATPase Yta7p was originally identified as an element that marked boundaries within chromatin, thereby influencing gene transcription by delineating active versus repressed regions. The protein has domains characteristic of the type-II family of AAA+–ATPases. It was previously noted that Yta7p becomes phosphorylated during S phase and that phosphorylation caused eviction of Yta7p from chromatin. However, the reason a barrier protein would need to be specifically modified during S phase remained elusive.
In this new study, Chacin et al. observed replication defects in yta7 mutants and hypothesized that Yta7p might play a heretofore-unknown role in DNA synthesis. To address this hypothesis, the authors purified the protein and demonstrated a hexameric structure similar to known segregases of the AAA+-ATPase family. Next, the authors established an in vitro assay to assess the activity of the purified protein on packaged nucleosomes. By means of impressive reconstitution experiments, they showed that Yta7p is recruited to acetylated chromatin but does not have activity on chromatin until Yta7p is specifically activated by phosphorylation.
This activating phosphorylation, they show, is performed by the S-CDK complex (CLB5-CDC28 kinase complex) specifically during S phase. Further, phosphorylation was then shown to stimulate the ATPase function of the Yta7p enzyme.
Using a similar set of reconstitution experiments, the authors then asked questions about the activity of activated Yta7p on naked DNA versus chromatin. They showed that Yta7p did not have an effect on either naked DNA or nonacetylated chromatin, but strongly stimulated active replication on acetylated nucleosomes. From this they propose a model whereby active Yta7p causes nucleosomes to disassemble so that origins of replication are accessible to the replication machinery.
Intriguingly, the YTA7 gene is conserved among eukaryotes, with the human homolog ATAD2 identified as an oncogene overexpressed in assorted cancers. The use of yeast to tease out the role of Yta7p in unpackaging nucleosomes ahead of DNA replication sheds light on the possible role of human ATAD2 in tumorigenesis. The simplicity of yeast once again affords insight into complicated systems within cell biology.
Categories: Research Spotlight
Tags: cancer, chromsome replication, DNA replication, Saccharomyces cerevisiae, yeast model for human disease
New Yeast-Based Assay for Classifying BRCA1 Variants
May 13, 2022
Lifetime risk of developing ovarian or breast cancer is increased by germline mutations in the BRCA1 gene. While specific pathogenic variants have been well studied, new sequencing technologies continue to identify variants of uncertain significance (VUS). These variants are comparatively rare and cannot easily be studied in humans. Thus, a recent study in the International Journal of Molecular Sciences by Bellè et al. demonstrates a means to assess pathogenicity of a given variant in a cell-based assay in yeast. The new technique complements previous techniques (one in yeast, others computational) to improve the accuracy and sensitivity of assessing pathogenicity for the numerous variants of BRCA1.
Belle et al. approached their study by noting that BRCA1 mainly affects DNA repair and genome stability, and that yeast has a full toolbox for studying these processes. The team previously demonstrated that pathogenic BRCA1 variants increase the rates of intra- and interchromosomal homologous recombination (HR) and also gene reversion (GR) in yeast.
For the current study, they developed a diploid strain that allows simultaneous assessment of intra- and interchromosomal HR by use of two mutated markers, one that repairs by intrachromosomal exchange and the other by interchromosomal exchange. When they induced BRCA1 variants from a plasmid, they were able to compare rates of HR (compared to the WT BRCA1 gene) by the simple use of plate assays.
Similarly, a haploid strain with a different mutated marker that repairs by gene reversion was used to assess rates of GR upon induction of a BRCA1 variant versus wild type.
After multiple replications of the experiments, the authors plotted Waterfall distributions of the results to arrive at breakpoint values at which a variant could be called benign versus pathogenic for each assay. These results were then analyzed for rates of false positives and false negatives with respect to previous data.
The combination of the two yeast-based assays (HR/GR and SCP, together called yBRCA1) was evaluated for performance and shown to be highly accurate and reliable.
As full concordance of results was obtained for three out of ten VUS between the yeast-based combined method and other non-yeast methods, the authors conclude that no one technique is suitable for making a clinical assessment but that multiple techniques will reduce numbers of VUS and resolving conflicting interpretations. Measurement of DNA repair in yeast is a particularly potent example of how yeast can provide a whole-cell, functional assay that sheds light on cell-division disorders.
Categories: Research Spotlight
Tags: breast cancer, cell-based assay, Saccharomyces cerevisiae, yeast model for human disease