DNA metylation and gene-expression in Chinese hamster ovary cells

Imperial Users onl

β-globin gene promoter generates 5' truncated transcripts in the embryonic foetal erythroid environment.

We report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly. Using hybrid constructs of these two genes we demonstrate that aberrant activity is conferred by sequences 5' of the beta-globin gene. Thus beta-globin promoter attached to the H2 coding sequence produces H2 transcripts with truncated 5' ends. By introducing a series of deletions in the beta-globin promoter we restrict these sequences to the -77/+28 base pair region spanning the CAAT element to the translation initiation site. These results are consistent with the lack of recognition of the beta-globin gene major cap site in Putko and K562 cells. We suggest that inactivity of the adult globin gene in the embryonic/fetal environment is at least in part conferred by sequences within the beta-globin gene promoter

Harnessing of the Nucleosome Remodeling Deacetylase complex controls lymphocyte development and prevents leukemogenesis

Cell fate decisions depend on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome remodeling and deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid-lineage determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active lymphoid differentiation genes. Loss in Ikaros DNA binding activity caused a local increase in Mi-2β chromatin remodeling and histone deacetylation and suppression of lymphoid gene expression. The NuRD complex also redistributed to transcriptionally poised non-Ikaros gene targets, involved in proliferation and metabolism, inducing their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks

Hyperthermia -a non-chemical control strategy against varroa

Το άκαρι βαρρόα (Varroa destructor Anderson & Trueman) είναι παγκοσμίως η σημαντικότερη παρασιτική ασθένεια των μελισσών του είδους Apis mellifera L. Προκειμένου να μελετηθεί εναλλακτικός τρόπος καταπολέμησης για τη βαρρόα αλλά και να περιοριστούν τα υπολείμματα των φυτοπροστατευτικών ουσιών στα προϊόντα της μέλισσας, χρησιμοποιήθηκε η μέθοδος της υπερθερμίας. Η μέθοδος πραγματοποιήθηκε σε 27 μελίσσια παρουσία γόνου σε αυτά οπότε χρησιμοποιήθηκε η θερμοκρασία των 42°C για 12 έως 480 λεπτά προκειμένου να μελετηθεί. Όλα τα μελίσσια στα οποία εφαρμόστηκε η μέθοδος αποτελούνταν από 10 κηρήθρες πληθυσμό (κατά προσέγγιση 10.000 μέλισσες σε κάθε μελίσσι) και γόνο όλων των σταδίων σε έκταση 5 με 8 κηρήθρες. Κατά τη διάρκεια των δοκιμών η τελική θερμοκρασία στο εσωτερικό της κυψέλης κυμάνθηκε από 42,3°C έως 46,5°C. Η αποτελεσματικότητα της μεθόδου στον έλεγχο της βαρρόα, σχετίζεται με τη διάρκεια της εφαρμογής. Στο χρονικό εύρος εφαρμογής από 12 έως 480 λεπτά, το ποσοστό πτώσης ακάρεων βαρρόα κυμάνθηκε από 29,8% έως 79,8% αντίστοιχα. Ένα μικρό ποσοστό νεκρών ακάρεων (4,7%) εντοπίστηκε στον σφραγισμένο γόνο μετά από εφαρμογή 60 λεπτών, ποσοστό που σταδιακά αυξήθηκε με την αύξηση του χρόνου εφαρμογής και έφτασε έως και 100% μετά τα 480 λεπτά. Η χρήση της συσκευής εφαρμογής της υπερθερμίας προκάλεσε ανησυχία στο μελίσσι χωρίς όμως να εμφανιστούν απώλειες μελισσών ή εκτεταμένη επιθετικότητα. Εφαρμογή της μεθόδου για χρονικό διάστημα μεγαλύτερο από 120 λεπτά, προκάλεσε νέκρωση μέρους του σφραγισμένου γόνου, ενώ το φαινόμενο εντάθηκε σε έκταση σε σχέση με το χρόνο εφαρμογής. Η μέθοδος της υπερθερμίας, ως εφαρμογή απευθείας σε μελίσσι παρουσία γόνου και πληθυσμού, είναι η πρώτη φορά που μελετάται ως τρόπος καταπολέμησης του ακάρεως βαρρόα παρέχοντας έτσι μία εναλλακτική λύση στους μελισσοκόμους για τη χρησιμοποίησή της απευθείας στο μελισσοκομείο και αποφεύγοντας τη χρήση χημικών σκευασμάτων. Ως μέθοδος θα μπορούσε να εφαρμοστεί σε περιορισμένη κλίμακα και γι’ αυτό προσφέρεται κυρίως για τους μελισσοκόμους εκείνους που έχουν μικρό αριθμό μελισσιών ή ασκούν βιολογική μελισσοκομία.Worldwide, the ectoparasitic mite varroa (Varroa destructor Anderson & Trueman) is potentially the main threatening parasite for Apis mellifera L. To find an alternative therapy for varroa and to limit the chemical residues in bee products, 27 bee colonies with their brood, were treated at 42°C for 12 to 480 minutes. All experimental colonies had 5-8 frames of brood and 10 frames of population (approximately 10.000 bees each colony). During the treatment the final temperature inside the hive varied from 42.3°C to 46.5°C. The effectiveness of hyperthermia to control the varroa population, depends on the duration of the therapy. When the time treatment was extended from 12 to 480 minutes, the falling mites ranged from 29.8% to 79.8%. A small number (4.7%) of dead mites was found in sealed brood after a 60-minute treatment, which gradually increased along with the treatment duration reaching 100% after 480 minutes. The use of the device irritated the bees but did not cause losses of honeybees, or excessive aggravation. Dead larvae inside sealed brood were observed when hyperthermia was applied for more than 120 minutes and increased along with the duration of heating. To our knowledge, in the presence of brood and adult bees, the hyperthermia method is used for the first time as alternative solution to limit the excessive use of chemical substances under field conditions. This method is proved to be most suitable for beekeepers with small amount of colonies

Restrictions in the T-cell repertoire of chronic lymphocytic leukemia: high-throughput immunoprofiling supports selection by shared antigenic elements

Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated

beta-Catenin induces T-cell transformation by promoting genomic instability

Deregulated activation of β-catenin in cancer has been correlated with genomic instability. During thymocyte development, β-catenin activates transcription in partnership with T-cell-specific transcription factor 1 (Tcf-1). We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination activating gene (RAG) and myelocytomatosis oncogene (Myc) activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG recombination events and resembled oncogenic translocations previously described in human TALL. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including near the translocation sites. Pretransformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG-induced DNA double strand breaks. These cells were able to survive despite DNA damage because activated β-catenin promoted an antiapoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas as a consequence of altered double strand break repair and increased survival of thymocytes with damaged DNA.link_to_OA_fulltex

A DNA Sequence Directed Mutual Transcription Regulation of HSF1 and NFIX Involves Novel Heat Sensitive Protein Interactions

BACKGROUND: Though the Nuclear factor 1 family member NFIX has been strongly implicated in PDGFB-induced glioblastoma, its molecular mechanisms of action remain unknown. HSF1, a heat shock-related transcription factor is also a powerful modifier of carcinogenesis by several factors, including PDGFB. How HSF1 transcription is controlled has remained largely elusive. METHODOLOGY/PRINCIPAL FINDINGS: By combining microarray expression profiling and a yeast-two-hybrid screen, we identified that NFIX and its interactions with CGGBP1 and HMGN1 regulate expression of HSF1. We found that CGGBP1 organizes a bifunctional transcriptional complex at small CGG repeats in the HSF1 promoter. Under chronic heat shock, NFIX uses CGGBP1 and HMGN1 to get recruited to this promoter and in turn affects their binding to DNA. Results show that the interactions of NFIX with CGGBP1 and HMGN1 in the soluble fraction are heat shock sensitive due to preferential localization of CGGBP1 to heterochromatin after heat shock. HSF1 in turn was found to bind to the NFIX promoter and repress its expression in a heat shock sensitive manner. CONCLUSIONS/SIGNIFICANCE: NFIX and HSF1 exert a mutual transcriptional repressive effect on each other which requires CGG repeat in HSF1 promoter and HSF1 binding site in NFIX promoter. We unravel a unique mechanism of heat shock sensitive DNA sequence-directed reciprocal transcriptional regulation between NFIX and HSF1. Our findings provide new insights into mechanisms of transcription regulation under stress

Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner

Chromatin organizer SATB1 and Wnt transducer β-catenin form a complex and regulate expression of GATA3 and TH2 cytokines in Wnt-dependent manner and orchestrate TH2 lineage commitment