Search

Abstract Background Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays. Results In general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. Conclusions The tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of tps2Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.

Abstract Background Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Results Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Conclusions Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

Background: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists., Results: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons., Conclusions: Given the profound early mesenchymal expression and MET signature of LGR6 + cells, together with the lineage tracing of mesenchymal LGR6 + cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association of Anatomists.)

Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or 'tubuloids'. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function.

Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and clustering of transcript abundance time series in synchronized cells revealed a requirement for Set1 for proper cell-cycle-dependent gene expression and Δset1 cells displayed delayed entry into S phase. Disruption of G1/S regulation in Δmbp1 and Δswi4 transcription factor mutants duplicated both benomyl resistance and suppression of ipl1-2 as was observed with Δset1 Taken together our results support a role for H3K4 methylation in the coordination of cell-cycle progression and proper assembly of the mitotic spindle during mitosis., (Copyright © 2017 by the Genetics Society of America.)

FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion., (© 2015 The Authors.)

The regulation of gene expression is, in large part, mediated by interplay between the general transcription factors (GTFs) that function to bring about the expression of many genes and site-specific DNA-binding transcription factors (STFs). Here, quantitative genetic profiling using the epistatic miniarray profile (E-MAP) approach allowed us to measure 48 391 pairwise genetic interactions, both negative (aggravating) and positive (alleviating), between and among genes encoding STFs and GTFs in Saccharomyces cerevisiae. This allowed us to both reconstruct regulatory models for specific subsets of transcription factors and identify global epistatic patterns. Overall, there was a much stronger preference for negative relative to positive genetic interactions among STFs than there was among GTFs. Negative genetic interactions, which often identify factors working in non-essential, redundant pathways, were also enriched for pairs of STFs that co-regulate similar sets of genes. Microarray analysis demonstrated that pairs of STFs that display negative genetic interactions regulate gene expression in an independent rather than coordinated manner. Collectively, these data suggest that parallel/compensating relationships between regulators, rather than linear pathways, often characterize transcriptional circuits.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Spontaneous mammary tumors are the most prevalent type of neoplasms in women as well as in female dogs. Although ovarian hormones estrogen and progesterone are known to play a key role in mammary tumorigenesis, conflicting reports have been obtained from in vivo and in vitro studies concerning the role of especially progesterone in mammary tumorigenesis. Prolonged exposure to high concentrations of progesterone during the unusually long luteal phase of the estrous cycle is suspected to be the key event in canine mammary tumorigenesis. Accordingly, previous studies have shown the development of mammary hyperplasia in dogs upon prolonged progestin administration. In this study, a dog-specific cDNA microarray was used to identify oncogenic determinants in progestin-induced canine hyperplasia (CMH) and spontaneous mammary tumors (CMC) by comparing expression profiles to those obtained from mammary glands of healthy dogs. The CMH profile showed elevated expression of genes involved in cell proliferation such as PCNA, NPY, RAN and also alterations in expression of transcription factors and cell adhesion molecules. Whereas in CMC, major alterations to the expression of genes involved in cell motility, cytoskeletal organization and extra cellular matrix production was evident besides differential expression of cell proliferation inducing genes. The overall gene expression profile of CMH was related to cell proliferation where as that of CMC was associated with both cell proliferation as well as neoplastic transformation. In conclusion, our findings support a strong cell proliferation inducing potential of progestins in the canine mammary gland. Moreover, deregulated genes identified in CMC are potentially involved in their malignant and may serve as prospective therapeutic targets.

In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.

Copper toxicity in the liver is mediated by free-radical generation, resulting in oxidative stress. To prevent toxic accumulation of copper, liver cells adapt to high copper levels. Here, we used microarray analysis to compare the adaptive responses on global gene expression in liver cells exposed to high copper levels in vitro and in vivo. In HepG2 cells we identified two clusters of upregulated genes over time, an "early" cluster that comprised metallothionein genes and a "late" cluster, highly enriched in genes involved in proteasomal degradation and in oxidative stress response. Concomitant with the "late" cluster, we detected a significant downregulation of several copper metabolism MURR1 domain (COMMD) genes that were recently implicated in copper metabolism and inhibition of nuclear transcription factor kappaB (NF-kappaB) signaling. As metal-induced oxidative stress increases NF-kappaB activity, our data suggest a role for reduced COMMD protein levels in prolonged activation of NF-kappaB, thus inducing cell survival. Mice exposed to a copper diet that highly exceeded normal daily intake accumulated only twofold more hepatic copper than control mice. Although a moderate, but significant upregulation of a set of 22 genes involved in immunity, iron and cholesterol metabolism was detected, these cannot account for direct mechanisms involved in copper excretion. In conclusion, we identified a novel set of genes that represent a delayed response to copper overload, thus providing insight into the adaptive transcriptional response to copper-induced oxidative stress.

Leukemic transformation often requires activation of protein kinase B (PKB/c-Akt) and is characterized by increased proliferation, decreased apoptosis, and a differentiation block. PKB phosphorylates and inactivates members of the FOXO subfamily of Forkhead transcription factors. It has been suggested that hyperactivation of PKB maintains the leukemic phenotype through actively repressing FOXO-mediated regulation of specific genes. We have found expression of the transcriptional repressor Id1 (inhibitor of DNA binding 1) to be abrogated by FOXO3a activation. Inhibition of PKB activation or growth factor deprivation also resulted in strong down-regulation of Id1 promoter activity, Id1 mRNA, and protein expression. Id1 is highly expressed in Bcr-Abl-transformed K562 cells, correlating with high PKB activation and FOXO3a phosphorylation. Inhibition of Bcr-Abl by the chemical inhibitor STI571 resulted in activation of FOXO3a and down-regulation of Id1 expression. By performing chromatin immunoprecipitation assays and promoter-mutation analysis, we demonstrate that FOXO3a acts as a transcriptional repressor by directly binding to the Id1 promoter. STI571 treatment, or expression of constitutively active FOXO3a, resulted in erythroid differentiation of K562 cells, which was inhibited by ectopic expression of Id1. Taken together our data strongly suggest that high expression of Id1, through PKB-mediated inhibition of FOXO3a, is critical for maintenance of the leukemic phenotype.

CD4+ T cells play a critical role in allergic diseases, both in the affected tissue as well as systemically. Our objective was to investigate the in vivo activation state of peripheral blood CD4+ T cells of atopic dermatitis (AD) patients by analyzing gene expression profiles of unstimulated CD4+ T cells. mRNA samples from blood CD4+ T cells, isolated from five AD patients and seven healthy controls (HC), were analyzed using oligonucleotide arrays. Differentially regulated genes were validated by quantitative PCR (Q-PCR) in a larger group of patients with AD, in a group of patients with allergic asthma (AA), and HC subjects. In addition, "typical" T helper type 1 (Th1)- and Th2-related genes were analyzed by Q-PCR. Microarray analysis revealed differential expression of 52 genes in AD patients. Q-PCR confirmed several differentially regulated genes in AD, including CCR10, CRTH2, C-JUN, and NR4A2. Two groups of genes with highly correlating gene expression levels involved in tissue homing and proliferation or apoptosis, respectively, were identified. No marked differences were found in gene expression levels of typical Th1 or Th2 genes in AD or in AA patients. This study demonstrates that peripheral blood, unstimulated CD4+ T cells in AD patients show differentially expressed genes involved in tissue homing, proliferation, and apoptosis. No marked expression differences of "typical" atopy genes were found.

Shear stress modulates gene expression in endothelial cells (ECs) partly through nitric oxide (NO), acting via enhanced cGMP formation by guanylyl cyclase (GC). We addressed non-cGMP-mediated transcriptional responses to shear stress in human umbilical ECs subjected to high-laminar shear stress (25 dyn/cm2; 150 minutes). RNA was isolated, reverse-transcribed, Cy3/5-labeled, and hybridized to 19 K human microarrays. High shear (n=6), high shear with 100 micromol/L L-NAME (n=3), and high shear with 10 micromol/L ODQ (GC inhibitor) in the perfusate (n=3) was compared with samples not subjected to flow. Among genes responding to high shear were HMOX1 (up) and PPARG (down). A high percentage of gene expression modulation by shear was absent during concomitant L-NAME or ODQ. Several transcriptional modulators were found (up: SOX5, SOX25, ZNF151, HOXD10; down: SOX11); a number of genes were regulated by shear and by shear with ODQ, but not regulated during L-NAME, indicating a nitric oxide synthase (NOS)-dependent, guanylyl cyclase (GC)-independent pathway. Several genes only responded to shear stress during L-NAME, others only responded to shear during ODQ. Upstream binding site analysis indicated shear stress and NO-dependent regulation of transcription via SOX5 and SOX9. Although NO importantly modulated the effect of shear stress on EC transcription, HMOX1 was consistently induced by shear stress, but not dependent on NOS or GC. Using bio-informatics software and databases, a promoter analysis identified SOX5 and SOX9 as potential, novel, shear-sensitive, and NO-dependent transcriptional regulators. The role of HMOX1 as a potential backup for NOS and the downstream role of SOXes should be explored.

Functional annotation of fully sequenced genomes is still a major issue. High-throughput data sets could be used to provide more and better functional annotations. However differences in data quality need to be taken into account. For this purpose these high-throughput data sets need to be integrated so that the data quality can be assessed, hypotheses can be prioritized and existing annotations can be improved and extended.

Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

Expression profiling using DNA microarrays is starting to come of age. The past year has seen significant advances in the number, scope and quality of studies that incorporate expression profiling experiments. Attention is starting to move on from making DNA microarrays to appropriate experimental design and sophisticated data analysis techniques.

The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II. TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAF(II)) subunits and each complex contains a subunit with histone acetyltransferase activity. These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping. Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes. We find that expression of most genes requires one or more of the common TAF(II) subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression. Among the subunits shared by TFIID and SAGA are three histone-like TAF(II)s, which have been proposed to form a sub-complex and mediate a common function in global transcription. Unexpectedly, we find that the histone-like TAF(II)s have distinct roles in expression of the yeast genome. Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAF(II)145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex.

Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally. Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4-10). Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast. Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes. Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding and the effects of loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.