RESUMO
Aureobasidium pullulans is a yeast-like fungus with remarkable phenotypic plasticity widely studied for its importance for the pharmaceutical and food industries. So far, genomic studies with strains from all over the world suggest they constitute a genetically unstructured population, with no association by habitat. However, the mechanisms by which this genome supports so many phenotypic permutations are still poorly understood. Recent works have shown the importance of sequencing yeast genomes from extreme environments to increase the repertoire of phenotypic diversity of unconventional yeasts. In this study, we present the genomic draft of A. pullulans strain from a Patagonian yeast diversity hotspot, re-evaluate its taxonomic classification based on taxogenomic approaches, and annotate its genome with high-depth transcriptomic data. Our analysis suggests this isolate could be considered a novel variant at an early stage of the speciation process. The discovery of divergent strains in a genomically homogeneous group, such as A. pullulans, can be valuable in understanding the evolution of the species. The identification and characterization of new variants will not only allow finding unique traits of biotechnological importance, but also optimize the choice of strains whose phenotypes will be characterized, providing new elements to explore questions about plasticity and adaptation.
Assuntos
Ascomicetos , Ascomicetos/genética , Saccharomyces cerevisiae , Aureobasidium , GenômicaRESUMO
The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ, hap5Δ, and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.
Assuntos
Maltose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Maltose/metabolismo , Cerveja , Glucose , CromatinaRESUMO
Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: ⢠Tausonia pullulans genome harbors a high number of genes coding for CAZymes. ⢠Among CAZy domains/families, the glycoside hydrolases are the most abundant. ⢠Secretome analysis in glucose or starch as main C sources identified 98 proteins. ⢠A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.
Assuntos
Glucana 1,4-alfa-Glucosidase , Proteômica , Basidiomycota , Cromatografia Líquida , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose , Hidrólise , Amido , Espectrometria de Massas em TandemRESUMO
Phytochrome (PHY)-mediated light and temperature perception has been increasingly implicated as important regulator of fruit development, ripening, and nutritional quality. Fruit ripening is also critically regulated by chromatin remodeling via DNA demethylation, though the molecular basis connecting epigenetic modifications in fruits and environmental cues remains largely unknown. Here, to unravel whether the PHY-dependent regulation of fruit development involves epigenetic mechanisms, an integrative analysis of the methylome, transcriptome and sRNAome of tomato fruits from phyA single and phyB1B2 double mutants was performed in immature green (IG) and breaker (BK) stages. The transcriptome analysis showed that PHY-mediated light perception regulates more genes in BK than in the early stages of fruit development (IG) and that PHYB1B2 has a more substantial impact than PHYA in the fruit transcriptome, in both analyzed stages. The global profile of methylated cytosines revealed that both PHYA and PHYB1B2 affect the global methylome, but PHYB1B2 has a greater impact on ripening-associated methylation reprogramming across gene-rich genomic regions in tomato fruits. Remarkably, promoters of master ripening-associated transcription factors (TF) (RIN, NOR, CNR, and AP2a) and key carotenoid biosynthetic genes (PSY1, PDS, ZISO, and ZDS) remained highly methylated in phyB1B2 from the IG to BK stage. The positional distribution and enrichment of TF binding sites were analyzed over the promoter region of the phyB1B2 DEGs, exposing an overrepresentation of binding sites for RIN as well as the PHY-downstream effectors PIFs and HY5/HYH. Moreover, phyA and phyB1B2 mutants showed a positive correlation between the methylation level of sRNA cluster-targeted genome regions in gene bodies and mRNA levels. The experimental evidence indicates that PHYB1B2 signal transduction is mediated by a gene expression network involving chromatin organization factors (DNA methylases/demethylases, histone-modifying enzymes, and remodeling factors) and transcriptional regulators leading to altered mRNA profile of ripening-associated genes. This new level of understanding provides insights into the orchestration of epigenetic mechanisms in response to environmental cues affecting agronomical traits.
RESUMO
Drought is one of the main environmental stresses that negatively impacts vegetative and reproductive yield. Water deficit responses are determined by the duration and intensity of the stress, which, together with plant genotype, will define the chances of plant survival. The metabolic adjustments in response to water deficit are complex and involve gene expression modulation regulated by DNA-binding proteins and epigenetic modifications. This last mechanism may also regulate the activity of transposable elements, which in turn impact the expression of nearby loci. Setaria italica plants submitted to five water deficit regimes were analyzed through a phenotypical approach, including growth, physiological, RNA-seq and sRNA-seq analyses. The results showed a progressive reduction in yield as a function of water deficit intensity associated with signaling pathway modulation and metabolic adjustments. We identified a group of loci that were consistently associated with drought responses, some of which were related to water deficit perception, signaling and regulation. Finally, an analysis of the transcriptome and sRNAome allowed us to identify genes putatively regulated by TE- and sRNA-related mechanisms and an intriguing positive correlation between transcript levels and sRNA accumulation in gene body regions. These findings shed light on the processes that allow S. italica to overcome drought and survive under water restrictive conditions.
Assuntos
Pequeno RNA não Traduzido , Setaria (Planta) , Adaptação Fisiológica/genética , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Pequeno RNA não Traduzido/metabolismo , Setaria (Planta)/genética , Estresse Fisiológico/genética , Água/metabolismoRESUMO
Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.
Assuntos
Neoplasias do Endométrio , Receptores de Progesterona , Linhagem Celular Tumoral , Cromatina , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Fator de Transcrição PAX2/genética , Progesterona , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
Global warming is predicted to exert negative impacts on plant growth due to the damaging effect of high temperatures on plant physiology. Revealing the genetic architecture underlying the heat stress response is therefore crucial for the development of conservation strategies, and for breeding heat-resistant plant genotypes. Here we investigated the transcriptional changes induced by heat in Nothofagus pumilio, an emblematic tree species of the sub-Antarctic forests of South America. Through the performance of RNA-seq of leaves of plants exposed to 20°C (control) or 34°C (heat shock), we generated the first transcriptomic resource for the species. We also studied the changes in protein-coding transcripts expression in response to heat. We found 5,214 contigs differentially expressed between temperatures. The heat treatment resulted in a down-regulation of genes related to photosynthesis and carbon metabolism, whereas secondary metabolism, protein re-folding and response to stress were up-regulated. Moreover, several transcription factor families like WRKY or ERF were promoted by heat, alongside spliceosome machinery and hormone signaling pathways. Through a comparative analysis of gene regulation in response to heat in Arabidopsis thaliana, Populus tomentosa and N. pumilio we provide evidence of the existence of shared molecular features of heat stress responses across angiosperms, and identify genes of potential biotechnological application.
Assuntos
Fagales/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Resposta ao Choque Térmico , Folhas de Planta/genética , Análise de Sequência de RNA , América do SulRESUMO
Cold environments impose challenges to organisms. Polyextremophile microorganisms can survive in these conditions thanks to an array of counteracting mechanisms. Naganishia vishniacii, a yeast species hitherto only isolated from McMurdo Dry Valleys, Antarctica, is an example of a polyextremophile. Here we present the first draft genomic sequence of N. vishniacii. Using comparative genomics, we unraveled unique characteristics of cold associated adaptations. 336 putative genes (total: 6183) encoding solute transfers and chaperones, among others, were absent in sister species. Among genes shared by N. vishniacii and its closest related species we found orthologs encompassing possible evidence of positive selection (dN/dS > 1). Genes associated with photoprotection were found in agreement with high solar irradiation exposure. Also genes coding for desaturases and genomic features associated with cold tolerance (i.e. trehalose synthesis and lipid metabolism) were explored. Finally, biases in amino acid usage (namely an enrichment of glutamine and a trend in proline reduction) were observed, possibly conferring increased protein flexibility. To the best of our knowledge, such a combination of mechanisms for cold tolerance has not been previously reported in fungi, making N. vishniacii a unique model for the study of the genetic basis and evolution of cold adaptation strategies.
Assuntos
Adaptação Fisiológica/genética , Basidiomycota/genética , Temperatura Baixa , Genoma Microbiano , Regiões Antárticas , Evolução Molecular , Genômica/métodosRESUMO
Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.
Assuntos
Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Feminino , Humanos , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Proteína Quinase 7 Ativada por Mitógeno/genética , Inclusão em Parafina , Isoformas de Proteínas , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Twenty-one psychrophilic yeast isolates related to the Camptobasidiaceae family in the Microbotryomycetes class were obtained from ice collected from cold environments worldwide. A new psychrophilic species from the recently described genus Cryolevonia, Cryolevania giraudoae is proposed to accommodate 18 isolates from Patagonia (Argentina) and Antarctica (holotype CRUB 2086T). In addition, a new psychrophilic species in the genus Camptobasidium is described as Camptobasidium gelus sp. nov. (holotype CBS 8941T), based on three isolates from glacial ice in the Russel glacier (Greenland ice sheet) and Antarctica. The strict psychrophilic profile is the salient feature of both novel species.