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Rad9 associates with Aft1 in vivo with its BRCT domain interacting with N-Aft1 in vitro. (A) In rad9 background, Aft1 was tagged with 9Myc epitopes. Rad9 tagged with flag epitope was inserted in the high copy plasmid pDB20 and this construct was inserted to rad9 strain in order to get rad9 Aft1–9Myc pDB20-Rad9-flag strain. rad9 Aft1–9Myc pDB20-flag strain was used as control. In SDS-PAGE and immunoblotting analysis, anti-Flag was used as a bait and probing was done with anti-Myc. Input and IP samples are shown. The co-IP experiment was repeated twice. (B) Protein extracts were prepared from wt cells grown in YPD / BPS-BCS, endogenously expressing Rad9–9Myc or Rad9–9Myc along with Aft1–3HA. Portions (1 / 40) of each extract (WCE) were analyzed by SDS-PAGE and immunoblotting using anti-HA or anti-Myc to detect Aft1–3HA and Rad9–9Myc, respectively. Remaining extracts were incubated with EZview anti-HA agarose beads and then flow-through (FT) as well as pulled-down (PD) proteins were analyzed by SDS-PAGE and immunoblotting using anti-Myc to detect Rad9–9Myc. (C) Bacterially expressed GST (negative control), GST-Nhp6a (positive control) (32), GST-N-Rad9 or GST-BRCT-Rad9 proteins, bound on glutathione agarose beads, were incubated with bacterially expressed 6xHis-N-Aft1 or 6xHis- C-Aft1 derivatives eluted from Ni-NTA agarose beads. Glutathione beads were then washed and proteins bound on them were analysed by SDS-PAGE and immunoblotting using anti-His antibody. The input lane contains 20% of the total amount of each 6xHis-tagged protein incubated with the beads. Left panel: coomassie blue gel showing the electrophoretic pattern of the GST-tagged (total amounts) as well as the 6xHis-tagged (input amounts) proteins used in the assay.
Source publication
DNA damage response and repair proteins are centrally involved in genome maintenance pathways. Yet, little is known about
their functional role under non-DNA damage-inducing conditions. Here we show that Rad9 checkpoint protein, known to mediate
the damage signal from upstream to downstream essential kinases, interacts with Aft1 transcription facto...
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Citations
... Although Aft1 and Aft2 share a core, Fe-S cluster-regulated DNA binding domain, Aft1 has extended sequences at both the N and C termini that are absent from Aft2 [20]. In addition to activating the iron regulon, Aft1 plays central roles in seemingly unrelated processes: it interacts with the kinetochore protein Iml3 to promote peri-centromeric cohesion [21], with Sit1 (Arn3) to regulate ferroxamine B uptake [22], and with the DNA damage checkpoint protein Rad9 to monitor fragile genomic sites [23]. ...
Energy status in all eukaryotic cells is sensed by AMP-kinases. We have previously found that the poly-histidine tract at the N-terminus of S. cerevisiae AMPK (Snf1) inhibits its function in the presence of glucose via a pH-regulated mechanism. We show here that in the absence of glucose, the poly-histidine tract has a second function, linking together carbon and iron metabolism. Under conditions of iron deprivation, when different iron-intense cellular systems compete for this scarce resource, Snf1 is inhibited. The inhibition is via an interaction of the poly-histidine tract with the low-iron transcription factor Aft1. Aft1 inhibition of Snf1 occurs in the nucleus at the nuclear membrane, and only inhibits nuclear Snf1, without affecting cytosolic Snf1 activities. Thus, the temporal and spatial regulation of Snf1 activity enables a differential response to iron depending upon the type of carbon source. The linkage of nuclear Snf1 activity to iron sufficiency ensures that sufficient clusters are available to support respiratory enzymatic activity and tests mitochondrial competency prior to activation of nuclear Snf1.
... FT5 (MATa ura3-52 trp1-∆63 his3-∆200 leu2::PET56) yeast strain was used to generate the sod1Δ ccs1∆ double deletion using the KanMX and LEU2 disruption cassettes, respectively, and mac1Δ sod1Δ double deletion using the KanMX and LEU2 disruption cassettes, respectively. FT5(MAC1-9MYC) (Andreadis et al. 2014) was used to generate MAC1-9MYC sod1Δ using a KanMX disruption cassette. FT5(SOD1-3HA) and FT5(MAC1-9MYC SOD1-3HA) strains were generated by C-terminal epitope tagging of the genomic SOD1 (Knop et al. 1999). ...
Although copper is an essential trace element for cell function and viability, its excess can lead to protein oxidation, DNA cleavage, and ultimate cell damage. Cells have established a variety of regulatory mechanisms to ensure copper ion homeostasis. In Saccharomyces cerevisiae, copper sensing and response to copper deficiency are regulated by the transcription factor Mac1. Our group has previously reported that in addition to copper, several chromatin proteins modulate Mac1 functionality. In this study, based on a synthetic growth deficiency phenotype, we showed that the Cu/Zn superoxide dismutase Sod1 plays an important role in Mac1 transcriptional activity, in unchallenged nutrient-rich growth conditions. Sod1 is a multipotent cytoplasmic and mitochondrial enzyme, whose main known function is to detoxify the cell from superoxide ions. It has been previously reported that Sod1 also enters the nucleus and affects the transcription of several genes, some of which are involved in copper homeostasis under Cu-depleted (Wood and Thiele in J Biol Chem 284:404-413, 2009) or only under specific oxidative stress conditions (Dong et al. Mol Cell Biol 33:4041-4050, 2013; Tsang et al. Nar Commun 8:3446, 2014). We have shown that Sod1 physically interacts with Mac1 transcription factor and is important for the transactivation as well as its DNA-binding activities. On the other hand, a constitutively active mutant of Mac1 is not affected functionally by the Sod1 ablation, pointing out that Sod1 contributes to the maintenance of the copper-unchelated state of Mac1. In conclusion, we showed that Sod1–Mac1 interaction is vital for Mac1 functionality, regardless of copper medium deficiency, in unchallenged growth conditions, and we suggest that Sod1 enzymatic activity may modify the redox state of the cysteine-rich motifs in the Mac1 DNA-binding and transactivation domains.
... Enrichments of genome coordinates and set overlaps were calculated as described in (41). The positional overlaps between gene coordinates were calculated with BedTools (42) and the enrichment was defined as the observed-overexpected ratio of the coordinates' overlap. ...
Nearly one third of Saccharomyces cerevisiae protein coding sequences correspond to duplicate genes, equally split between small-scale duplicates (SSD) and whole-genome duplicates (WGD). While duplicate genes have distinct properties compared to singletons, to date, there has been no systematic analysis of their positional preferences. In this work, we show that SSD and WGD genes are organized in distinct gene clusters that occupy different genomic regions, with SSD being more peripheral and WGD more centrally positioned close to centromeric chromatin. Duplicate gene clusters differ from the rest of the genome in terms of gene size and spacing, gene expression variability and regulatory complexity, properties that are also shared by singleton genes residing within them. Singletons within duplicate gene clusters have longer promoters, more complex structure and a higher number of protein–protein interactions. Particular chromatin architectures appear to be important for gene evolution, as we find SSD gene-pair co-expression to be strongly associated with the similarity of nucleosome positioning patterns. We propose that specific regions of the yeast genome provide a favourable environment for the generation and maintenance of small-scale gene duplicates, segregating them from WGD-enriched genomic domains. Our findings provide a valuable framework linking genomic innovation with positional genomic preferences.
... Enrichments of genome coordinates and set overlaps were calculated as described in (Andreadis et al. 2014). The positional overlaps between gene coordinates were calculated with BedTools (Quinlan and Hall 2010) and the enrichment was defined as the observed-overexpected ratio of coordinate overlap. ...
Gene duplication is a major source of genomic innovation in all eukaryotes, with large proportions of genes being the result of either small-scale (SSD) or genome-wide duplication (WGD) events. In the model unicellular eukaryote Saccharomyces cerevisiae , of which nearly one third of the genome corresponds to gene duplicates, the two modes of duplication have been shown to follow different evolutionary fates, with SSD genes being more prone to acquire novel functionalities (neofunctionalization) and WGD more likely to retain different parts of the original ancestral function (subfunctionalization). Having previously described aspects of functional compartmentalization for the genes of S. cerevisiae , in this work we set out to investigate the existence of positional preferences of gene duplicates. We found that SSD and WGD genes are organized in distinct gene clusters that are, furthermore, segregated, occupying different regions, with SSD being more peripheral and WGD more centrally positioned close to centromeric chromatin.
Duplicate gene clusters differ from the rest of the genome in terms of gene size and spacing, gene expression variability and regulatory complexity. What is more interesting, some of these properties are also shared by singleton genes residing in duplicate-rich regions in a position-dependent manner. Our analysis further reveals particular chromatin architectures in the promoters of duplicate genes, which are generally longer, with less pronounced nucleosome-free regions, strong structural constraints and a larger number of regulatory elements. Such structural features appear to be important for gene evolution as we find SSD gene-pair co-expression to be strongly associated with the similarity of nucleosome positioning patterns.
We propose that specific regions of the yeast genome provide a favourable environment for the generation and maintenance of small-scale gene duplicates. The existence of such genomic “niches” is supported by the enrichment of these regions in singleton genes bearing similarities with gene “relics”, remnants of recent duplications that have reverted to single gene status. Our findings provide a valuable framework for the study of genomic innovation and suggest taking into account positional preferences in the study of gene emergence and fixation in experimentally and naturally evolving populations.
... We obtained domains of focal deregulation (DFD), as described in [24], and used the deduced domains of coordinated expression (DCE) from [25] to examine whether genes affected by AS tend to overlap with them to a greater extent than the one expected by chance. We performed a spatial overlap enrichment analysis implementing a permutation test as described in [26] and separately for various categories of DCEs and AS events (alternative acceptor/donor, exon skipping and intron retention). We found that AS events do not tend to overlap with domains enriched in deregulated genes, as suggested by the small overlaps of AS-affected genes with the DFDs (Figure 5, Table S3). ...
In addition to increasing the complexity of the transcriptional output, alternative RNA splicing can lead to the reduction of mRNA translation or the production of non-functional or malfunctional proteins, thus representing a vital component of the gene regulation process. Herein, we set out to detect and characterize alternative splicing events that occur in whole-blood samples of patients with Systemic Lupus Erythematosus (SLE) as compared to healthy counterparts. Through the implementation of a computational pipeline on published RNA-sequencing data, we identified extensive changes in the transcription dynamics affecting a large number of genes. We found a predominance of intron retention events, with the majority introducing premature stop codons, suggestive of gene repression, in both inactive and active SLE patient samples. Alternative splicing affected a distinct set of genes from the ones detected as differentially expressed in the same comparisons, while alternatively spliced genes tended to reside in genome areas associated with increased gene co-expression. Functional analysis of genes affected by alternative splicing pointed towards particular functions related to metabolism and histone acetylation as of potential interest. Together, our findings underline the importance of incorporating alternative splicing analyses in the context of molecular characterization of complex diseases such as SLE.
... It is known that, in physiological conditions, Rad9 binds chromatin by recognizing distinct histone methylation marks although its functional role at non-damaged DNA sites remains unclear (Gilbert et al. 2001;Granata et al. 2010;Hammet et al. 2007). Additionally, in a previous genome-wide study, our group found that, in the absence of exogenously induced DNA damage, Rad9 localizes on fragile genomic regions via its direct interaction with the multifunctional transcriptional activator Aft1, thereby ensuring rapid and effective cellular response to possible DNA damage events (Andreadis et al. 2014). Here, we demonstrate Rad9 association with chromatin in the absence of DNA damage, via its direct interaction with different transcription complexes. ...
... Our findings provide solid biochemical evidence for Rad9 association with a DNA-binding transactivator on active promoters as well as with initiating and processive RNA Pol II transcription complex. Rad9 was known to be recruited on chromatin only under DNA damage conditions via histone modifications whereas we have demonstrated that, in undamaged cells, it associates with Aft1 transcriptional activator on fragile genomic regions (Andreadis et al. 2014). Here, we show that Rad9 can also interact with chromatin, in the absence of DNA damage, via its direct association with different transcription complexes. ...
... Furthermore, the pattern of RNA Pol II on CTR1 gene remained unchanged when RAD9 was deleted whereas the combination of rad9Δ with mutant factors involved in elongation such as Hir1 or Spt16 did not reveal any synthetic effect on CTR1 expression or any growth defect (our unpublished observations). We have previously observed a slight growth defect for the rad9Δ strain, only in the presence of a-Azauracil (interfering with transcriptional elongation) (Andreadis et al. 2014). ...
While it is known that ScRad9 DNA damage checkpoint protein is recruited to damaged DNA by recognizing specific histone modifications, here we report a different way of Rad9 recruitment on chromatin under non DNA damaging conditions. We found Rad9 to bind directly with the copper-modulated transcriptional activator Mac1, suppressing both its DNA binding and transactivation functions. Rad9 was recruited to active Mac1-target promoters (CTR1, FRE1) and along CTR1 coding region following the association pattern of RNA polymerase (Pol) II. Hir1 histone chaperone also interacted directly with Rad9 and was partly required for its localization throughout CTR1 gene. Moreover, Mac1-dependent transcriptional initiation was necessary and sufficient for Rad9 recruitment to the heterologous ACT1 coding region. In addition to Rad9, Rad53 kinase also localized to CTR1 coding region in a Rad9-dependent manner. Our data provide an example of a yeast DNA-binding transcriptional activator that interacts directly with a DNA damage checkpoint protein in vivo and is functionally restrained by this protein, suggesting a new role for Rad9 in connecting factors of the transcription machinery with the DNA repair pathway under unchallenged conditions.
... For co-immunoprecipitation [29] the co-expressed cell lysate was incubated with an anti-sucinh polysera antibody overnight at 4°C. Further, it was incubated with protein A-Sepharose beads for 3 h at 4°C. ...
Human sucrase enzyme is a key therapeutic target for type 2 diabetes. While sugarcane sucrase inhibitor (sucinh) modulates invertase activity thereby accumulates sucrose. Molecular level understanding of sucinh towards mammalian α-glucosidases is scarce. The interaction of sucinh with human sucrase was identified and the association of these proteins was confirmed using co-purification, co-immunoprecipitation and pull-down assay. In addition, microscale thermophoresis assay showed that sucinh has a tight binding with sucrase (Kd = 4.77 nM) and a better affinity over acarbose. Collectively, in vitro, ex vivo and in silico data revealed that sucinh is selective for intestinal sucrase. The M region (H5/6 loop) of sucinh identified at the protein-protein interface is shown to have high affinity over N and C regions. Whereas, the biolayer luminescent imaging and microscale thermophoresis on the synthetic peptide of 28-amino acids of M region has a weak dose-dependent binding with sucrase. However, the synthetic peptide did not show substantial inhibition of sucrase and amylase activities at low concentration. Naturally derived carbohydrate mimics were shown to have a positive impact at the in vitro conditions. The insights obtained in this study give clues towards a new class of bioactive therapeutic peptides for α-glucosidases. A new horizon towards polypeptides derived from food sources emerge as a promising strategy for dietary interventions for prediabetic conditions.
... Overlaps between DFDs and differentially expressed genes, gene clusters or other sets of genomic coordinates were reported as Jaccard Indexes and assessed statistically through a permutation test, performed as described in [39]. ...
Systemic Lupus Erythematosus (SLE) is the prototype of autoimmune diseases, characterized by extensive gene expression perturbations in peripheral blood immune cells. Circumstantial evidence suggests that these perturbations may be due to altered epigenetic profiles and chromatin accessibility but the relationship between transcriptional deregulation and genome organization remains largely unstudied. We developed a genomic approach that leverages patterns of gene coexpression from genome-wide transcriptome profiles in order to identify statistically robust Domains of Co-ordinated gene Expression (DCEs). By implementing this method on gene expression data from a large SLE patient cohort, we identify significant disease-associated alterations in gene co-regulation patterns, which also correlate with the SLE activity status. Low disease activity patient genomes are characterized by extensive fragmentation leading to DCEs of smaller size. High disease activity genomes display excessive spatial redistribution of co-expression domains with expanded and newly-appearing (emerged) DCEs. Fragmentation and redistribution of gene coexpression patterns correlate with SLE-implicated biological pathways and clinically relevant endophenotypes such as kidney involvement. Notably, genes lying at the boundaries of split DCEs of low activity genomes are enriched in the interferon and other SLE susceptibility signatures, suggesting the implication of DCE fragmentation at early disease stages. Interrogation of promoter-enhancer interactions from various immune cell subtypes shows that a significant percentage of nested connections are disrupted by a DCE split or depletion in SLE genomes. Collectively, our results underlining an important role for genome organization in shaping gene expression in SLE, could provide valuable insights into disease pathogenesis and the mechanisms underlying disease flares.
Significance
Although widespread gene expression changes have been reported in Systemic Lupus Erythematosus (SLE), attempts to link gene deregulation with genome structure have been lacking. Through a computational framework for the segmentation of gene expression data, we reveal extensive fragmentation and reorganization of gene co-regulation domains in SLE, that correlates with disease activity states. Gene co-expression domains pertaining to biological functions implicated in SLE such as the interferon pathway, are being disrupted in patients, while others associated to severe manifestations such as nephritis, emerge in previously uncorrelated regions of the genome. Our results support extensive genome re-organization underlying aberrant gene expression in SLE, which could assist in the early detection of disease flares in patients that are in remission.
Graphical Abstract
... Overlaps between DFDs and differentially expressed genes, gene clusters or other sets of genomic coordinates were reported as Jaccard Indexes and assessed statistically through a permutation test, performed as described in [39]. ...
Genes in linear proximity often share regulatory inputs, expression and evolutionary patterns, even in complex eukaryote genomes with extensive intergenic sequences. Gene regulation, on the other hand, is effected through the co-ordinated activation (or suppression) of genes participating in common biological pathways, which are often transcribed from distant loci. Existing approaches for the study of gene expression focus on the functional aspect, taking positional constraints into account only marginally. In this work we propose a novel concept for the study of gene expression, through the combination of topological and functional information into bipartite networks. Starting from genome-wide expression profiles, we define extended chromosomal regions with consistent patterns of differential gene expression and then associate these domains with enriched functional pathways. By analyzing the resulting networks in terms of size, connectivity and modularity we can draw conclusions on the way genome organization may underlie the gene regulation program. Implementation of this approach in a detailed RNASeq profiling of sustained Tnf stimulation of mouse synovial fibroblasts, allowed us to identify unexpected regulatory changes taking place in the cells after 24 h of stimulation. Bipartite network analysis suggests that the cytokine response set by Tnf, progresses through two distinct transitions. An early generalization of the inflammatory response, that is followed by a late shutdown of immune-related functions and the redistribution of expression to developmental and cell adhesion pathways and distinct chromosomal regions. We show that the incorporation of topological information may provide additional insights in the complex propagation of Tnf activation.
... For instance, Aft2 protects the cells from toxic doses of the metalloid selenite by indirectly repressing the low-affinity phosphate transport system, which is a key entry point for selenite [130]. Aft1 has been identified in genetic screens as a partner of the chromosome segregation machinery and as an actor of the DNA damage response [131][132][133][134]. ...
Iron is an essential element to most microorganisms, yet an excess of iron is toxic. Hence, living cells have to maintain a tight balance between iron uptake and iron consumption and storage. The control of intracellular iron concentrations is particularly challenging for pathogens because mammalian organisms have evolved sophisticated high-affinity systems to sequester iron from microbes and because iron availability fluctuates among the different host niches. In this review, we present the current understanding of iron homeostasis and its regulation in the fungal pathogen Candida glabrata. This yeast is an emerging pathogen which has become the second leading cause of candidemia, a life-threatening invasive mycosis. C. glabrata is relatively poorly studied compared to the closely related model yeast Saccharomyces cerevisiae or to the pathogenic yeast Candida albicans. Still, several research groups have started to identify the actors of C. glabrata iron homeostasis and its transcriptional and post-transcriptional regulation. These studies have revealed interesting particularities of C. glabrata and have shed new light on the evolution of fungal iron homeostasis.
