The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger

Abstract Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. Key points • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

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Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR

Applied Microbiology and Biotechnology ◽

10.1007/s00253-018-8753-7 ◽

2018 ◽

Vol 102 (6) ◽

pp. 2723-2736 ◽

Cited By ~ 17

Author(s):

Ebru Alazi ◽

Tim Knetsch ◽

Marcos Di Falco ◽

Ian D. Reid ◽

Mark Arentshorst ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Niger ◽

Galacturonic Acid

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158193 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8193

Author(s):

Daniel Pérez-Cremades ◽

Ana B. Paes ◽

Xavier Vidal-Gómez ◽

Ana Mompeón ◽

Carlos Hermenegildo ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Regulatory Network ◽

Mirna Target ◽

Target Genes ◽

Functional Characterization ◽

Human Endothelial Cells ◽

Mrna Targets ◽

Canonical Pathways

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

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STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

International Journal of Molecular Sciences ◽

10.3390/ijms22010460 ◽

2021 ◽

Vol 22 (1) ◽

pp. 460

Author(s):

Huan Ou-Yang ◽

Shinn-Chih Wu ◽

Li-Ying Sung ◽

Shiao-Hsuan Yang ◽

Shang-Hsun Yang ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Fluorescent Protein ◽

Mouse Embryos ◽

Cell Nuclei ◽

Cell Stage ◽

Promoter Assay ◽

Maternal To Zygotic Transition ◽

Mouse Zygotes ◽

Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

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The AML-associated K313 mutation enhances C/EBPα activity by leading to C/EBPα overexpression

Cell Death and Disease ◽

10.1038/s41419-021-03948-6 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Ian Edward Gentle ◽

Isabel Moelter ◽

Mohamed Tarek Badr ◽

Konstanze Döhner ◽

Michael Lübbert ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activity ◽

Target Gene ◽

Wild Type ◽

Elevated Expression ◽

Factor C ◽

Acute Myeloid

AbstractMutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

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HbxB Is a Key Regulator for Stress Response and β-Glucan Biogenesis in Aspergillus nidulans

Microorganisms ◽

10.3390/microorganisms9010144 ◽

2021 ◽

Vol 9 (1) ◽

pp. 144

Author(s):

Sung-Hun Son ◽

Mi-Kyung Lee ◽

Ye-Eun Son ◽

Hee-Soo Park

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Aspergillus Nidulans ◽

Deletion Mutant ◽

Phenotypic Analysis ◽

Spore Viability ◽

Fungal Development ◽

Expression Of Genes ◽

Trehalose Biosynthesis

Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with β-glucan degradation and decreased the amount of β-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, β-glucan degradation, and sterigmatocystin production in A.nidulans conidia.

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Expression Analyses of Soybean VOZ Transcription Factors and the Role of GmVOZ1G in Drought and Salt Stress Tolerance

International Journal of Molecular Sciences ◽

10.3390/ijms21062177 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2177 ◽

Cited By ~ 1

Author(s):

Bo Li ◽

Jia-Cheng Zheng ◽

Ting-Ting Wang ◽

Dong-Hong Min ◽

Wen-Liang Wei ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Salt Stress ◽

Abiotic Stress ◽

Stress Tolerance ◽

Zinc Finger ◽

Hairy Roots ◽

Yeast Cells ◽

Salt Stress Tolerance ◽

Drought And Salt Stress

Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.

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The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation

Toxins ◽

10.3390/toxins13060431 ◽

2021 ◽

Vol 13 (6) ◽

pp. 431

Author(s):

Longxue Ma ◽

Xu Li ◽

Xiaoyun Ma ◽

Qiang Yu ◽

Xiaohua Yu ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Environmental Factors ◽

Health Risks ◽

Sexual Development ◽

Regulatory Mechanism ◽

Food Storage ◽

Economic Losses ◽

Transcriptional Level ◽

Conidia Production

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

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Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6293 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6293-6304 ◽

Cited By ~ 96

Author(s):

Vesco Mutskov ◽

Delphine Gerber ◽

Dimitri Angelov ◽

Juan Ausio ◽

Jerry Workman ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Molecular Biology ◽

Binding Sites ◽

Cross Linking ◽

Uv Laser ◽

Histone Tail ◽

Histone Tails ◽

Nucleosomal Dna ◽

Core Histones

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

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