Coordinated changes of histone modifications and HDAC mobilization regulate the induction of MHC class II genes by Trichostatin A

The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the Major Histocompatibility Class II (MHC II) DRA gene in a way independent of the master coactivator CIITA. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHC II expression, we used chromatin immunoprecipitation (ChIP) assays to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. We found that a dramatic increase in promoter linked histone acetylation is followed by an increase in Histone H3 lysine 4 methylation and a decrease of lysine 9 methylation. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA increases the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHC II family and the adjacent histone cluster that are located in chromosome 6p21-22 locus are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response

A novel SATB1 protein isoform with different biophysical properties

Intra-thymic T cell development is coordinated by the regulatory actions of SATB1 genome organizer. In this report, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. More importantly, we characterized a novel SATB1 protein isoform and described its distinct biophysical behavior, implicating potential functional differences compared to the commonly studied isoform. SATB1 utilized its prion-like domains to transition through liquid-like states to aggregated structures. This behavior was dependent on protein concentration as well as phosphorylation and interaction with nuclear RNA. Notably, the long SATB1 isoform was more prone to aggregate following phase separation. Thus, the tight regulation of SATB1 isoforms expression levels alongside with protein post-translational modifications, are imperative for SATB1’s mode of action in T cell development. Our data indicate that deregulation of these processes may also be linked to disorders such as cancer

miR-16 Targets Transcriptional Corepressor SMRT and Modulates NF-kappaB-Regulated Transactivation of Interleukin-8 Gene

The signaling pathways associated with the Toll-like receptors (TLRs) and nuclear factor-kappaB (NF-κB) are essential to pro-inflammatory cytokine and chemokine expression, as well as initiating innate epithelial immune responses. The TLR/NF-κB signaling pathways must be stringently controlled through an intricate network of positive and negative regulatory elements. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the stability and/or translation of protein-coding mRNAs. Herein we report that miR-16 promotes NF-κB-regulated transactivation of the IL-8 gene by suppression of the silencing mediator for retinoid and thyroid hormone receptor (SMRT). LPS stimulation activated miR-16 gene transcription in human monocytes (U937) and biliary epithelial cells (H69) through MAPK-dependent mechanisms. Transfection of cells with the miR-16 precursor promoted LPS-induced production of IL-8, IL-6, and IL-1α, without a significant effect on their RNA stability. Instead, an increase in NF-κB-regulated transactivation of the IL-8 gene was confirmed in cells following transfection of miR-16 precursor. Importantly, miR-16 targeted the 3′-untranslated region of SMRT and caused translational suppression of SMRT. LPS decreased SMRT expression via upregulation of miR-16. Moreover, functional manipulation of SMRT altered NF-κB-regulated transactivation of LPS-induced IL-8 expression. These data suggest that miR-16 targets SMRT and modulates NF-κB-regulated transactivation of the IL-8 gene

Klotho Lacks a Vitamin D Independent Physiological Role in Glucose Homeostasis, Bone Turnover, and Steady-State PTH Secretion In Vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo

The Msx1 Homeoprotein Recruits G9a Methyltransferase to Repressed Target Genes in Myoblast Cells

Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development

Interaction Analysis between HLA-DRB1 Shared Epitope Alleles and MHC Class II Transactivator CIITA Gene with Regard to Risk of Rheumatoid Arthritis

HLA-DRB1 shared epitope (SE) alleles are the strongest genetic determinants for autoantibody positive rheumatoid arthritis (RA). One of the key regulators in expression of HLA class II receptors is MHC class II transactivator (CIITA). A variant of the CIITA gene has been found to associate with inflammatory diseases

Critical Role of TCF-1 in Repression of the IL-17 Gene

Overwhelming activation of IL-17, a gene involved in inflammation, leads to exaggerated Th17 responses associated with numerous autoimmune conditions, such as experimental autoimmune encephalomyelitis (EAE). Here we show that TCF-1 is a critical factor to repress IL-17 gene locus by chromatin modifications during T cell development. Deletion of TCF-1 resulted in increased IL-17 gene expression both in thymus and peripheral T cells, which led to enhanced Th17 differentiation. As a result, TCF-1-/- mice were susceptible to Th17-dependent EAE induction. Rag1-/- mice reconstituted with TCF-1-/- T cells were also susceptible to EAE, indicating TCF-1 is intrinsically required to repress IL-17. However, expression of wild-type TCF-1 or dominant negative TCF-1 did not interfere with Th17 differentiation in mature T cells. Furthermore, expression of TCF-1 in TCF-1-/- T cells could not restore Th17 differentiation to wild-type levels, indicating that TCF-1 cannot affect IL-17 production at the mature T cell stage. This is also supported by the normal up-regulation or activation in mature TCF-1-/- T cells of factors known to regulate Th17 differentiation, including RORγt and Stat3. We observed hyperacetylation together with trimethylation of Lys-4 at the IL-17 locus in TCF-1-/- thymocytes, two epigenetic modifications indicating an open active state of the gene. Such epigenetic modifications were preserved even when TCF-1-/- T cells migrated out of thymus. Therefore, TCF-1 mediates an active process to repress IL-17 gene expression via epigenetic modifications during T cell development. This TCF-1-mediated repression of IL-17 is critical for peripheral T cells to generate balanced immune responses

Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms

c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

The development of natural Foxp3+ CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25+GITRhiFoxp3−CD4+ nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation

Transcriptional vegulation of MHC class II genes from the class II transactivator, CIITA

During my PhD studies I worked on the mechanisms regulating the expression of Major Histocompatibility Complex class II genes. MHC class II antigens are dimeric membrane glycoproteins that recognize and present antigenic peptides to T-lymphocytes. Their expression is highly regulated in transcriptional level and their stimulation needs the existence of an enhanceosome, which is composed on highly conserved sequences (HXY) of the proximal promoter, as well as the expression of CIITA, which is the major transcriptional regulator of the MHC class II genes. CIITA can positively regulate the expression of MHC class II genes upon its recruitment through its carboxy-terminal domain onto the class II enhanceosome and with its amino-terminal domain interacting with various other transcriptional coactivators. We showed that transcriptional coactivators such as CBP, pCAF and GCN5 interact physically with CIITA in vitro and in vivo and positively regulate the expression of class II antigens. CIITA not only interacts with these coactivators but it is being acetylated in discrete lysine residues that reside in an amino-terminal nuclear localization signal. Acetylation of CIITA results in increased nuclear accumulation of the protein and enhancement of transcriptional initiation. The subcellular localization of CIITA is also affected from its self-association. Self association of CIITA results in subcellular redistribution of the protein and its functional complementation. It’s the first time, in the constraints of this study that the MHC class II enhanceosome was characterized using recombinant in vitro expressed proteins and not whole-cell extracts and it was shown the cooperative binding of all complexes onto the class II proximal promoter as well as the specificity in binding. We showed that the in vitro reconstituted enhanceosome can specifically recruit CIITA on the proximal promoter as well as other components of the basal transcription machinery. Using the technique of chromatin immunoprecipitation we showed the order of recruitment of all the factors needed for the induction of an MHC class II gene-DRA after the induction of cells with IFN-γ. It is an important result that a transcriptional transactivator such as CIITA can regulate the initiation and elongation of transcription regulating the levels of phosphorylation of specific serine residues of RNA polymerase II. This induction of DRA from IFN-γ is an inducible system of transcription that resembles the order of recruitment and induction of IFN-β gene upon virus induction, in that they both need an enhanceosome, preassembled for class II or inducible in IFN-β. Someone can take information concerning transcriptional regulation of genes in general, from the comparison of the two systems. It is more important trying to understand the transcription of class II genes and induction of expression in solid tumors where they are not normally expressed.Στα πλαίσια της παρούσας διδακτορικής διατριβής μελετήσαμε τους μηχανισμούς ρύθμισης των τάξης ΙΙ αντιγόνων του Κυρίου Συμπλόκου Ιστοσυμβατότητας. Τα τάξης ΙΙ αντιγόνα είναι διμερείς μεμβρανικές γλυκοπρωτεΐνες με σκοπό την παρουσίαση αντιγόνων στα Τ-λεμφοκύτταρα. Η ρύθμιση της έκφρασής τους είναι αυστηρά ρυθμιζόμενη στο μεταγραφικό επίπεδο και η επαγωγή τους απαιτεί την ύπαρξη ενός ενισχυοσώματος που δημιουργείται σε συντηρημένα στοιχεία (Η,Χ,Υ) του εγκύς υποκινητή καθώς και την έκφραση του παράγοντα CIITA, που είναι και ο κύριος μεταγραφικός ενεργοποιητής των τάξης ΙΙ γονιδίων. Το CIITA μπορεί να ρυθμίζει θετικά την έκφραση των MHC τάξης ΙΙ γονιδίων αφού με την καρβοξυτελική του περιοχή στρατολογείται στο τάξης ΙΙ ενισχυόσωμα και με την αμινοτελική του περιοχή μπορεί να αλληλεπιδρά με άλλους μεταγραφικούς συνενεργοποιητές και να επάγει τη μεταγραφή. Δείξαμε οτι οι μεταγραφικοί συνενεργοποιητές CBP, pCAF και GCN5 αλληλεπιδρούν με την αμινοτελική περιοχή του CIITA τόσο in vitro όσο και in vivo και μπορούν να ρυθμίζουν θετικά την έκφραση των τάξης ΙΙ αντιγόνων. Το CIITA όχι μόνο αλληλεπιδρά με τους παραπάνω συνενεργοποιητές αλλά ακετυλιώνεται απ’αυτούς σε διακριτά αμινοξικά κατάλοιπα λυσινών που εδράζονται σε ένα αμινοτελικό σήμα πυρηνικού εντοπισμού. Η ακετυλίωση του CIITA έχει σαν αποτέλεσμα την αύξηση της πυρηνικής του εντόπισης και την ενδυνάμωσή του για μεταγραφική ενεργοποίηση. Η υποκυτταρική κατανομή του CIITA καθορίζεται επίσης και από γεγονότα αλληλεπίδρασής του με τον εαυτό του. Η αλληλεπίδραση του CIITA με τον εαυτό του οδηγεί σε κυτταρική ανακατανομή και λειτουργική συμπλήρωση. Ο χαρακτηρισμός του τάξης ΙΙ ενισχυοσώματος έγινε για πρώτη φορά στα πλαίσια αυτής της εργασίας χρησιμοποιώντας ανασυνδιασμένες πρωτεΐνες και όχι πρωτεϊνικά εκχυλίσματα και δείχθηκε η συνεργατικότητα στην πρόσδεση των συμπλόκων στον εγκύς υποκινητή καθώς και η εξειδίκευση στην πρόσδεση. Δείξαμε οτι το in vitro ανασυστημένο τάξης ΙΙ ενισχυόσωμα μπορεί να στρατολογεί ειδικά στον υποκινητή τόσο το CIITA όσο και άλλα μέρη της βασικής μεταγραφικής μηχανής. Χρησιμοποιώντας την τεχνική ανοσοκατακρήμνισης χρωματίνης δείξαμε την αλληλουχία στρατολόγησης των διαφόρων παραγόντων που απαιτούνται για την επαγωγή του τάξης ΙΙ DRA γονιδίου μετά την επαγωγή των κυττάρων από IFN-γ. Πολλή σημαντικό αποτέλεσμα αποτελεί η απόδειξη οτι ένας συνενεργοποιητής όπως το CIITA μπορεί να ρυθμίζει την έναρξη και επιμήκυνση της μεταγραφής ρυθμίζοντας τα επίπεδα φωσφορυλίωσης συγκεκριμένων καταλοίπων σερίνης της RNA Πολυμεράσης ΙΙ. Το σύστημα επαγωγής του γονιδίου DRA από IFN-γ είναι ένα επαγόμενο σύστημα μεταγραφής που ομοιάζει με την αλληλουχία των γεγονότων επαγωγής του γονιδίου της IFN-β μετά την προσθήκη ιού, στο ότι απαιτείται ένα ενισχυόσωμα, που στην περίπτωση των τάξης ΙΙ είναι προσχηματισμένο ενώ στην περίπτωση της IFN-β είναι επαγόμενο. Πληροφορίες που αφορούν τη μεταγραφική ρύθμιση των γονιδίων γενικότερα μπορεί να αντλήσει κανείς συγκρίνοντας τα δύο συστήματα. Πιο σημαντικό γεγονός κρίνεται όμως η προσπάθεια κατανόησης της μεταγραφής των τάξης ΙΙ γονιδίων και η προσπάθεια επαγωγής τους σε συμπαγείς όγκους όπου και η έκφραση ελλείπει