Modeling the Transcriptional Regulatory Network That Controls the Early Hypoxic Response in Candida albicans

ABSTRACTWe determined the changes in transcriptional profiles that occur in the first hour following the transfer ofCandida albicansto hypoxic growth conditions. The impressive speed of this response is not compatible with current models of fungal adaptation to hypoxia that depend on the depletion of sterol and heme. Functional analysis using Gene Set Enrichment Analysis (GSEA) identified the Sit4 phosphatase, Ccr4 mRNA deacetylase, and Sko1 transcription factor (TF) as potential regulators of the early hypoxic response. Cells mutated in these and other regulators exhibit a delay in their transcriptional responses to hypoxia. Promoter occupancy data for 29 TFs were combined with the transcriptional profiles of 3,111in vivotarget genes in a Network Component Analysis (NCA) to produce a model of the dynamic and highly interconnected TF network that controls this process. With data from the TF network obtained from a variety of sources, we generated an edge and node model that was capable of separating many of the hypoxia-upregulated and -downregulated genes. Upregulated genes are centered on Tye7, Upc2, and Mrr1, which are associated with many of the gene promoters that exhibit the strongest activations. The connectivity of the model illustrates the high redundancy of this response system and the challenges that lie in determining the individual contributions of specific TFs. Finally, treating cells with an inhibitor of the oxidative phosphorylation chain mimics most of the early hypoxic profile, which suggests that this response may be initiated by a drop in ATP production.

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RNA sequencing reveals an additional Crz1-binding motif in promoters of its target genes in the human fungal pathogen Candida albicans

Cell Communication and Signaling ◽

10.1186/s12964-019-0473-9 ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 3

Author(s):

Huihui Xu ◽

Tianshu Fang ◽

Raha Parvizi Omran ◽

Malcolm Whiteway ◽

Linghuo Jiang

Keyword(s):

Candida Albicans ◽

Rna Sequencing ◽

Microarray Analysis ◽

Target Genes ◽

Binding Motif ◽

Cellular Transport ◽

Gene Promoters ◽

Calcium Stress

Abstract Background The calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated. Methods An inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2 M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively. Results RNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5′-GGAGGC(G/A)C(T/A)G-3′], which is different from the putative CaCrz1-binding motif [5′-G(C/T)GGT-3′] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5′-GNGGC(G/T)CA-3′]. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression. Conclusions CaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5′-TGAGGGACTG-3′ site in its promoter.

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Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013728 ◽

2020 ◽

Vol 295 (39) ◽

pp. 13617-13629

Author(s):

Clément Immarigeon ◽

Sandra Bernat-Fabre ◽

Emmanuelle Guillou ◽

Alexis Verger ◽

Elodie Prince ◽

...

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Direct Interaction ◽

Mediator Complex ◽

Loss Of Function ◽

Transcriptional Responses ◽

Rna Pol Ii ◽

Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

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Activation of HIF1α Rescues the Hypoxic Response and Reverses Metabolic Dysfunction in the Diabetic Heart

10.2337/figshare.16539774 ◽

2021 ◽

Author(s):

Maria da Luz Sousa Fialho ◽

Ujang Purnama ◽

Kaitlyn MJH Dennis ◽

Claudia N Montes Aparicio ◽

Marcos Castro-Guarda ◽

...

Keyword(s):

Insulin Resistance ◽

Glucose Metabolism ◽

Target Genes ◽

Metabolic Phenotype ◽

Diabetic Heart ◽

Acid Oxidation ◽

Adaptation To Hypoxia ◽

Metabolic Dysfunction ◽

Hypoxic Response ◽

Human Cardiomyocytes

Type 2 diabetes (T2D) impairs Hypoxia-Inducible Factor (HIF)1α activation, a master transcription factor that drives cellular adaptation to hypoxia. Reduced activation of HIF1α contributes to the impaired post-ischaemic remodelling observed following myocardial infarction in T2D. Molidustat is a HIF stabiliser currently undergoing clinical trials for the treatment of renal anaemia associated with chronic kidney disease, however, it may provide a route to pharmacologically activate HIF1α in the T2D heart. In human cardiomyocytes, molidustat stabilised HIF1α and downstream HIF target genes, promoting anaerobic glucose metabolism. In hypoxia, insulin resistance blunted HIF1α activation and downstream signalling, but this was reversed by molidustat. In T2D rats, oral treatment with molidustat rescued the cardiac metabolic dysfunction caused by T2D, promoting glucose metabolism and mitochondrial function, whilst suppressing fatty acid oxidation and lipid accumulation. This resulted in beneficial effects on post-ischemic cardiac function, with the impaired contractile recovery in T2D heart reversed by molidustat treatment. In conclusion, pharmacological HIF1α stabilisation can overcome the blunted hypoxic response induced by insulin resistance. In vivo this corrected the abnormal metabolic phenotype and impaired post-ischaemic recovery of the diabetic heart. Therefore, molidustat may be an effective compound to further explore the clinical translatability of HIF1α activation in the diabetic heart.

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Direct Protein Interactions Are Responsible for Ikaros-GATA and Ikaros-Cdk9 Cooperativeness in Hematopoietic Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00296-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3064-3076 ◽

Cited By ~ 10

Author(s):

Stefania Bottardi ◽

Lionel Mavoungou ◽

Vincent Bourgoin ◽

Nazar Mashtalir ◽

El Bachir Affar ◽

...

Keyword(s):

Zinc Finger ◽

Protein Interactions ◽

Transcriptional Activation ◽

Target Genes ◽

Transcription Elongation ◽

Hematopoietic Cells ◽

Elongation Factor ◽

Gene Promoters ◽

Zinc Finger Domain

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstratedin vivoin COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteinsin vivoand perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

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Activation of HIF1α Rescues the Hypoxic Response and Reverses Metabolic Dysfunction in the Diabetic Heart

10.2337/figshare.16539774.v1 ◽

2021 ◽

Author(s):

Maria da Luz Sousa Fialho ◽

Ujang Purnama ◽

Kaitlyn MJH Dennis ◽

Claudia N Montes Aparicio ◽

Marcos Castro-Guarda ◽

...

Keyword(s):

Insulin Resistance ◽

Glucose Metabolism ◽

Target Genes ◽

Metabolic Phenotype ◽

Diabetic Heart ◽

Acid Oxidation ◽

Adaptation To Hypoxia ◽

Metabolic Dysfunction ◽

Hypoxic Response ◽

Human Cardiomyocytes

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Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

10.1101/2020.04.03.023895 ◽

2020 ◽

Author(s):

Clément Immarigeon ◽

Sandra Bernat-Fabre ◽

Emmanuelle Guillou ◽

Alexis Verger ◽

Elodie Prince ◽

...

Keyword(s):

Dna Binding ◽

Rna Polymerase Ii ◽

Zinc Finger ◽

Target Gene ◽

Target Genes ◽

Mediator Complex ◽

Transcriptional Responses ◽

Evolutionarily Conserved

AbstractThe evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

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Histone Deacetylase 3 (HDAC3), a Component of N-CoR-TBL1/R1 Chromatin Remodeling Co-Repressor Complex, Is Recruited to the Target Gene Promoters by PML-RARα To Repress the Transcription In Vivo.

Blood ◽

10.1182/blood.v106.11.2750.2750 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2750-2750

Author(s):

Akihiro Tomita ◽

Akihide Atsumi ◽

Hitoshi Kiyoi ◽

Tomoki Naoe

Keyword(s):

Gene Expression ◽

Retinoic Acid ◽

Target Gene ◽

Target Genes ◽

Gene Promoters ◽

Transcription Repression ◽

Histone Deacetylase 3 ◽

Repressor Complex ◽

Endogenous Target

Abstract PML-RARα is a chimeric transcription factor deeply associated with acute promyelocytic leukemia (APL). PML-RARα plays an important role in the aberrant transcription repression on the target genes of wild type retinoic acid receptors (RARα). Pharmacological concentration of all-trans retinoic acid (ATRA) induces transcription de-repression on several target genes, and results in terminal differentiation of APL cells. However, the detailed mechanisms of transcription repression by PML-RARα in vivo are still unclear. Here we demonstrated that histone deacetylase 3 (HDAC3), one component of the N-CoR (nuclear receptor co-repressor)-TBL1/R1 (transducin beta-like protein 1/relating protein) transcription repressor protein complex, is a key regulator of the transcription repression by PML-RARα in vivo. Using immunoprecipitation (IP) assay, we first demonstrated that PML-RARα physically interacted with N-CoR/HDAC3 in vivo in the absence of ligand. The interaction was dissociated by adding ATRA in the dose dependent manner. Next we showed, using chromatin immunoprecipitation (ChIP) assay, that N-CoR/HDAC3 co-repressor complex was recruited to the endogenous target gene promoters (RARβ and CYP26) through PML-RARα. The neighboring histone H4 was de-acetylated and the gene expression was significantly repressed. When HDAC3 protein is knocked down by RNA interference in PML-RARα-presenting cells, the endogenous target gene expression was significantly activated. Almost the same results were also obtained when performing the luciferase reporter assay using RARβ and CYP26 promoter reporter vectors. Previously, we have shown that N-CoR-TBLR1 is recruited to the target gene promoter through PML-RARα in the absence of ligand, resulting in the transcription repression. Consistent with these data, it is strongly suggested that N-CoR/HDAC3/TBLR1 co-repressor complex is closely related to the aberrant transcription regulation by PML-RARα in APL cells. Furthermore, we also confirmed that PLZF-RARα, which is expressed in ATRA resistant APL cells, interacted with N-CoR/HDAC3/TBLR1 in ligand independent manner. These insights provide not only the basic mechanism of transcription repression by leukemia-related chimeric transcription factors, but also the new molecular targets for the transcription therapy for leukemia.

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Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact To Repress Target Gene Promoters.

Blood ◽

10.1182/blood.v110.11.1240.1240 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1240-1240

Author(s):

Kevin R. Petrie ◽

Fabien Guidez ◽

Jun Zhu ◽

Gareth Owen ◽

Yat Peng Chew ◽

...

Keyword(s):

Transcription Factor ◽

Stem Cell ◽

Transcriptional Repression ◽

Cell Biology ◽

Target Genes ◽

Retinoic Acid Receptor ◽

Gene Promoters ◽

Cell Commitment

Abstract Translocations of the retinoic acid receptor alpha (RARA) locus with the PLZF or PML genes lead to expression of oncogenic PLZF-RARα or PML-RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (POZ) and DNA binding (zinc-finger) domains. Additionally, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction between PML or E2F1 with Rb, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. The interaction between PLZF and Rb is further underlined by chromatin immunoprecipitation analysis of PLZF binding to genomic DNA, which shows that PLZF associates with genes controlling cell proliferation known to be regulated by Rb and E2F (for example cdc6). Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to have roles in stem cell biology and, taken together, these data provide a plausible scenario in which interactions between PLZF and Rb function in stem cell commitment or maintenance and self-renewal. The oncogenic PLZF-RARα fusion also interacts with Rb, suggesting that deregulation of Rb function may be a factor in the molecular pathogenesis of PLZF-RARα associated acute promyelocytic leukemia.

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Do unliganded thyroid hormone receptors have physiological functions?

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310009 ◽

2003 ◽

Vol 31 (1) ◽

pp. 9-20 ◽

Cited By ~ 37

Author(s):

O Chassande

Keyword(s):

Thyroid Hormone ◽

Hormone Receptors ◽

Target Genes ◽

Thyroid Hormone Receptors ◽

Intrinsic Activity ◽

Vertebrate Development ◽

Transcriptional Responses ◽

Embryonic And Fetal Development

Thyroid hormone (TH) is required for the development of vertebrates and exerts numerous homeostatic functions in adults. TH acts through nuclear receptors which control the transcription of target genes. Unliganded and liganded thyroid hormone receptors (TRs) have been shown to exert opposite effects on the transcription of target genes in vitro. However, the occurance of an aporeceptor activity in vivo and its potential physiological significance has not been clearly addressed. Several data generated using experimental hypothyroidism and thyrotoxicosis in wild type and TR knockout mice support the notion that apoTRs have an intrinsic activity in several tIssues. ApoTRs, and in particular TRalpha1, are predominant during the early stages of vertebrate development and must be turned into holoTRs for post-natal development to proceed normally. However, the absence of striking alterations of embryonic and fetal development in mice devoid of TRs indicates that apoTRs do not play a fundamental role. During development, as well as in adults, apoTRs rather appears as a system which increases the range of transcriptional responses to moderate variations of T3.

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A shift in the ligand responsiveness of thyroid hormone receptor alpha induced by heterodimerization with retinoid X receptor alpha.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.219 ◽

1996 ◽

Vol 16 (1) ◽

pp. 219-227 ◽

Cited By ~ 12

Author(s):

F X Claret ◽

T Antakly ◽

M Karin ◽

F Saatcioglu

Keyword(s):

Thyroid Hormone ◽

Dna Binding ◽

Conformational Changes ◽

Transcriptional Activation ◽

Target Genes ◽

Thyroid Hormone Receptor ◽

Transcriptional Responses ◽

Receptor Alpha

Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R alpha forms stable heterodimers with RXR alpha in living cells. Most important, we describe a new role for RXR alpha in modulating ligand-dependent T3R alpha activity: heterodimerization with RXR alpha greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R alpha. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R alpha plus RXR alpha than by T3R alpha alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R alpha homodimers but does not affect DNA binding by T3R alpha:RXR alpha heterodimers. We provide evidence that increased activities of T3R alpha at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR alpha-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.

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