Activation Of Target Promoters Research Articles

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Sustained MYB Activity Is Necessary for Oncogenic Transcription in KMT2A-Rearranged Acute Lymphoblastic Leukemia through Enhancer-Promoter Interactions and Epigenetic Modifications at Enhancers

Aberrant enhancer activity is a hallmark of many cancers including KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive leukemia subtype with an event-free survival of 19-45% in infant-ALL (<1 year of age) and 50-59% in childhood-ALL cases. Targeting enhancer activity may provide a novel therapeutic strategy for this hard-to-treat disease. Transcription factors are key effectors of enhancer activity. They recognise and bind to specific DNA sequences at enhancers, driving the expression of target genes from these distal loci. MYB is an important hematopoietic transcription factor that is commonly overexpressed in KMT2A-rearranged leukemias and is required for leukemia survival, where loss of MYB has been shown to abrogate the leukemia phenotype. However, the precise mechanism by which MYB contributes to enhancer function, and the stages of enhancer activation at which it acts are unknown. We employed a well-characterized chromatin anchoring system to interrogate MYB function and determine whether it can recruit specific enhancer activities de novo. By fusing the MYB transactivation domain (MYB TA) to the Tet repressor (TetR) DNA binding domain, MYB TA is directed to an array of Tet operator ( TetO) sequences incorporated into a neutral genomic region devoid of native gene regulatory elements. We found that MYB TA alone is sufficient to recruit key enhancer-associated proteins, including P300, Brd4 and Mediator to the TetO locus. We observed MYB TA-dependent deposition of H3K27ac, increased chromatin accessibility and transcription not just at the TetO locus, but remarkably, also at regions more than 50kb away. Using the chromosome conformation capture (3C) technique NG Capture-C, we observed MYB TA-induced DNA interactions between TetO and the distal sites of transcription, as well as more long-range interactions up to 400kb distal to where MYB TA is bound. Together, these observations suggest that MYB TA binding alone is sufficient to establish an enhancer-like regulatory element, which activates distant cryptic promoters. This activity is dependent on the continued presence of MYB TA, as disruption of MYB TA binding results in a rapid loss of transcription and acquired chromatin features, implicating MYB in enhancer maintenance as well as initiation. To confirm our observations in a disease context, we tested the requirement for MYB at endogenous enhancers in SEM cells, a model of KMT2A-rearranged ALL. By stably introducing a degradation tag (FKBP12 F36V) to the endogenous MYB gene, we were able to rapidly induce protein degradation and observe the immediate effects on chromatin features. Using transient transcriptome sequencing, we noted significant transcriptional downregulation of known MYB target genes, including BCL2, MYC, LMO4 and CDK6. We also observed decreases in enhancer H3K27ac levels and eRNA transcription, most marked at MYB-bound loci. To investigate the requirement for MYB in maintaining physical proximity between enhancer and promoter, we performed the base pair-resolution 3C technique Micro-Capture-C (MCC). MCC revealed highly punctate interactions between enhancer elements and their target promoters. At BCL2 and MYC, MYB degradation results in a reduction in interactions with the promoter at discrete regions within the enhancers. This argues for highly localised MYB-dependent activity at enhancers, driving interaction with and activation of distal target promoters. Overall, our data argue for a role for MYB in establishing enhancers de novo, indicating that MYB overexpression may directly result in onco-enhancer activity at key genes in vivo. At a subset of MYB-bound enhancers, the continued presence of MYB is absolutely required to maintain enhancer activity and oncogene upregulation. Further exploration of associated co-factors that mediate MYB-dependent enhancer activity may identify novel targets for therapeutic exploitation.

Flavone inhibits Staphylococcus aureus virulence via inhibiting the sae two component system

The rising prevalence of antibiotic resistance in Staphylococcus aureus calls for the development of innovative antimicrobial agents targeting novel pathways. S. aureus generates various virulence factors that compromise host defense mechanisms. Flavone, a core structure of flavonoids, has been shown to diminish the production of staphyloxanthin and alpha-hemolysin. Nonetheless, the influence of flavone on the majority of other virulence factors in S. aureus and its underlying molecular mechanism remain elusive. In this study, we examined the impact of flavone on the transcriptional profile of S. aureus using transcriptome sequencing. Our findings revealed that flavone substantially downregulated the expression of over 30 virulence factors implicated in immune evasion by the pathogen. Gene set enrichment analysis of the fold change-ranked gene list in relation to the Sae regulon indicated a robust association between flavone-induced downregulation and membership in the Sae regulon. Through the analysis of Sae target promoter-gfp fusion expression patterns, we observed a dose-dependent inhibition of Sae target promoter activity by flavone. Moreover, we discovered that flavone protected human neutrophils from S. aureus-mediated killing. Flavone also decreased the expression of alpha-hemolysin and other hemolytic toxins, resulting in a reduction in S. aureus' hemolytic capacity. Additionally, our data suggested that the inhibitory effect of flavone on the Sae system operates independently of its capacity to lower staphyloxanthin levels. In conclusion, our study proposes that flavone exhibits a broad inhibitory action on multiple virulence factors of S. aureus by targeting the Sae system, consequently diminishing the bacterium's pathogenicity.

Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50

Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.

A gain-of-function mutation in CITED2 is associated with congenital heart disease

CITED2 is a transcription co-activator that interacts with TFAP2 and CBP/ P300 transcription factors to regulate the proliferation and differentiation of the cardiac progenitor cells. It acts upstream to NODAL-PITX2 pathways and regulates the left-right asymmetry. Both human genetic and model organism studies have shown that altered expression of CITED2 causes various forms of congenital heart disease. Therefore, we sought to screen the coding region of CITED2 to identify rare genetic variants and assess their impact on the structure and function of the protein. Here, we have screened 271 non-syndromic, sporadic CHD cases by Sanger’s sequencing method and detected a non-synonymous variant (c.301C>T, p.P101S) and two synonymous variants (c.21C>A, p.A7A; c.627C>G, p.P209P). The non-synonymous variant c.301C>T (rs201639244) is a rare variant with a minor allele frequency of 0.00011 in the gnomAD browser and 0.0018 in the present study. in vitro analysis has demonstrated that p.P101S mutation upregulates the expression of downstream target genes Gata4, Mef2c, Nfatc1&2, Nodal, Pitx2, and Tbx5 in P19 cells. Luciferase reporter assay also demonstrates enhanced activation of downstream target promoters. Further, in silico analyses implicate that increased activity of mutant CITED2 is possibly due to phosphorylation of Serine residue by proline-directed kinases. Homology modeling and alignment analysis have also depicted differences in hydrogen bonding and tertiary structures of wild-type versus mutant protein. The impact of synonymous variations on the mRNA structure of CITED2has been analyzed by Mfold and relative codon bias calculations. Mfold results have revealed that both the synonymous variants can alter the mRNA structure and stability. Relative codon usage analysis has suggested that the rate of translation is attenuated due to these variations. Altogether, our results from genetic screening as well as in vitro and in silico studies support a possible role of nonsynonymous and synonymous mutations in CITED2contributing to pathogenesis of CHD.

P53 pulse modulation differentially regulates target gene promoters to regulate cell fate decisions.

The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features—thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.

The wheat TabZIP2 transcription factor is activated by the nutrient starvation-responsive SnRK3/CIPK protein kinase.

The understanding of roles of bZIP factors in biological processes during plant development and under abiotic stresses requires the detailed mechanistic knowledge of behaviour of TFs. Basic leucine zipper (bZIP) transcription factors (TFs) play key roles in the regulation of grain development and plant responses to abiotic stresses. We investigated the role and molecular mechanisms of function of the TabZIP2 gene isolated from drought-stressed wheat plants. Molecular characterisation of TabZIP2 and derived protein included analyses of gene expression and its target promoter, and the influence of interacting partners on the target promoter activation. Two interacting partners of TabZIP2, the 14-3-3 protein, TaWIN1 and the bZIP transcription factor TaABI5L, were identified in a Y2H screen. We established that under elevated ABA levels the activity of TabZIP2 was negatively regulated by the TaWIN1 protein and positively regulated by the SnRK3/CIPK protein kinase WPK4, reported previously to be responsive to nutrient starvation. The physical interaction between the TaWIN1 and the WPK4 was detected. We also compared the influence of homo- and hetero-dimerisation of TabZIP2 and TaABI5L on DNA binding. TabZIP2 gene functional analyses were performed using drought-inducible overexpression of TabZIP2 in transgenic wheat. Transgenic plants grown under moderate drought during flowering, were smaller than control plants, and had fewer spikes and seeds per plant. However, a single seed weight was increased compared to single seed weights of control plants in three of four evaluated transgenic lines. The observed phenotypes of transgenic plants and the regulation of TabZIP2 activity by nutrient starvation-responsive WPK4, suggest that the TabZIP2 could be the part of a signalling pathway, which controls the rearrangement of carbohydrate and nutrient flows in plant organs in response to drought.

Activation of CK1ɛ by PP2A/PR61ɛ is required for the initiation of Wnt signaling.

Canonical Wnt signaling induces the stabilization of β-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 co-receptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ɛ, γ and GSK3. Among these, activation of CK1ɛ, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ɛ is activated by the PP2A/PR61ɛ phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ɛ with Frizzled-bound PR61ɛ regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ɛ and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.

A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors

Extracytoplasmic function (ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The Myxococcus xanthus genome reveals ∼45 putative ECF-σ factors, but for the overwhelming majority, the specific signals or mechanisms for selective activation and regulation remain unknown. One well-studied ECF-σ, CarQ, binds to its anti-σ, CarR, and is inactive in the dark but drives its own expression from promoter P(QRS) on illumination. This requires the CarD/CarG complex, the integration host factor (IHF) and a specific CarD-binding site upstream of P(QRS). Here, we show that DdvS, a previously uncharacterized ECF-σ, activates its own expression in a CarD/CarG-dependent manner but is inhibited when specifically bound to the N-terminal zinc-binding anti-σ domain of its cognate anti-σ, DdvA. Interestingly, we find that the autoregulatory action of 11 other ECF-σ factors studied here depends totally or partially on CarD/CarG but not IHF. In silico analysis revealed possible CarD-binding sites that may be involved in direct regulation by CarD/CarG of target promoter activity. CarD/CarG-linked ECF-σ regulation likely recurs in other myxobacteria with CarD/CarG orthologous pairs and could underlie, at least in part, the global regulatory effect of the complex on M. xanthus gene expression.

Enhancer–promoter interference and its prevention in transgenic plants

Biotechnology has several advantages over conventional breeding for the precise engineering of gene function and provides a powerful tool for the genetic improvement of agronomically important traits in crops. In particular, it has been exploited for the improvement of multiple traits through the simultaneous introduction or stacking of several genes driven by distinct tissue-specific promoters. Since transcriptional enhancer elements have been shown to override the specificity of nearby promoters in a position- and orientation-independent manner, the co-existence of multiple enhancers/promoters within a single transgenic construct could be problematic as it has the potential to cause the mis-expression of transgene product(s). In order to develop strategies with, which to prevent such interference, a clear understanding of the mechanisms underlying enhancer-mediated activation of target promoters, as well as the identification of DNA sequences that function to block these interactions in plants, will be necessary. To date, little is known concerning enhancer function in plants and only a very limited number of enhancer-blocking insulators that operate in plant species have been identified. In this review, we discuss the current knowledge surrounding enhancer-promoter interactions, as well as possible means of minimizing such interference during plant transformation experiments.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Abstract 3953: C-Kit and Mitf signaling: Novel involvement of the PI3K/Akt and p38 pathways

Abstract The development of melanocyte from melanoblast is regulated by the tyrosine kinase receptor c-Kit and the basic-helix-loop-heliz-leucine zipper transcription factor Mitf. It is established that Mitf activity is controlled by c-Kit activation and subsequent phosphorylation of the Erk/Mapk pathway. Serine phosphorylation (activation) of Mitf not only leads to upregulation of Mitf target gene expression but also ubiquitin dependent proteosomal degradation of Mitf. There is little knowledge surrounding the dysregulation between c-Kit and Mitf signaling that is often found in melanocyte progression to melanoma. The aim of our study is, therefore, to identify the signal transduction pathways involved in c-Kit mediated regulation of Mitf. In our study, we found c-Kit tyrosine phosphorylation sites and novel signaling pathways involved in the regulation of Mitf. Phosphorylation of c-Kit at Tyr721 has been shown to activate the PI3K pathway and Tyr568 and Tyr568/570 are responsible for Src kinase mediated activation of Erk/Mapk pathway. We demonstrate that Y721F, Y568F and Y568/570F mutation of c-Kit prevented Mitf mobility shift, indicating that these tyrosine phosphorylation sites are involved in Mitf regulation. Further, we observe that treatment of c-Kit and Mitf transfected HEK293T cells with LY294002 (PI3K inhib.), Akt IV (Akt inhib.), SU6656 (Src kinase inhib.), SB203580 (p38 inhib.) or U0126 (Mek inhib.) prevented c-Kit mediated Mitf mobility shift. Similar results were also observed in the murine melanocyte, Melan- A cell line where Mitf and c-Kit are endogenously expressed. In order to further verify c-Kit's role as Mitf regulator, we examined tyrosinase (downstream target of Mitf) promoter activity using a luciferase reporter construct. Ligand stimulation of c-Kit increased the Mitf target promoter activity. In conclusion, for the first time, we demonstrate that the signal transduction between c-Kit and Mitf is dependent on, 1) c-Kit Tyr721/PI3K/Akt pathway, 2) p38 pathway and 3) c-Kit Tyr568 or Tyr568/570 / Src kinase mediated Erk pathway. C-Kit activation leading to downstream were also shown to promote the transcriptional activity of Mitf. Our results reveal novel mechanisms by which c-Kit signaling regulates Mitf, with implication for understanding of both melanocyte development and melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3953.

Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex

The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter ( NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-α-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

Pathogenic mechanisms of tooth agenesis linked to paired domain mutations in human PAX9

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.

Gene-specific Requirement of a Nuclear Protein, IκB-ζ, for Promoter Association of Inflammatory Transcription Regulators

Expression of many inflammatory genes is induced through activation of the transcription factor NF-kappaB. In contrast to the advanced understanding of cytoplasmic control of NF-kappaB activation, its regulation in the nucleus has not been fully understood despite its importance in selective gene expression. We previously identified an inducible nuclear protein, IkappaB-zeta, and demonstrated that this molecule is indispensable for the expression of a group of NF-kappaB-regulated genes. In this study, we established a unique gene induction system, in which IkappaB-zeta is expressed independently of inflammatory stimuli, to specifically investigate the molecular basis underlying IkappaB-zeta-mediated gene activation. We show that in the presence of IkappaB-zeta other primary response genes are dispensable for the expression of the target secondary response genes. ChIP analyses revealed that IkappaB-zeta is required for stimulus-induced recruitment of NF-kappaB onto the target promoter in a gene-specific manner. Surprisingly, IkappaB-zeta is also necessary for the gene-selective promoter recruitment of another inflammatory transcription factor, C/EBPbeta, and the chromatin remodeling factor Brg1. We propose a new gene regulatory mechanism underlying the selective expression of inflammatory genes.

Novel auto-inducing expression systems for the development of whole-cell biocatalysts

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes.

Sequences in Intron 51 of the von Willebrand Factor Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice

In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5'-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in non-endothelial cells and interacts with endothelial and non-endothelial specific complexes that contain YY1. We demonstrate that beta-actin is associated with YY1 specifically in the nucleus of non-endothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

CR6-Interacting Factor 1 Represses the Transactivation of Androgen Receptor by Direct Interaction

CR6-interacting factor 1 (CRIF1) was previously identified as a nuclear protein that interacts with members of the Gadd45 family and plays a role as a negative regulator in cell growth. However, the nuclear function of CRIF1 remains largely unknown. In this study, we demonstrate that CRIF1 acts as a novel corepressor of the androgen receptor (AR) in prostatic cells. Transient transfection studies show that CRIF1 specifically represses AR transcriptional activation of target promoters in a dose-dependent manner. Additionally, CRIF1 is recruited with AR to the endogenous AR target promoters. In vivo and in vitro protein interaction assays reveal that CRIF1 directly interacts with AR via the activation function-1 domain of AR. Interestingly, both the N-terminal and C-terminal half-regions of CRIF1 are independently capable of interacting with and repressing the transactivation of AR. CRIF1 represses AR transactivation through competition with AR coactivators. In addition, the CRIF1-mediated inhibition of AR transactivation involves the recruitment of histone deacetylase 4. Down-regulation of CRIF1 by small interfering RNA increases the transactivation of AR and the mRNA level of the AR target gene prostate-specific antigen, whereas the overexpression of CRIF1 decreases the prostate-specific antigen mRNA level. Finally, the overexpression of CRIF1 inhibits the androgen-induced proliferation and cell cycle progression of prostate cancer cells. Taken together, these results suggest that CRIF1 acts as an AR corepressor and may play an important role in the regulation of AR-positive growth of prostate cancer.

Sequences in Intron 51 of the VWF Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice.

The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons. VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences −487 to +247 that functions as an endothelial specific promoter in vitro. Subsequently a number of transacting factors including, NF1, Oct 1, Ets, GATA6, NFY and Ebp4 that positively and negatively regulate the activity of this promoter were identified by us and others. However, in vivo analysis of the promoter demonstrated that this promoter fragment (sequences −487 to +247) targets activation of fused heterologous transgenes (LacZ and amyloid precursor proteins) specifically and exclusively to brain vascular endothelial cells of transgenic mice. A longer VWF promoter fragment, including 2182 bp of the 5′ flanking sequences, the first exon and the first intron was reported by Aird et al to activate LacZ transgene expression in endothelial cells of the heart and muscle as well as brain of transgenic mice. Considering that endogenous VWF expression is observed in almost all endothelial cells, these results suggested that additional VWF gene sequences were required for transcriptional activation of the VWF promoter in vascular endothelial cells of multiple other organs in vivo; and that distinct regions of the VWF gene are required to achieve promoter activity in endothelial cells of distinct organs. To identify additional cis acting elements within the VWF gene that may participate in its transcriptional regulation generally and/or in distinct organs, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells. This region was shown to interact with YY1 transcription factor in a manner that forms endothelial and non-endothelial specific complexes. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site significantly increased a heterologous SV40 promoter activity specifically in endothelial cell and that this increase was dependent on the presence of an intact YY1 binding site. In contrast, the HSS sequences significantly decreased the SV40 promoter activity in non-endothelial cells. These results suggested that the HSS sequences may participate in activation of gene expression in an YY1 dependent manner in endothelial cells, while repress gene expression in non-endothelial cells. Nevertheless the HSS sequences did not significantly affect the homologous VWF promoter activity that was analyzed by in vitro transfection analyses. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (−487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene participate in organ specific regulation of VWF gene expression, an observation that could not be determined by in vitro analysis. These analyses suggest that the HSS sequences contain cis-acting elements that are specifically necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.