Fonctional studies of TBP & its paralogue TRF2 in vivo

L'initiation de la transcription par l'ARN polymérase II requiert la formation d'un complexe multiprotéique sur l'ADN. Ce complexe comprend, outre la pol.II, les facteurs généraux de transcription TFIIA, -B, -D, -E, -F et -H ainsi que le médiateur. TFIID est constitué par la TATA Binding Protéine (TBP) et 13 à 14 facteurs associés (TAFs). TBP participant à la transcription par les trois ARN polymérases nucléaires, Il est souvent considéré comme le facteur universel . Le caractère universel de TBP a été remis en cause par la découverte de TRF1, TRF2 et TRF3, des paralogues de TBP. Des études antérieures, dans différents organismes modèles, ont montré que TRF2 est impliqué dans l'embryogenèse et la spermatogenèse. Mais ses fonctions précises sont toujours incertaines. Lors de mes travaux de thèse, j ai étudié la fonction et la localisation cellulaire de TRF2. Alors que TBP peut être identifié dans tous les compartiments nucléaires, ceci dans toutes les lignées somatiques examinées, TRF2 présente une localisation variable. Dans les cellules HeLa et Cos, ce facteur est concentré dans les nucléoles tout au long du cycle cellulaire. TRF2 est relocalisé dans le nucléoplasme suite à un stress oxydatif et non suite aux traitements apoptotiques ou mitogéniques testés. Un traitement des noyaux par la RNase A ou l'arrêt de la transcription par la pol I provoquent également une relocalisation rapide de TRF2 vers le nucléoplasme. Dans les cellules NIH 3T3, la protéine TRF2 adopte une localisation nucléoplasmique. L'expression constitutive de TRF2 réduit la prolifération et augmente la proportion de cellules en phase S. Nos résultats infirment l'hypothèse d'un passage de TRF2 du cytoplasme au noyau pour réguler la transition G2/M. L'ensemble de nos observations suggère qu un ou plusieurs ARN(s), probablement du type snoARN, maintient TRF2 dans le nucléole d'où il est libéré lors d'un stress oxydatif. Dans un deuxième projet, nous avons développé un système d'étude in vivo des mutations de TBP. Nous avons généré des fibroblastes murins embryonnaires tbplox/-, dans lesquels TBP peut être invalidé par une activité recombinase Cre. L'absence de TBP s'avère létale pour ces cellules ; elle peut néanmoins être compensée par le TBP humain en transfection transitoire ou stable. Des tests de complémentation ont donc pu être réalisés pour différents mutants du TBP humain, jusqu alors seulement caractérisés in vitro. La plupart des résidus rapportés comme cruciaux pour l'interaction de TBP avec ses partenaires ne semblent pas nécessaires au maintien de l'intégrité cellulaire. L'expression de certains mutants entraîne toutefois une diminution de la prolifération cellulaire. Ce système offr une nouvelle approche pour mieux comprendre les fonctions de TBP in vivo.The initiation of transcription by RNA polymerase II requires the formation of a multiprotein complex at the basal promoter around the mRNA start site. In addition to polymerase II, this complex comprises the general transcription factors TFIIA, B, D, E, F and H and the mediator complex. TFIID consists of the TATA binding protein, TBP and 13-14 TBP-associated factors, TAFs. TBP has been shown to be involved in transcription by all three nuclear RNA polymerases, however this universal requirement has been called into question by the discovery of the TBP-related factors TRF1, 2 and 3. Previous studies in different model organisms have shown that TRF2 is involved in embryogenesis and in spermatogenesis, but several questions concerning its function remained subject to debate. During my thesis, I have studied the function and intracellular localization of TRF2. These experiments show that TRF2 localises to the nucleolus in HeLa and Cos cells throughout the cell cycle, while TBP is nucleoplasmic and nucleolar. TRF2 is released from the nucleolus by oxidative stress, but not by the other tested apoptotic or mitogenic stimuli. TRF2 is also rapidly released from the nucleolus by arrest of pol I transcription or treatment of the nuclei by RNase A. In contrast, in NIH 3T3 cells, TRF2 is nucleoplasmic indicating that nucleolar localization is cell specific. Constitutive expression of TRF2 in 3T3 cells results in reduced proliferation and an increase the S-phase population. Our results do not support the idea that TRF2 is cytoplasmic and imported into the nucleus to regulate the G2/M checkpoint. Together our results rather suggest that one or several RNAs, perhaps snoRNAs, direct nucleolar localisation of TRF2 where it is released in response to oxidative, but not other stress stimuli. In a second project, we have developed a novel system to evaluate the effect of TBP mutations in vivo. Previous studies have generated a large collection of amino acid substitutions on the surface of TBP. The effect of these mutations has been evaluated in vitro binding assays with its known partners and in vitro transcription assays with pol I, II and III promoters. However, the effect of these mutations has not been studied in vivo in living cells. We have generated tbplox/- mouse embryonic fibroblasts in which TBP can be inactivated by expression of the Cre recombinase. The tbp-/- fibroblasts die rapidly, but the loss of endogenous mouse TBP can be complemented by expression of wild-type human TBP. We have tested the ability of various TBP mutants to complement for the loss of endogenous TBP in either transient or stable expression assays where we have generated viable tbp-/- cell lines expressing mutant TBPs. Our results show that TBP mutants which show severely compromised function in vitro can support cell viability. However, cells expressing several mutants show defective proliferation. This system offers a novel approach to better understand the function of TBP in vivo

Fonctional studies of TBP & its paralogue TRF2 in vivo

L'initiation de la transcription par l'ARN polymérase II requiert la formation d'un complexe multiprotéique sur l'ADN. Ce complexe comprend, outre la pol.II, les facteurs généraux de transcription TFIIA, -B, -D, -E, -F et -H ainsi que le médiateur. TFIIDThe initiation of transcription by RNA polymerase II requires the formation of a multiprotein complex at the basal promoter around the mRNA start site. In addition to polymerase II, this complex comprises the general transcription factors TFIIA, B, D, E

Etudes fonctionnelles de TBP et de son paralogue TRF2 in vivo

L initiation de la transcription par l ARN polymérase II requiert la formation d un complexe multiprotéique sur l ADN. Ce complexe comprend, outre la pol.II, les facteurs généraux de transcription TFIIA, -B, -D, -E, -F et -H ainsi que le médiateur. TFIID est constitué par la TATA Binding Protéine (TBP) et 13 à 14 facteurs associés (TAFs). TBP participant à la transcription par les trois ARN polymérases nucléaires, Il est souvent considéré comme le facteur universel . Le caractère universel de TBP a été remis en cause par la découverte de TRF1, TRF2 et TRF3, des paralogues de TBP. Des études antérieures, dans différents organismes modèles, ont montré que TRF2 est impliqué dans l embryogenèse et la spermatogenèse. Mais ses fonctions précises sont toujours incertaines. Lors de mes travaux de thèse, j ai étudié la fonction et la localisation cellulaire de TRF2. Alors que TBP peut être identifié dans tous les compartiments nucléaires, ceci dans toutes les lignées somatiques examinées, TRF2 présente une localisation variable. Dans les cellules HeLa et Cos, ce facteur est concentré dans les nucléoles tout au long du cycle cellulaire. TRF2 est relocalisé dans le nucléoplasme suite à un stress oxydatif et non suite aux traitements apoptotiques ou mitogéniques testés. Un traitement des noyaux par la RNase A ou l arrêt de la transcription par la pol I provoquent également une relocalisation rapide de TRF2 vers le nucléoplasme. Dans les cellules NIH 3T3, la protéine TRF2 adopte une localisation nucléoplasmique. L expression constitutive de TRF2 réduit la prolifération et augmente la proportion de cellules en phase S. Nos résultats infirment l hypothèse d un passage de TRF2 du cytoplasme au noyau pour réguler la transition G2/M. L ensemble de nos observations suggère qu un ou plusieurs ARN(s), probablement du type snoARN, maintient TRF2 dans le nucléole d où il est libéré lors d un stress oxydatif. Dans un deuxième projet, nous avons développé un système d étude in vivo des mutations de TBP. Nous avons généré des fibroblastes murins embryonnaires tbplox/-, dans lesquels TBP peut être invalidé par une activité recombinase Cre. L absence de TBP s avère létale pour ces cellules ; elle peut néanmoins être compensée par le TBP humain en transfection transitoire ou stable. Des tests de complémentation ont donc pu être réalisés pour différents mutants du TBP humain, jusqu alors seulement caractérisés in vitro. La plupart des résidus rapportés comme cruciaux pour l interaction de TBP avec ses partenaires ne semblent pas nécessaires au maintien de l intégrité cellulaire. L expression de certains mutants entraîne toutefois une diminution de la prolifération cellulaire. Ce système offre une nouvelle approche pour mieux comprendre les fonctions de TBP in vivo.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Cell-specific Nucleolar Localization of TBP-related Factor 2

TATA-binding protein (TBP)-related factor 2 (TRF2) is one of four closely related RNA polymerase II transcription factors. We compared the intracellular localizations of TBP and TRF2 during the cell cycle and mitosis in HeLa cells. We show that during interphase, endogenous or exogenously expressed TRF2 is located almost exclusively in the nucleolus in HeLa or Cos cells. TRF2 localization is not affected by stress or mitotic stimuli, but TRF2 is rapidly released from the nucleolus upon inhibition of pol I transcription or treatment by RNase. These results suggest that localization of HeLa TRF2 requires a nucleolar-associated RNA species. In contrast, in 3T3 fibroblast cells, exogenously expressed TRF2 localizes to the nucleoplasm. Constitutive expression of ectopic TRF2 in 3T3 cells leads to a prolonged S phase of the cell cycle and reduced proliferation. Together with previous data, our results highlight the cell-specific localization and functions of TRF2. Furthermore, we show that during cell division, HeLa TRF2 and TBP are localized in the mitotic cytoplasm and TRF2 relocalizes into the nascent nucleoli immediately after mitosis, whereas TBP reassociates with the chromatin. Although partially contradictory results have been reported, our data are consistent with a model where only small proportion of the cellular TBP remains associated with specific promoter loci during mitosis

Emerging Themes from EBV and KSHV microRNA Targets

EBV and KSHV are both gamma-herpesviruses which express multiple viral microRNAs. Various methods have been used to investigate the functions of these microRNAs, largely through identification of microRNA target genes. Surprisingly, these related viruses do not share significant sequence homology in their microRNAs. A number of reports have described functions of EBV and KSHV microRNA targets, however only three experimentally validated target genes have been shown to be targeted by microRNAs from both viruses. More sensitive methods to identify microRNA targets have predicted approximately 60% of host targets could be shared by EBV and KSHV microRNAs, but by targeting different sequences in the host targets. In this review, we explore the similarities of microRNA functions and targets of these related viruses

CRISPR Design Tool and KO Protocol

<p>Excel tool with documentation for designing sgRNAs from the Casellas Lab, NIH. In addition, we have included the full protocol for making KOs in CH12 and mouse ES cells. Philippe found an error in previously updated tool which falsely called some TRUE off-targets as not off-targets. New tool has been fixed - sorry for the inconvenience.</p

KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation.

Kaposi's sarcoma (KS) is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3' untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC) by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis

KSHV and miR-K2 decrease anoikis of endothelial cells.

<p><b>(A)</b> At 7 dpi, HUVECs were transferred to a polyHEMA coated or a control 12-well plate. After 24 and 48 hours in the 12-well plate, cell viability was measured using WST-1 and calcein AM. Results are presented as the ratio of the viability signals polyHEMA/control (CNT) relative to mock-infected cells. Average and SD values were calculated from five independent experiments, with statistically significant data (p<0.05) indicated by an asterisk. <b>(B)</b> Representative immunodetection of TPM1 in total protein extract of HUVECs transfected 48 hours with the indicated RNA. <b>(C)</b> Quantification of TPM1 proteins at 48 hpt presented as the average change in TPM1 protein expression level normalized to actin and relative to levels of miR-Neg or si-Neg transfected cells and based on four independent experiments. <b>(D)</b> HUVECs transfected for 24 hours with the indicated RNA were transferred to a polyHEMA coated or a control 12-well plate. After 24 and 48 hours in the 12-well plate, cell viability was measured with the same process as in 5A.</p

miR-K2 and miR-K5 down-regulate the expression of HMW-TPM1.

<p>HUVECs were transfected with individual KSHV miRNA. After 48 hours, expression of TPM1 and actin was analyzed by quantitative Western blot. <b>(A)</b> Representative images for TPM1 and actin expression are shown. <b>(B)</b> The average change in TPM1 protein expression levels was normalized to actin and to TPM1 levels in cells transfected with miR-Neg (based on four independent experiments). <b>(C)</b> HUVECs were transfected with a LNA control (LNA-Neg), inhibitor of miR-K2 (LNAαK2), or inhibitor of miR-K5 (LNAαK5). At 24 hpt, the cells were infected with KSHV for 48 more hours. After 72 hpt (2 dpi), total cell lysates were harvested and the expression of TPM1 was analyzed by quantitative immunoblot. Results are presented as the average change in TPM1 protein expression levels normalized to actin and relative to the levels of LNA-Neg in <i>de novo</i> infected cells. Average and SD values were calculated from four independent experiments. <b>(D)</b> Relative KSHV miRNA expression level determined by Taqman qPCR in <i>de novo</i> infected HUVEC. (E) Relative protein level changes from cells infected with wild-type KSHV or mutant version lacking miR-K2 (Kd2) or mirR-K5 (Kd5). (F) Immunoblot for data in (E). (G) Relative miRNA level changes from cells infected with mutant viruses used in (E) and (F).</p