Transcriptomic Analysis of Extensive Changes in Metabolic Regulation in Kluyveromyces lactis Strains

ABSTRACT Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

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Analysis of the Mechanism by Which Glucose Inhibits Maltose Induction of MAL Gene Expression in Saccharomyces

Genetics ◽

10.1093/genetics/154.1.121 ◽

2000 ◽

Vol 154 (1) ◽

pp. 121-132

Author(s):

Zhen Hu ◽

Yingzi Yue ◽

Hua Jiang ◽

Bin Zhang ◽

Peter W Sherwood ◽

...

Keyword(s):

Gene Expression ◽

Glucose Repression ◽

Protein A ◽

The Other ◽

Maltose Permease ◽

Inducer Exclusion ◽

Glucose Inhibition ◽

Independent Mechanism ◽

Mal Genes

Abstract Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

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Gene Expression Profile and Co-Expression Network of Pearl Gentian Grouper under Cold Stress by Integrating Illumina and PacBio Sequences

Animals ◽

10.3390/ani11061745 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1745

Author(s):

Ben-Ben Miao ◽

Su-Fang Niu ◽

Ren-Xie Wu ◽

Zhen-Bang Liang ◽

Bao-Gui Tang ◽

...

Keyword(s):

Gene Expression ◽

Cold Stress ◽

Differential Expression Analysis ◽

Endocrine System ◽

Control Group ◽

Regulation Mechanism ◽

Rna Seq ◽

Cold Tolerant ◽

Stress Signals ◽

Membrane Changes

Pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) is a fish of high commercial value in the aquaculture industry in Asia. However, this hybrid fish is not cold-tolerant, and its molecular regulation mechanism underlying cold stress remains largely elusive. This study thus investigated the liver transcriptomic responses of pearl gentian grouper by comparing the gene expression of cold stress groups (20, 15, 12, and 12 °C for 6 h) with that of control group (25 °C) using PacBio SMRT-Seq and Illumina RNA-Seq technologies. In SMRT-Seq analysis, a total of 11,033 full-length transcripts were generated and used as reference sequences for further RNA-Seq analysis. In RNA-Seq analysis, 3271 differentially expressed genes (DEGs), two low-temperature specific modules (tan and blue modules), and two significantly expressed gene sets (profiles 0 and 19) were screened by differential expression analysis, weighted gene co-expression networks analysis (WGCNA), and short time-series expression miner (STEM), respectively. The intersection of the above analyses further revealed some key genes, such as PCK, ALDOB, FBP, G6pC, CPT1A, PPARα, SOCS3, PPP1CC, CYP2J, HMGCR, CDKN1B, and GADD45Bc. These genes were significantly enriched in carbohydrate metabolism, lipid metabolism, signal transduction, and endocrine system pathways. All these pathways were linked to biological functions relevant to cold adaptation, such as energy metabolism, stress-induced cell membrane changes, and transduction of stress signals. Taken together, our study explores an overall and complex regulation network of the functional genes in the liver of pearl gentian grouper, which could benefit the species in preventing damage caused by cold stress.

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Influence of Mutations in Hexose-Transporter Genes on Glucose Repression in Kluyveromyces Lactis

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1997.t01-1-00248.x ◽

1997 ◽

Vol 249 (1) ◽

pp. 248-257 ◽

Cited By ~ 42

Author(s):

Jorg Weirich ◽

Paola Goffrini ◽

Petra Kuger ◽

Iliana Ferrero ◽

Karin D. Breunig

Keyword(s):

Glucose Repression ◽

Kluyveromyces Lactis ◽

Hexose Transporter

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A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5331 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5331-5339 ◽

Cited By ~ 104

Author(s):

R Herrera ◽

H S Ro ◽

G S Robinson ◽

K G Xanthopoulos ◽

B M Spiegelman

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Adipocyte Differentiation ◽

Binding Protein ◽

Point Mutations ◽

Chloramphenicol Acetyltransferase ◽

Lipid Binding ◽

Direct Role ◽

New Genes ◽

Chloramphenicol Acetyltransferase Gene

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

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Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2632-2638.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2632-2638 ◽

Cited By ~ 15

Author(s):

Claudia Donnini ◽

Francesca Farina ◽

Barbara Neglia ◽

Maria Concetta Compagno ◽

Daniela Uccelletti ◽

...

Keyword(s):

Glucose Repression ◽

Kluyveromyces Lactis ◽

Kinase Domain ◽

Arxula Adeninivorans ◽

Nucleotide Position ◽

Heterologous Proteins ◽

Reporter Protein ◽

Gene Encoding ◽

Defective Mutant ◽

Highly Sensitive

ABSTRACT The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

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Evolution of mouse circadian enhancers from transposable elements

Genome Biology ◽

10.1186/s13059-021-02409-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Julius Judd ◽

Hayley Sanderson ◽

Cédric Feschotte

Keyword(s):

Gene Expression ◽

Transposable Elements ◽

Binding Sites ◽

Mouse Liver ◽

Integration Site ◽

Point Mutations ◽

Regulatory Elements ◽

Circadian Gene ◽

Binding Motifs ◽

Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

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Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3058-3066.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3058-3066

Author(s):

W Zachariae ◽

K D Breunig

Keyword(s):

Carbon Source ◽

Binding Site ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Transcriptional Activator ◽

Optimal Growth ◽

Source Control ◽

Poor Growth ◽

Protein Protein Interaction ◽

Metabolic Genes

The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose.

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Conservation of Histone Binding and Transcriptional Repressor Functions in a Schizosaccharomyces pombeTup1p Homolog

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8461 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8461-8468 ◽

Cited By ~ 34

Author(s):

Yukio Mukai ◽

Eri Matsuo ◽

Sharon Y. Roth ◽

Satoshi Harashima

Keyword(s):

Glucose Repression ◽

Kluyveromyces Lactis ◽

Chimeric Protein ◽

Evolutionary Conservation ◽

Histone Binding ◽

Amino Terminal ◽

Corepressor Complex ◽

Related Proteins ◽

Repressor Activity

ABSTRACT The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned fromKluyveromyces lactis (TUP1) andSchizosaccharomyces pombe (tup11 +). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11 + gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, anS. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiaecorresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11 + in combination with a disruption of tup12 +, anotherTUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1 +, suggesting thatS. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species.

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Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3442 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3442-3449 ◽

Cited By ~ 16

Author(s):

M S Donoviel ◽

N Kacherovsky ◽

E T Young

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Copy Number ◽

Glucose Repression ◽

Regulatory Protein ◽

Regulatory Region ◽

Upstream Regulatory Region ◽

Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

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Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

Cholesterol ◽

10.1155/2015/682904 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Zahra Tavoosi ◽

Hemen Moradi-Sardareh ◽

Massoud Saidijam ◽

Reza Yadegarazari ◽

Shiva Borzuei ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Syndrome ◽

Mononuclear Cells ◽

Fasting Blood Glucose ◽

Control Group ◽

Regulation Mechanism ◽

Healthy Individuals ◽

Peripheral Blood Mononuclear ◽

Significant Difference ◽

Abca1 Expression

ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran) during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75%) compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P<0.05). Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression.

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