New & Noteworthy
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
Role of Npl3p in regulating senescence rates
May 06, 2022
Telomeres, regions of repetitive DNA at the terminal ends of linear chromosomes, function as protective caps essential to maintain chromosomal structural integrity. Telomeres shorten with every cell cycle and eventually activate replicative senescence (a checkpoint-mediated cell cycle arrest) once they reach a critical length. Regulation of telomere length is essential as an uncontrolled shortening of telomeres can cause organismal aging, and the inability to trigger senescence can result in tumor development. In budding yeast, regulatory factors such as TERRA (telomere repeat containing RNA), a RNAPII-transcribed long non-coding RNA at all telomeres, and R-loops promote Homology-Directed Repair (HDR) at critically short telomeres. Whereas at long telomeres, RNase H2 and Rat1p are recruited and function to degrade TERRA and R loops during S phase.
An interesting new study by Perez-Martinez L et al. in EMBO identifies a telomere-binding protein, Npl3p, to stabilize R-loops at critically short telomeres to prevent premature senescence. The authors propose that at short telomeres, TERRA recruits Npl3p, and the bound protein stabilizes R-loops by limiting access to degrading enzymes like Rat1p and RNAse H2. As a result, the stabilized R-loops promote HDR, and telomeres are elongated to prevent early senescence. Conversely, the absence of Npl3p causes R-loop instability and a defective HDR mechanism, unable to constrain the senescence rates.
The authors additionally show that like TERRA and R-loops, Npl3p levels are regulated in a cell-cycle-dependent manner, with increased accumulation during the early S phase followed by a decline in the late S phase. The study also points to the fact that several proteins or complexes, including Npl3p and Tlc1p, accumulate strongly at short telomeres and in the presence of RNAse A and RNAse H, this interaction is lost.
The study highlights Npl3p as an essential factor in studying the diseases associated with dysregulation of telomere length and senescence rates.
Categories: Research Spotlight
Tags: Homology-Directed Repair, Saccharomyces cerevisiae, senescence, telomere, telomere length
Degradation of Mmr1 protein vital for mitochondrial dynamics
April 29, 2022
Regulating mitochondrial dynamics during cell division is crucial for maintaining homeostasis in eukaryotic cells. In budding yeast, proteins such as Myo2p and Mmr1p are essential to transport mitochondria into the growing bud, which post cell division, exists as an independent daughter cell. Improper inheritance and/or distribution of mitochondria can generate reactive oxygen species (ROS) toxicity in daughter cells.
An interesting new study by Obara K et al. in Nature Communications shows that degradation of Mmr1p is crucial in maintaining mitochondrial homeostasis in budding yeast. During cell division, Mmr1p bridges mitochondria and Myo1p and assists in its transportation to the bud via actin cables. Once the transportation is complete, kinases such as Ste20p and Cla4p phosphorylate Mmr1, leading to its recognition and poly-ubiquitination by E3 ligases, Dma1p/Dma2p. The proteasome degrades the poly-ubiquitinated and phosphorylated Mmr1p, and the mitochondria are released from Myo2p and distributed in the bud. Once released, Myo2p translocates to the bud neck.
The study shows that in the absence of Dma1p/Dma2p, the transported mitochondria are not released from the actin-myosin machinery but in fact, become expanded or deformed and accumulate at the bud tip and then at the bud neck (due to translocation of Myo2p). Double mutants with altered mitochondrial morphology exhibit elevated respiratory activity and increased generation of ROS, rendering cells hypersensitive to oxidative stress
Additionally, the authors show that bud-localized kinases, Ste20p, and Cla4p phosphorylate (most probably) the serine residue at amino acid 414 of Mmr1p and regulate its degradation only after the mitochondria are transported to the growing bud, a crucial step in proper mitochondrial distribution.
Thus, the study highlights the importance of Mmr1p, Dma1p, and Dma2p in maintaining mitochondrial morphology and distribution required to sustain cell homeostasis in yeast cells.
Categories: Research Spotlight
Tags: cell division, cell homeostatis, mitochondrial inheritance, Saccharomyces cerevisiae
Yeast Lifespan Impacted by rDNA Copy Number
April 22, 2022
Replicative lifespan (RLS), determined by the number of daughter cells a mother cell produces before death, has proved to be an effective model for studying aging in budding yeast. The chromatin-associated proteins Sir2p and Fob1p have been shown to modulate ribosomal DNA (rDNA) and impact the formation of extrachromosomal rDNA circles (ERCs), an accumulation of which is linked to a shorter lifespan.
A recent study by Hotz M et al. in PNAS has shown that chromosomal rDNA copy number (CN) positively correlates with RLS in budding yeast. The authors performed whole-genome sequencing (WGS) of 13 wild-type strains and analyzed the lifespan data, which showed an increased rDNA CN along with enhanced RLS. Additionally, the data showed that the rDNA CN explains the majority (~ 70%) of RLS variation observed in almost identical wild-type strains.
To understand this correlation, the authors analyzed ERC levels in aging cells and fob1-Δ strains, in which ERC levels are low. Together, the analysis concluded that ERCs are inversely correlated with rDNA CN. Exploring further, the authors found that cells with lower rDNA CN showed improved accessibility of the upstream activating factor (UAF) complex binding site at the SIR2 locus. This change in chromatin accessibility reduces the expression of SIR2, causing higher ERC levels and thus a shortened lifespan, implicating both Sir2p levels and ERCs as the underlying cause of the CN-RLS correlation.
Additionally, the authors analyzed the CN-RLS relationship in a set of mutant strains (such as hda2-Δ, upb8-Δ, gpa2-Δ, etc.), all known to increase lifespan. The data showed that while some mutants appeared to impact the CN-RLS relationship, rDNA CN strongly influenced the RLS of these mutant strains (except for fob1-Δ).
Thus, the study demonstrates how rDNA CN impacts yeast lifespan by regulating certain aging factors and highlights rDNA copy number as an essential parameter to examine in aging studies.
Categories: Research Spotlight
Tags: replicative life span, Saccharomyces cerevisiae, yeast model for aging
Yeast Reveals an Alzheimer’s Clue
April 15, 2022
In a recent issue of EMBO Molecular Medicine, an intriguing study with potential implications for Alzheimer’s disease (AD) by Ring et al. used yeast to look at why human amyloid beta 42 (Abeta42) kills cells. Upon overexpression in yeast, human Abeta42 protein oligomerizes into aggregates that translocate to mitochondria, where the aggregates cause oxidative stress and eventual necrotic-like cell death. Functional mitochondria are required for this Abeta42-mediated death, and a combined genetic and proteomic approach in yeast identified the HSP40-type chaperone Ydj1p as critical for stabilizing the oligomers and escorting them to mitochondria.
The effect of ydj1Δ deletion was to lower toxicity, with the effect specific to Abeta42 and not to a different type of induced cell death in a yeast model for Parkinson’s disease. Further, Ydj1p protein directly interacted with Abeta42 in a co-immunoprecipitation experiment.
Yeast YDJ1 is homologous to human DnaJA1, which, when expressed in yeast, re-established the toxicity of Abeta42 to a ydj1Δ strain, indicating functional complementation. The human protein directly interacted with Abeta42 in a murine model for Alzheimer’s disease and also displayed dysregulation in post mortem brain samples of AD patients.
In a fly model for AD, deletion of Droj2 (the Drosophila melanogaster ortholog of YDJ1 and DnaJA1) not only reduced the toxicity of Abeta42 but significantly improved the short-term olfactory memory loss associated with Abeta42 expression. Together, the authors convincingly demonstrate the strong evolutionary conservation of this particular chaperone and its effects on exacerbating Abeta42-mediated toxicity, cell death, and memory loss in relevant model systems. The use of yeast to identify this key factor indicates the power of a simplified and tractable system and will hopefully lead to progress in treating a terrible disease.
Categories: Research Spotlight
Tags: alzheimer's, Cell toxicity, Saccharomyces cerevisiae, yeast model for human disease
The Ties That Bind Mitochondria and Aging
April 08, 2022
Continuing our theme of highlighting lifespan in yeast, this week’s study by Liu et al. in eLife looks at a possible mechanism for age-related loss of mitochondrial quality. As loss of mitochondrial quality is linked to the rate of building new mitochondria, and as mitochondrial biogenesis depends on the import of new building blocks through the mitochondrial membranes, the abundance of transporters is an important determinant. The authors wondered whether cells were “smart” enough to coregulate the level of transporters with the level of new building blocks.
To get at this question, they overexpressed the translocases of the outer membrane (TOM) proteins to ask if the overabundance of a particular one caused the overabundance of representative mitochondrial proteins, i.e. displayed a regulatory role. Of these, only TOM70 overexpression (OE) increased the abundance of the four representative mitochondrial proteins. They further showed that TOM70 OE led to increased levels of mitochondrial DNA, likely via Mip1p, the mitochondrial DNA polymerase gamma. As further evidence of the role of TOM70 in regulation, the tom70∆ knockout led to the reverse effect from OE, i.e. reduction in levels of both mitochondrial proteins and mtDNA. Tom70p is a well-conserved receptor within the TOM complex, but this role in regulation of mitochondrial biogenesis is a novel finding.
The authors then examined a number of possible intermediate signaling possibilities between Tom70p and the increased abundance of their target proteins. By disrupting various transcription factors and assessing reactive oxygen species, they saw only partial blockage by loss of any one signaling partner, and thus concluded that Tom70p works via multiple pathways.
As TOM70 appears to have a role in regulating new mitochondria, and mitochondrial defects are well established as an indicator of age, the authors asked about connections between TOM70 and aging. They first noted that Tom70p levels go down over time across many organisms. Is this a cause or an association? To counteract the wild-type reductions in Tom70p levels, they overexpressed TOM70 and then examined age-related phenotypes. Higher levels of Tom70p reversed age-associated loss of mitochondrial membrane potential, age-related reductions in other mitochondrial proteins, and, indeed, extended the lifespan of yeast. Further, the tom70∆ knockout once more showed the reverse effects, leading to accelerated aging and reduced lifespan.
Using the remarkable power of yeast, the authors were then able to study the mechanics of the reduction in Tom70p over time. They found that mRNA levels reduce over time due to loss of transcriptional activity, which was rescuable by changes made to the TOM70 promoter. Further, they showed the degradation of Tom70p increased over time due to increased levels of the protein Dnm1p, involved in sorting proteins for degradation under age-related vacuole deacidification. Thus, TOM70 mRNA levels decrease and Tom70p protein becomes less stable, providing redundancy in reduction of a protein that activates mitochondrial biogenesis. With the relative ease possible in yeast, the authors made important headway in revealing these mechanistic links between mitochondrial quality and aging.
Categories: Research Spotlight
Tags: mitochondrial biogenesis, mitochondrial import, mitochondrial quality, mitochondrial regulation, Saccharomyces cerevisiae, yeast model for aging