Intrinsic cooperativity potentiates parallel cis-regulatory evolution.

Convergent evolutionary events in independent lineages provide an opportunity to understand why evolution favors certain outcomes over others. We studied such a case where a large set of genes-those coding for the ribosomal proteins-gained cis-regulatory sequences for a particular transcription regulator (Mcm1) in independent fungal lineages. We present evidence that these gains occurred because Mcm1 shares a mechanism of transcriptional activation with an ancestral regulator of the ribosomal protein genes, Rap1. Specifically, we show that Mcm1 and Rap1 have the inherent ability to cooperatively activate transcription through contacts with the general transcription factor TFIID. Because the two regulatory proteins share a common interaction partner, the presence of one ancestral cis-regulatory sequence can 'channel' random mutations into functional sites for the second regulator. At a genomic scale, this type of intrinsic cooperativity can account for a pattern of parallel evolution involving the fixation of hundreds of substitutions.

Genome-wide determinants of sequence-specific DNA binding of general regulatory factors.

General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. It is assumed that sequence recognition via direct base readout suffices for specificity and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-base pair (bp) resolution using several ChIP-exo-based assays. We find that computationally predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the nonunique portions of GRF motifs and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels.

A novel MADS-box transcription factor PstMCM1-1 is responsible for full virulence of Puccinia striiformis f. sp. tritici.

In many eukaryotes, transcription factor MCM1 gene plays crucial roles in regulating mating processes and pathogenesis by interacting with other co-factors. However, little is known about the role of MCM1 in rust fungi. Here, we identified two MCM1 orthologs, PstMCM1-1 and PstMCM1-2, in the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). Sequence analysis indicated that both PstMCM1-1 and PstMCM1-2 contain conserved MADS domains and that PstMCM1-1 belongs to a group of SRF-like proteins that are evolutionarily specific to rust fungi. Yeast two-hybrid assays indicated that PstMCM1-1 interacts with transcription factors PstSTE12 and PstbE1. PstMCM1-1 was found to be highly induced during early infection stages in wheat and during pycniospore formation on the alternate host barberry (Berberis shensiana). PstMCM1-1 could complement the lethal phenotype and mating defects in a mcm1 mutant of Saccharomyces cerevisiae. In addition, it partially complemented the defects in appressorium formation and plant infection in a Magnaporthe oryzae Momcm1 mutant. Knock down of PstMCM1-1 resulted in a significant reduction of hyphal extension and haustorium formation and the virulence of Pst on wheat. Our results suggest that PstMCM1-1 plays important roles in the regulation of mating and pathogenesis of Pst most likely by interacting with co-factors.

Inositol phosphate multikinase dependent transcriptional control.

Production of lipid-derived inositol phosphates including IP4 and IP5 is an evolutionarily conserved process essential for cellular adaptive responses that is dependent on both phospholipase C and the inositol phosphate multikinase Ipk2 (also known as Arg82 and IPMK). Studies of Ipk2, along with Arg82 prior to demonstrating its IP kinase activity, have provided an important link between control of gene expression and IP metabolism as both kinase dependent and independent functions are required for proper transcriptional complex function that enables cellular adaptation in response to extracellular queues such as nutrient availability. Here we define a promoter sequence cis-element, 5'-CCCTAAAAGG-3', that mediates both kinase-dependent and independent functions of Ipk2. Using a synthetic biological strategy, we show that proper gene expression in cells lacking Ipk2 may be restored through add-back of two components: IP4/IP5 production and overproduction of the MADS box DNA binding protein, Mcm1. Our results are consistent with a mechanism by which Ipk2 harbors a dual functionality that stabilizes transcription factor levels and enzymatically produces a small molecule code, which together coordinate control of biological processes and gene expression.

Decoding common and divergent cellular functions of the domains of forkhead transcription factors Fkh1 and Fkh2.

Forkhead transcription factors play a key role in embryonic patterning during development. In Saccharomyces cerevisiae, two forkhead transcription factors, Fkh1 and Fkh2, regulate the transcription of CLB2 cluster genes important for mitosis. Fkh1 reduces, whereas Fkh2 elevates, the transcription of CLB2 cluster genes. However, the mechanism for this observation remains unclear. Fkh1 and Fkh2 each contain a forkhead domain (DNA-binding domain, DBD) and a forkhead-associated domain (FHAD), whereas Fkh2 possesses an extra C' domain containing six consensus cyclin-dependent kinase phosphorylation sites. In the present study, roles of these domains in protein complexes, the regulation of cell growth and CLB2 cluster genes and protein interactions were investigated using various domain mutants. The result showed that the DBD was vital for ternary complex formation with Mcm1, whereas the FHAD was central for the regulation of cell growth and CLB2 cluster transcription and for interactions with Ndd1 and Clb2. However, the Fkh2 C' domain was dispensable for the above functions. Both DBDs and FHADs had functional divergences in the cell, and Ndd1 functioned via its phosphorylated form. These data provide important insights into the functional mechanism of Fkh1 and Fkh2 in cell cycle control.

Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast.

Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. (1)H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes.

The MADS-box transcription factor FgMcm1 regulates cell identity and fungal development in Fusarium graminearum.

In eukaryotic cells, MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors. In this study, we functionally characterized the FgMCM1 MADS-box gene in Fusarium graminearum, the causal agent of wheat and barley head blight. Deletion of FgMCM1 resulted in the loss of perithecium production and phialide formation. The Fgmcm1 mutant was significantly reduced in virulence, deoxynivalenol biosynthesis and conidiation. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1 and Fst12, two transcription factors important for sexual reproduction. Whereas Fgmcm1 mutants were unstable and produced stunted subcultures, Fgmcm1 mat1-1-1 but not Fgmcm1 fst12 double mutants were stable. Furthermore, spontaneous suppressor mutations occurred frequently in stunted subcultures to recover growth rate. Ribonucleic acid sequencing analysis indicated that a number of sexual reproduction-related genes were upregulated in stunted subcultures compared with the Fgmcm1 mutant, which was downregulated in the expression of genes involved in pathogenesis, secondary metabolism and conidiation. We also showed that culture instability was not observed in the Fvmcm1 mutants of the heterothallic Fusarium verticillioides. Overall, our data indicate that FgMcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F. graminearum.

Following gene duplication, paralog interference constrains transcriptional circuit evolution.

Most models of gene duplication assume that the ancestral functions of the preduplication gene are independent and can therefore be neatly partitioned between descendant paralogs. However, many gene products, such as transcriptional regulators, are components within cooperative assemblies; here, we show that a natural consequence of duplication and divergence of such proteins can be competitive interference between the paralogs. Our example is based on the duplication of the essential MADS-box transcriptional regulator Mcm1, which is found in all fungi and regulates a large set of genes. We show that a set of historical amino acid sequence substitutions minimized paralog interference in contemporary species and, in doing so, increased the molecular complexity of this gene regulatory network. We propose that paralog interference is a common constraint on gene duplicate evolution, and its resolution, which can generate additional regulatory complexity, is needed to stabilize duplicated genes in the genome.

Bck2 acts through the MADS box protein Mcm1 to activate cell-cycle-regulated genes in budding yeast.

The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G1/S-specific transcription factors SBF (Swi4, Swi6) and MBF (Mbp1, Swi6), yet the mechanism of gene activation by Bck2 has remained obscure. We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment.

Structure and evolutionary origins of the CMG complex.

The CMG (Cdc45-MCM-GINS) complex is the eukaryotic replicative helicase, the enzyme that unwinds double-stranded DNA at replication forks. All three components of the CMG complex are essential for its function, but only in the case of MCM, the molecular motor that harnesses the energy of ATP hydrolysis to catalyse strand separation, is that function clear. Here, we review current knowledge of the three-dimensional structure of the CMG complex and its components and highlight recent advances in our understanding of its evolutionary origins.

[The identification of Saccharomyces cerevisiae genes leading to synthetic lethality of prion [PSI+] with Sup45 mutations].

Previously, we proposed a test system allowing to perform search for genes that influence the properties of the Sup35 and Sup45 protein. This test is based on the phenomenon of lethality of diploids that combine mutations in SUP45 gene with [PSI+] prion. Lethality of this combination depends on the type of sup45 mutation, and the properties of the prion. [PSI+] variant, which is a strong suppressor ([PSI+]s), showing synthetic lethality with all the nonsense mutations and some missense sup45 mutations in the heterozygote state. The presence of extra copies of a gene under test that affects the phenotypic manifestation of prion [PSI+] or translation termination factors properties, leads to the increase or decrease in diploid lethality. Gene library screening using this test system allowed us to establish the effect of ten fragments of genomic DNA of yeast on synthetic lethality. Deletion analysis of these regions has led to the identification of the HLJ1 and TEF2 genes, as affecting Sup35 protein prionization and/or the efficiency of translation termination.

Protein kinase C regulates late cell cycle-dependent gene expression.

The control of the cell cycle in eukaryotes is exerted in part by the coordinated action of a series of transcription factor complexes. This is exemplified by the Mcm1p-Fkh2p-Ndd1p complex in Saccharomyces cerevisiae, which controls the cyclical expression of the CLB2 cluster of genes at the G(2)/M phase transition. The activity of this complex is positively controlled by cyclin-dependent kinase (CDK) and polo kinases. Here, we demonstrate that the protein kinase Pkc1p works in the opposite manner to inhibit the activity of the Mcm1p-Fkh2p-Ndd1p complex and the expression of its target genes. In particular, Pkc1p causes phosphorylation of the coactivator protein Ndd1p. Reductions in Pkc1p activity and the presence of Pkc1p-insensitive Ndd1p mutant proteins lead to changes in the timing of CLB2 cluster expression and result in associated late cell cycle defects. This study therefore identifies an important role for Pkc1p in controlling the correct temporal expression of genes in the cell cycle.

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase.

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1.

Overexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeast Candida albicans. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 for MDR1 expression. We found that Mcm1 was dispensable for MDR1 upregulation by H2O2 but was required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induce MDR1 expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

Same partners, different dance: involvement of DNA replication proteins in centrosome regulation.

Genome replication is the most fundamental element of the continuity of life. In eukaryotes, DNA replication is regulated by an elegant network of many different protein factors to ensure the timely and accurate copying of their entire genome once per cell cycle. The replication factors include the maintenance (MCM) proteins, Cdt1, Cdc6, Cdc7, Cdc45, and geminin. All of these proteins are involved in the regulation of DNA replication at the initiation step. Interestingly, recent studies have shown that some of these replication proteins also localize to the centrosome, often throughout the entire cell cycle. These centrosomally localized replication proteins appear to play essential roles in the regulation of centrosome biogenesis, suggesting that genome replication and segregation are regulated interdependently. In this review, we summarize and discuss the inter-dependent regulation played by some of the replication proteins.

King of the castle: competition between repressors and activators on the Mcm1 platform.

Darieva and coworkers (Darieva et al., 2010) have shown that the repressor Yox1 and the activator Fkh2-Ndd1 associate with opposite sides of the dimeric Mcm1 transcription factor but nevertheless compete for binding; the outcome determines expression of late mitotic genes in yeast.

SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa).

To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

The glutamate switch is present in all seven clades of AAA+ protein.

Recent work has identified a "glutamate switch" in six of the seven clades of AAA+ ATPases. The glutamate switch acts to transduce information regarding substrate binding to the ATPase active site. We provide biochemical evidence that a highly conserved threonine residue acts as a glutamate switch in the replicative helicase, MCM, and, thus, reveal that the glutamate switch is a feature common to all seven AAA+ clades.

Mammalian MCM loading in late-G(1) coincides with Rb hyperphosphorylation and the transition to post-transcriptional control of progression into S-phase.

BACKGROUND: Control of the onset of DNA synthesis in mammalian cells requires the coordinated assembly and activation of the pre-Replication Complex. In order to understand the regulatory events controlling preRC dynamics, we have investigated how the timing of preRC assembly relates temporally to other biochemical events governing progress into S-phase. METHODOLOGY/PRINCIPAL FINDING: In murine and Chinese hamster (CHO) cells released from quiescence, the loading of the replicative MCM helicase onto chromatin occurs in the final 3-4 hrs of G(1). Cdc45 and PCNA, both of which are required for G(1)-S transit, bind to chromatin at the G(1)-S transition or even earlier in G(1), when MCMs load. An RNA polymerase II inhibitor (DRB) was added to synchronized murine keratinocytes to show that they are no longer dependent on new mRNA synthesis 3-4 hrs prior to S-phase entry, which is also true for CHO and human cells. Further, CHO cells can progress into S-phase on time, and complete S-phase, under conditions where new mRNA synthesis is significantly compromised, and such mRNA suppression causes no adverse effects on preRC dynamics prior to, or during, S-phase progression. Even more intriguing, hyperphosphorylation of Rb coincides with the start of MCM loading and, paradoxically, with the time in late-G(1) when de novo mRNA synthesis is no longer rate limiting for progression into S-phase. CONCLUSIONS/SIGNIFICANCE: MCM, Cdc45, and PCNA loading, and the subsequent transit through G(1)-S, do not depend on concurrent new mRNA synthesis. These results indicate that mammalian cells pass through a distinct transition in late-G(1) at which time Rb becomes hyperphosphorylated and MCM loading commences, but that after this transition the control of MCM, Cdc45, and PCNA loading and the onset of DNA replication are regulated at the post-transcriptional level.

Role of Plc1p in regulation of Mcm1p-dependent genes.

In budding yeasts, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) and several inositol polyphosphate kinases represent a nuclear pathway for synthesis of inositol polyphosphates (InsPs), which are involved in several aspects of DNA and RNA metabolism, including transcriptional regulation. Plc1p-produced inositol trisphosphate (InsP(3)) is phosphorylated by Ipk2p/Arg82p to yield InsP(4)/InsP(5). Ipk2p/Arg82p is also a component of ArgR-Mcm1p complex that regulates transcription of genes involved in arginine metabolism. The role of Ipk2p/Arg82p in this complex is to stabilize the essential MADS box protein Mcm1p. Consequently, ipk2Delta cells display reduced levels of Mcm1p and attenuated expression of Mcm1p-dependent genes. Because plc1Delta cells display aberrant expression of several groups of genes, including genes involved in stress response, the objective of this study was to determine whether Plc1p also affects expression of Mcm1p-dependent genes. Here we report that not only ipk2Delta, but also plc1Delta cells display decreased expression of Mcm1p-dependent genes. However, Plc1p is not involved in stabilization of Mcm1p and affects transcription of Mcm1p-dependent genes by a different mechanism, probably involving regulation of chromatin remodeling complexes.