Stepwise evolution of Elk-1 in early deuterostomes

Metazoans have multiple ETS paralogues with overlapping or indiscriminate biological functions. Elk- 1, one of three mammalian Ternary Complex Factors (TCFs), is a well-conserved, ETS domain-containing transcriptional regulator of mitogen-responsive genes that operates in concert with Serum Response Factor (SRF). Nonetheless, its genetic role remains unresolved because the elk-1 gene could be deleted from the mouse genome seemingly without adverse effect. Here we have explored the evolution of Elk-1 to gain insight into its conserved biological role. We identified antecedent Elk-1 proteins in extant early metazoans and used amino acid sequence alignments to chart the appearance of domains characteristic of human Elk-1. We then performed biochemical studies to determine whether putative domains apparent in the Elk-1 protein of a primitive hemichordate were functionally orthologous to those of human Elk-1. Our findings imply the existence of primordial Elk-1 proteins in primitive deuterostomes that could operate as mitogen-responsive ETS transcription factors but not as TCFs. The role of TCF was acquired later, but presumably prior to the whole genome duplications in the basal vertebrate lineage. Thus its evolutionary origins link Elk-1 to the appearance of mesoderm

Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation.

Multisite phosphorylation regulates many transcription factors, including the serum response factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events had remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate, and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity

Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)

Stem rust resistance in wheat is suppressed by a subunit of the mediator complex

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat. Stem rust is an important disease of wheat and resistance present in some cultivars can be suppressed by the SuSr-D1 locus. Here the authors show that SuSr-D1 encodes a subunit of the Mediator Complex and that nonsense mutations are sufficient to abolish suppression and confer stem rust resistance

SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes

We thank Ms. Kathy Kyler for her kind help in English editing of the manuscript.Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3’-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.Yeshttp://www.plosone.org/static/editorial#pee

In vivo and in vitro immunological study in patients with recurrent condylomata acuminata

Condylomata acuminata often have the tendency to recur, even after a seemingly satisfactory treatment. The exact phenomenon of virus recurrence and latency remains obscure. In the present study we tried to approach the subject with regard to its virological, immunohistochemical and immunological aspects. Thirty male Greek patients, aged 24.7±4.7 years, suffering from recurrent condylomata acuminata and 30 healthy males of similar age used as controls, were included in the virologic and immunologic examinations. Eighteen patients were immunohistochemically examined. Skin biopsies from the lesion and the apparently healthy surrounding skin were obtained from these patients. Southern Blot hybridization analysis has been used with clean viral DNA as a probe. The following DNA probes were used: HPV-6, 11, 16, 18, 31, 33, 35, 39, 45, 51 and 56. The results of the hybridization analysis were as follows: a. All biopsies (30) originating from condylomata acuminata were tested positive for HPV DNA. b. No viral DNA was found in the apparently healthy area, except in one case. c. The vast majority of the biopsy samples from the lesions tested positive for HPV-6/-11 types, and only 1 biopsy was negative for these two types. The prevailing type was HPV-6a although -in all- 6 sub-types of HPV-6 are known today. d. Contrary to neoplasias and invasive carcinomas -where the distribution of the various HPV types seems to be associated with the geographical area - no such association is seen in condylomata acuminata. e. The duration of the disease and the structure of the lesions is not associated with the types and sub-types of the virus. Indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunoenzyme assays were used for the immunohistochemical examination of epidermic dendritic cells. Our findings point towards a local immunodeficiency, since both LC and HLA-DR cells in general present statistically significant differences between the lesion and the apparently healthy neighbouring epidermis. A reduction in the epidermic dendritic cells has been observed in the lesion compared with the apparently healthy neighbouring epidermis. Immediate hypersensitivity parameters (total IgE, phadia top and prick tests) and delayed hypersensitivity against seven recli antigens (Multi-test) were studied. Significantly higher immediate hypersensitivity activity was shown in the patient group. Qualitative evaluation of delayed type hypersensitivity showed that controls had a positive test 16 times more often than patients. The hypothesis of a CD4+ Th-2 lympohocyte predominance in recurrent condylomata, owed to longstanding or repetitive antigenic stimulation seems to adequately explain the findings of the present study.Τα οξυτενή κονδυλώματα έχουν συχνά την τάση να υποτροπιάζουν ακόμα και μετά από φαινομενικά επαρκή θεραπεία. Το φαινόμενο των υποτροπών και της λανθάνουσας διαβίωσης του ιού παραμένει σκοτεινό. Στην παρούσα μελέτη έγινε προσπάθεια προσέγγισης του φαινομένου ιολογικά, ανοσοϊστοχημικά και ανοσολογικά. Στον ιολογικό και ανοσολογικό έλεγχο έλαβαν μέρος 30 άνδρες Έλληνες ασθενείς ηλικίας 24.7±4.7 χρόνια που έπασχαν από υποτροπιάζοντα οξυτενή κονδυλώματα και 30 υγιείς άνδρες παρόμοιας ηλικίας σαν μάρτυρες. Ανοσοϊστοχημικά, ελέγχθηκαν 18 ασθενείς. Στους ασθενείς αυτούς έγινε λήψη τεμαχίου δέρματος από την περιοχή της βλάβης και από τη φαινομενικά υγιή περιβλαβική περιοχή. Ο υβριδισμός του DNA του ιού έγινε με τη μέθοδο southern blot χρησιμοποιώντας σαν ανιχνευτές καθαρό ιϊκό DNA. Χρησιμοποιήθηκαν DNA ανιχνευτές (probes) των τύπων HPV-6, 11, 16, 18, 31, 33, 35, 39, 45, 51 και 56. Τα αποτελέσματα του μοριακού υβριδισμού έδειξαν ότι: α. Όλες οι βιοψίες (30), που προήλθαν από οξυτενή κονδυλώματα ήταν θετικές για HPV DNA. β. Εκτός από μία βιοψία, δε βρέθηκε DNA του ιού στη φαινομενικά υγιή περιοχή, γ. Το συντριπτικά μεγαλύτερο ποσοστό των δειγμάτων από τις βλάβες ήταν θετικά για τους τύπους HPV-6/-11, ενώ μόνο μία βιοψία ήταν αρνητική γι' αυτούς τους τύπους. Ο επικρατέστερος τύπος ήταν ο HPV-6a, παρόλο που σήμερα είναι γνωστοί έξι υπότυποι για τον HPV-6. δ. Σε αντίθεση με τις νεοπλασίες και τους διηθητικούς καρκίνους όπου η εικόνα κατανομής των διαφόρων HPV τύπων φαίνεται να εξαρτάται από τη γεωγραφική περιοχή, το φαινόμενο αυτό δεν συναντάται στα οξυτενή κονδυλώματα. ε. Η διάρκεια της νόσο« και η έκταση των βλαβών δεν έχει σχέση με το είδος των τόπων και υπότυπων του ιού. Για τον ανοσοϊστοχημικό έλεγχο των κυττάρων του Langerhans χρησιμοποιήθηκαν οι ανοσοενζυμικές μέθοδοι της έμμεσης ανοσοϋπεροξειδάσης και της αλκαλικής φωσφατάσης-αντιαλκαλικής φωσφατάσης (ΑΡΑΑΡ). Τα ευρήματα της μελέτης, τονίζουν την εστιακή ανοσοανεπάρκεια, αφού τόσο τα κύτταρα του Langerhans όσο και γενικότερα τα HLA-DR+ δενδριτικά κύτταρα παρουσιάζουν στατιστικά σημαντικότατες διαφορές μεταξύ της βλάβης και του φαινομενικά υγιούς δέρματος. Υπάρχει ελάττωση των δενδριτικών επιδερμιδικών κυττάρων στη βλάβη σε σχέση με το φαινομενικά γειτονικό υγιές. Η διερεύνηση της άμεσης υπερευαισθησίας έγινε με τον έλεγχο ολικής IgE, Phadia-top και Prick-tests, ενώ η επιβραδυνόμενη υπερευαισθησία ελέγχθηκε με σειρά αναμνηστικών αντιγόνων (Multi-test). Βρέθηκε αύξηση των παραμέτρων της άμεσης υπερευαισθησίας και μείωση της επιβραδυνόμενης υπερευαισθησίας, σαν μία in vivo ένδειξη της κυτταρικής ανοσίας στα υποτροπιάζοντα οξυτενή κονδυλώματα. Ποσοτική εκτίμηση της επιβραδυνόμενης υπερευαισθησίας έδειξε ότι οι μάρτυρες παρουσιάζουν θετική δοκιμασία 16 φορές πιο συχνά απ' ότι οι ασθενείς. Τα ευρήματα αυτά φαίνεται να εξηγούνται ικανοποιητικά από την υπόθεση της επικράτησης των CD4+ Th-2 λεμφοκυττάρων στα επίμονα ή υποτροπιάζοντα οξυτενή κονδυλώματα

Recurrent condylomata acuminata: How routine immediate and delayed hypersensitivity parameters might provide a clue to their immunopathogenesis

In 30 male patients suffering from recurrent condylomata acuminata, immediate hypersensitivity parameters (total IgE, PTT and prick tests) and delayed hypersensitivity against seven recall antigens (multi test) were studied. Thirty healthy male volunteers, matched in age, were the controls. Significantly higher immediate hypersensitivity activity was shown in the patient group. Qualitative evaluation of delayed type hypersensitivity showed that controls had a positive test 16 times more often than patients. A rather homogeneous suppression of delayed type hypersensitivity was found in the patient group mainly as regards the presumably most common antigens vs. the control group. This suppression was proved to be related to disease duration. The hypothesis of a CD4+ Th-2 lymphocyte predominance in recurrent condylomata, owed to longstanding or repetitive antigenic stimulation seems to adequately explain the findings of the present study

Complexity in transcription control at the activation domain-mediator interface.

International audienceTranscript elongation by polymerase II paused at the Egr1 promoter is activated by mitogen-activated protein kinase phosphorylation of the ternary complex factor (TCF) ELK1 bound at multiple upstream sites and subsequent phospho-ELK1 interaction with mediator through the MED23 subunit. Consequently, Med23 knockout (KO) nearly eliminates Egr1 (early growth response factor 1) transcription in embryonic stem (ES) cells, leaving a paused polymerase at the promoter. Med23 KO did not, however, eliminate Egr1 transcription in fibroblasts. Chromatin immunoprecipitation analysis and direct visualization of fluorescently labeled TCF derivatives and mediator subunits revealed that three closely related TCFs bound to the same control regions. The relative amounts of these TCFs, which responded differently to the loss of MED23, differed in ES cells and fibroblasts. Transcriptome analysis suggests that most genes expressed in both cell types, such as Egr1, are regulated by alternative transcription factors in the two cell types that respond differently to the same signal transduction pathways