Jitendra K. Thakur

A nuclear receptor-like pathway regulating multidrug resistance in fungi

Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa)

Auxin exerts pleiotropic effects on plant growth and development by regulating the expression of early auxin-responsive genes of auxin/indoleacetic acid (Aux/IAA), small auxin-up RNA, and GH3 classes. These genes have been studied extensively in dicots like soybean and Arabidopsis. We had earlier characterized a cDNA of the first monocot member of Aux/IAA family from rice. The achievement of the large scale rice genome sequencing combined with the availability of full-length cDNA sequences from Knowledge-based Oryza Molecular Biological Encyclopedia provided us the opportunity to draw up the first comprehensive list of Aux/IAA genes in a monocot. By screening the available databases, we have identified 31 Aux/IAA genes having high sequence identity within the conserved domains I, II, III, and IV. The genomic organization as well as chromosomal location of all the Oryza sativa indoleacetic acid (OsIAA) genes is reported. The rice Aux/IAA proteins can be classified in two groups (A and B) on the basis of their phylogenetic relationship with Arabidopsis Aux/IAA proteins. An evolutionary pattern of the rice Aux/IAA genes has been discussed by analyzing their structure (exon/intron organization) and duplications. Interestingly, the duplication of rice Aux/IAA genes was found to be associated with chromosomal block duplication events in rice. The in-silico analysis has been complemented with real-time polymerase chain reaction analysis to quantify transcript levels of all Aux/IAA family members. OsIAA genes showed differential and overlapping organ-specific expression patterns in light- and dark-grown seedlings/plants. Although auxin enhanced the transcript abundance of most of the OsIAA genes, the effect was more pronounced on OsIAA9, 14, 19, 20, 24, and 31. These results provide a foundation for future studies on elucidating the precise role of rice Aux/IAA genes in early steps of auxin signal transduction.

Mediator Subunit Gal11p/MED15 Is Required for Fatty Acid-dependent Gene Activation by Yeast Transcription Factor Oaf1p

The yeast zinc cluster transcription factor Oaf1p activates transcription of target genes in response to direct binding of fatty acids in a manner analogous to the vertebrate nuclear receptor peroxisome proliferator-activated receptorα (PPARα). PPARs and other metazoan nuclear receptors productively engage several distinct LXXLL motif-containing co-activators, including p160 family members and the TRAP220/MED1 subunit of the Mediator co-activator, to promote ligand-dependent gene activation. Yeast, however, does not appear to harbor LXXLL motif co-activators, and the mechanism of fatty acid-dependent gene activation by the yeast PPARα analog Oaf1p is unknown. Here we show that the yeast Mediator subunit Gal11p/MED15 and its activator-targeted KIX domain plays a critical role in fatty acid-dependent transcriptional regulation of fatty acid β-oxidation and peroxisomal genes by Oaf1p and for the ability of yeast to utilize fatty acids as a sole carbon source. Moreover, structural studies by NMR spectroscopy reveal that the Oaf1p activation domain interacts with the Gal11p/MED15 KIX domain in a manner similar to the yeast zinc cluster family member and xenobiotic receptor Pdr1p, revealing that the Gal11p/MED15 KIX domain is a key target of several ligand-dependent transcription factors in yeast. Together with previous work showing that the Caenorhabditis elegans Gal11p/MED15 homolog MDT-15 plays a critical role in regulation of fatty acid metabolism by the nematode PPAR-like nuclear receptor NHR-49, the findings presented here provide evidence for an ancient and essential role of a Mediator co-activator subunit in regulation of fatty acid metabolism by nuclear receptor-like transcription factors in eukaryotes.

Nuclear receptor-like transcription factors in fungi

Members of the metazoan nuclear receptor superfamily regulate gene expression programs in response to binding of cognate lipophilic ligands. Evolutionary studies using bioinformatics tools have concluded that lower eukaryotes, such as fungi, lack nuclear receptor homologs. Here we review recent discoveries suggesting that members of the fungal zinc cluster family of transcription regulators represent functional analogs of metazoan nuclear receptors. These findings indicate that nuclear receptor-like ligand-dependent gene regulatory mechanisms emerged early during eukaryotic evolution, and provide the impetus for further detailed studies of the possible evolutionary and mechanistic relationships of fungal zinc cluster transcription factors and metazoan nuclear receptors. Clinical implications of the discovery of nuclear receptor-like transcription factors in pathogenic fungi will also be discussed.

Molecular recognition by the KIX domain and its role in gene regulation

The kinase-inducible domain interacting (KIX) domain is a highly conserved independently folding three-helix bundle that serves as a docking site for transcription factors, whereupon promoter activation and target specificity are achieved during gene regulation. This docking event is a harbinger of an intricate multi-protein assembly at the transcriptional apparatus and is regulated in a highly precise manner in view of the critical role it plays in multiple cellular processes. KIX domains have been characterized in transcriptional coactivators such as p300/CREB-binding protein and mediator of RNA polymerase II transcription subunit 15, and even recQ protein-like 5 helicases in various organisms. Their targets are often intrinsically disordered regions within the transactivation domains of transcription factors that attain stable secondary structure only upon complexation with KIX. In this article, we review the KIX domain in terms of its sequence and structure and present the various implications of its ability to act as a transcriptional switch, the mechanistic basis of molecular recognition by KIX, its binding specificity, target promiscuity, combinatorial potential and unique mode of regulation via allostery. We also discuss the possible roles of KIX domains in plants and hope that this review will accelerate scientific interest in KIX and pave the way for novel avenues of research on this critical domain.

A POLYCOMB group gene of rice (Oryza sativa L. subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development

The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes.

Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants

Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediators involvement in these processes.

Analysis of differential expression of Mediator subunit genes in Arabidopsis.

Mediator is a conserved eukaryotic multiprotein complex required by RNA polymerase II for transcription of its target genes. Till date, there is no report explaining the signals that affect the overall concentration of individual Med subunits. In this report, we have analyzed the effect of different phytohormones and stresses on the transcript level of Med genes in Arabidopsis. Hormones like auxin and JA, and cold stress did not show significant effect. ABA moderately increased the transcript abundance of more than 70% of AtMed genes analyzed in this study. However, there was noticeable change in the transcript level of several AtMed genes in response to BR. Stresses like high light, dark and salt also caused significant change in the transcript abundance of many AtMed genes. These data reveal that different environmental cues can affect stoichiometric concentration of Med subunits by affecting the transcription of their respective genes. This may, in turn, affect the overall arrangement of functional Mediator complex. This also suggests that some subunits may have some specific functions to play in response different signals.

The interplay of HuR and miR-3134 in regulation of AU rich transcriptome

MicroRNAs and AU Rich element (ARE)-mediated degradation of transcripts are thought to be two independent means of gene regulation at the post-transcriptional level. However, since their site of action is the same (3‘UTR of mRNA), there exists a high probability that specific miRNAs may bind to AREs and, thus, interact with ARE-binding proteins (ARE-BPs) to regulate transcript levels. In this study, we have characterized AREs as potential targets of hsa-miR-3134. An analysis of the global gene expression profile of breast cancer cell line MCF7 overexpressing miR-3134 revealed the presence of at least one AUUUA element in the 3′-UTRs of 63% of miR-3134 regulated protein coding genes. Quantitative RT-PCR or 3′UTR luciferase assays show that miR-3134 mediates an up to 4–8-fold increase in the levels of ARE bearing transcripts-SOX9, VEGFA, and EGFR, while mutated miR-3134 shows a decreased effect. The miR-3134-mediated increase in transcript levels was unaffected by treatment with transcription inhibitor (actinomycin D), indicating that miR-3134 enhances transcript stability. To investigate a possible interplay between miR-3134 and a prototype ARE-BP, HuR, we compared their overexpression transcriptome profiles. Interestingly, up to 80% of miR-3134-regulated genes were also regulated by HuR. Overexpression studies of HuR alone or in combination with miR-3134 shows that wt miR-3134 but not a mutated miR-3134 promotes stabilization of HuR-regulated transcripts SOX9, VEGFA, and EGFR as confirmed by qRT-PCR or RNA-immunoprecipitation experiments. Overall, this report suggests that collaboration between ARE-binding microRNAs and ARE-binding proteins could be a general mechanism of 3′-UTR mediated regulation of gene expression in human cells.