Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes.

The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.

Targeted Gene Repression Technologies for Regenerative Medicine, Genomics, and Gene Therapy

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

Rescue of gene expression by modified REST decoy oligonucleotides in a cellular model of Huntington's disease

Transcriptional dysfunction is a prominent hallmark of Huntington's disease (HD). Several transcription factors have been implicated in the aetiology of HD progression and one of the most prominent is repressor element 1 (RE1) silencing transcription factor (REST). REST is a global repressor of neuronal gene expression and in the presence of mutant Huntingtin increased nuclear REST levels lead to elevated RE1 occupancy and a concomitant increase in target gene repression, including brain-derived neurotrophic factor. It is of great interest to devise strategies to reverse transcriptional dysregulation caused by increased nuclear REST and determine the consequences in HD. Thus far, such strategies have involved RNAi or mutant REST constructs. Decoys are double-stranded oligodeoxynucleotides corresponding to the DNA-binding element of a transcription factor and act to sequester it, thereby abrogating its transcriptional activity. Here, we report the use of a novel decoy strategy to rescue REST target gene expression in a cellular model of HD. We show that delivery of the decoy in cells expressing mutant Huntingtin leads to its specific interaction with REST, a reduction in REST occupancy of RE1s and rescue of target gene expression, including Bdnf. These data point to an alternative strategy for rebalancing the transcriptional dysregulation in HD.

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated here, led us to the hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. In this study, we investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods we were able to detect deviations from the wild-type patterns of the anterior-most pair-rule stripes in different genetic backgrounds, which were consistent with Hkb-mediated repression. Moreover, we developed an image processing tool that, for the most part, confirmed our assumptions. Using an hkb misexpression system, we further detected specific repression on anterior stripes. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. We further discuss that Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes. (C) 2011 Elsevier Inc. All rights reserved.

Functional requirements of histone deacetylases in Tup1-Ssn6 repression

The corepressor complex Tup1-Ssn6 regulates many classes of genes in yeast including cell type specific, glucose repressible, and DNA damage inducible. Tup1 and Ssn6 are recruited to target promoters through their interactions with specific DNA binding proteins such as α2, Mig1, and Crt1. Most promoters that are repressed by this corepressor complex exhibit a high degree of nucleosomal organization. This chromatin domain occludes transcription factor access to the promoter element resulting in gene repression. Previous work indicated that Tup1 interacts with underacetylated isoforms of H3 and H4, and that mutation of these histones synergistically compromises repression. These studies predict that Tup1-hypoacetyalted histone interaction is important to the repression mechanism, and in vivo hyperacetylation might compromise the corepressors ability to repress target genes. ^ One way to alter histone acetylation levels in vivo is to alter the balance between histone acetyltransferases and histone deacetylases. To date five histone deacetylases (HDACs) have been identified in yeast Rpd3, Hos1, Hos2, Hos3 and Hda1. Deletion of single or double HDAC genes had little to no effect on Tup1-Ssn6 repression, but simultaneous deletion of three specific activities Rpd3, Hos1, and Hos2 abolished repression in vivo. Promoter regions of Tup1-Ssn6 target genes in these triple deacetylase mutant cells are dramatically hyperacetylated in both H3 and H4. Examination of bulk histone acetylation levels showed that this specific HDAC triple mutant combination (rpd3 hos1 hos2) caused a dramatic and concomitant hyperacetylation of both H3 and H4. The loss of repression in the rpd3 hos1 hos2 cells, but not in other mutants, is consistent with previous observations, which indicate that histones provide redundant functions in the repression mechanism and that high levels of acetylation are required to prevent Tup1 binding. Investigation into a potential direct interaction between the Tup1-Ssn6 corepressor complex and one or more HDAC activities showed that both Rpd3 and Hos2 interact with the corepressor complex in vivo. These findings indicate that Tup1-Ssn6 repression involves the recruitment of histone deacetylase activities to target promoters, where they locally deacetylate histone residues promoting Tup1-histone tail interaction to initiate and/or maintain the repressed state. ^

Involvement of histone H3 methylation in Tup1-Ssn6 repression

The Tup1-Ssn6 complex regulates the expression of diverse classes of genes in Saccharomyces cerevisiae including those regulated by mating type, DNA damage, glucose, and anaerobic stress. The complex is recruited to target genes by sequence-specific repressor proteins. Once recruited to particular promoters, it is not completely clear how it functions to block transcription. Repression probably occurs through interactions with both the basal transcriptional machinery and components of chromatin. Tup1 interactions with chromatin are strongly influenced by acetylation of histories H3 and H4. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Like acetylation, histone methylation is involved in regulation of gene expression. The possible role of histone methylation in Tup1 repression is not known. Here we examined possible roles of histone methyltransferases in Tup1-Ssn6 functions. We found that like other genes, Tup1-Ssn6 target genes exhibit increases in the levels of histone H3 lysine 4 methylation upon activation. However, deletion of individual or multiple histone methyltransferases (HMTs) and other SET-domain containing genes has no apparent effect on Tup1-Ssn6 mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Since deletion of SET2 does not affect Tup1-Ssn6 repression, Ssn6 may utilize Set2 in other contexts to regulate gene repression. In order examine if the two components of the Tup1-Ssn6 complex have independent functions in the cell, we identified genes differentially expressed in tup1Δ and ssn6Δ mutants using DNA microarrays. Our data indicate that ∼4% of genes in the cell are regulated by Ssn6 independently of Tup1. In addition, expression of genes regulated by Tup1-Ssn6 seems to be differently affected by deletion of Ssn6 and deletion of Tup1, which indicates that these components might have separate functions. Our data shed new light on the classical view of Tup1-Ssn6 functions, and indicate that Ssn6 might have repressive functions as well. ^

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor β through E2F binding sites

Transforming growth factor β (TGF-β) causes growth arrest in most cell types. TGF-β induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G1. It is possible, however, that the E2F-RB complex induced by TGF-β may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-β treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-β. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-β treatment. Consistent with their repression of TGF-β-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15INK4B gene effectively inhibited its induction by TGF-β. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-β induction of p15INK4B gene when tethered to p15INK4B promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-β. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-β treatment, which may constitute an integral part of the TGF-β-induced growth arrest program.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Defense and counterdefense in the plant world

An important role of RNA interference (RNAi)-like pathways in plants is defense against viral infection. Viruses can overcome this defense by expressing proteins that suppress the pathway. A new study of Agrobacterium tumefaciens infection reveals that this plant pathogen, although a bacterium, also induces and then suppresses the host RNAi response. © 2006 Nature Publishing Group.

G9a histone methyltransferase contributes to imprinting in the mouse placenta

Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.

Multifaceted roles of peroxisome proliferator-activated receptors (PPARs) at the cellular and whole organism levels.

Chronic disorders, such as obesity, diabetes, inflammation, non-alcoholic fatty liver disease and atherosclerosis, are related to alterations in lipid and glucose metabolism, in which peroxisome proliferator-activated receptors (PPAR)α, PPARβ/δ and PPARγ are involved. These receptors form a subgroup of ligand-activated transcription factors that belong to the nuclear hormone receptor family. This review discusses a selection of novel PPAR functions identified during the last few years. The PPARs regulate processes that are essential for the maintenance of pregnancy and embryonic development. Newly found hepatic functions of PPARα are the mediation of female-specific gene repression and the protection of the liver from oestrogen induced toxicity. PPARα also controls lipid catabolism and is the target of hypolipidaemic drugs, whereas PPARγ controls adipocyte differentiation and regulates lipid storage; it is the target for the insulin sensitising thiazolidinediones used to treat type 2 diabetes. Activation of PPARβ/δ increases lipid catabolism in skeletal muscle, the heart and adipose tissue. In addition, PPARβ/δ ligands prevent weight gain and suppress macrophage derived inflammation. In fact, therapeutic benefits of PPAR ligands have been confirmed in inflammatory and autoimmune diseases, such as encephalomyelitis and inflammatory bowel disease. Furthermore, PPARs promote skin wound repair. PPARα favours skin healing during the inflammatory phase that follows injury, whilst PPARβ/δ enhances keratinocyte survival and migration. Due to their collective functions in skin, PPARs represent a major research target for our understanding of many skin diseases. Taken altogether, these functions suggest that PPARs serve as physiological sensors in different stress situations and remain valuable targets for innovative therapies.

Étude de la collaboration entre les facteurs de transcription hématopoïétiques lors du développement et de la différenciation des cellules érythroïdes

La régulation transcriptionnelle des gènes est cruciale pour permettre le bon fonctionnement des cellules. Afin que les cellules puissent accomplir leurs fonctions, les gènes doivent être exprimés adéquatement dans le bon type cellulaire et au stade de développement et de différenciation approprié. Un dérèglement dans l’expression de un ou plusieurs gènes peut entraîner de graves conséquences sur le destin de la cellule. Divers éléments en cis (ex : promoteurs et enhancers) et en trans (machinerie transcriptionnelle et facteurs de transcription) sont impliqués dans la régulation de la transcription. Les gènes du locus humain beta-globine (hub) sont exprimés dans les cellules érythroïdes et sont finenement régulés lors du développement et de la différenciation. Des mutations dans différentes régions du locus causent entre autres les beta-thalassémies. Nous avons utilisé ce modèle bien caractérisé afin d’étudier différents mécanismes de régulation favorisés par les facteurs de transcription qui sont exprimés dans les cellules érythroïdes. Nous nous sommes intéressés à l’importance de l’élément en cis HS2 du Locus control region. Cet élément possède plusieurs sites de liaison pour des facteurs de transcription impliqués dans la régulation des gènes du locus hub. Nos résultats montrent que HS2 possède un rôle dans l’organisation de la chromatine du locus qui peut être dissocié de son rôle d’enhancer. De plus, HS2 n’est pas essentiel pour l’expression à haut niveau du gène beta alors qu’il est important pour l’expression des gènes gamma. Ceci suggère que le recrutement des différents facteurs au site HS2 lors du développement influence différement les gènes du locus. Dans un deuxième temps, nous avons investigué l’importance de HS2 lors de la différenciation des cellules érythroïdes. Il avait été rapporté que l’absence de HS2 influence grandement la potentialisation de la chromatine du gène beta. La potentialisation dans les cellules progénitrices favorise l’activation transcriptionnelle du gène dans les cellules matures. Nous avons caractérisé le recrutement de différents facteurs de transcription au site HS2 et au promoteur beta dans les cellules progénitrices hématopoïétiques (CPH) ainsi que dans les cellules érythroïdes matures. Nos résultats montrent que le facteur EKLF est impliqué dans la potentialisation de la chromatine et favorise le recrutement des facteurs BRG1, p45 et CBP dans les CPH. L’expression de GATA-1 dans les cellules érythroïdes matures permet le recrutement de GATA-1 au locus hub dans ces cellules. Ces données suggèrent que la combinaison de EKLF et GATA-1 est requise pour permettre une activation maximale du gène beta dans les cellules érythroïdes matures. Un autre facteur impliqué dans la régulation du locus hub est Ikaros. Nous avons étudié son recrutement au locus hub et avons observé que Ikaros est impliqué dans la répression des gènes gamma. Nos résultats montrent aussi que GATA-1 est impliqué dans la répression de ces gènes et qu’il interagit avec Ikaros. Ensemble, Ikaros et GATA-1 favorisent la formation d’un complexe de répression aux promoteurs gamma. Cette étude nous a aussi permis d’observer que Ikaros et GATA-1 sont impliqués dans la répression du gène Gata2. De façon intéressante, nous avons caractérisé le mécanisme de répression du gène Hes1 (un gène cible de la voie Notch) lors de la différenciation érythroïde. Similairement à ce qui a été observé pour les gènes gamma, Hes1 est aussi réprimé par Ikaros et GATA-1. Ces résultats suggèrent donc que la combinaison de Ikaros et GATA-1 est associée à la répression de plusieurs de gènes dans les cellules érythroïdes. Globalement cette thèse rapporte de nouveaux mécanismes d’action de différents facteurs de transcription dans les cellules érythroïdes. Particulièrement, nos travaux ont permis de proposer un modèle pour la régulation des gènes du locus hub lors du développement et de la différenciation. De plus, nous rapportons pour la première fois l’importance de la collaboration entre les facteurs Ikaros et GATA-1 dans la régulation transcriptionnelle de gènes dans les cellules érythroïdes. Des mutations associées à certains des facteurs étudiés ont été rapportées dans des cas de beta-thalassémies ainsi que de leucémies. Nos travaux serviront donc à avoir une meilleure compréhension des mécanismes d’action de ces facteurs afin de potentiellement pouvoir les utiliser comme cibles thérapeutiques.