Diploma of Thorough Studies

« Biology of the Blood cells »

TLS, a protein of the spliceosome, is implied in the mechanism of action of the rétinoïque acid

through the regulations post-transcriptionnelle

and transcriptionnelle

Eric Corvec

University Paris 7 Denis Diderot

July 2002

Cellular laboratory of Biology Hématopoïétique

EMI 00-03, Univ. Paris 7 EA 316, Saint-Louis UF474

University institute of Hematology, Saint-Louis Hospital

75475 Paris Cedex 10

Director of the laboratory and Master of training course

Christine Chomienne

Laurent Delva

I cordially thank Christine Chomienne for having allowed myself to carry out this DEA.

I in particular thank Laurent Delva who me provided assistance and councils, and without which this work could not have been possible.

I thank Gilles Despouy for his moral and scientific support.

I thank Nicole Balitrand for his advised councils.

I thank Sylvie Deshaie for all that it brought to me during the year.

Thank you friendly in Alexandra Mazharian for her good mood.

Lastly, I would like to thank all the team for the LBCH for their reception and their sympathy.

SUMMARY

The regulations transcriptionnelle and post-transcriptionnelle are strongly implied in the diversification of the proteinic expression.

The fact that TLS is surexprimée in 60% of the LAM and that it interacts with RXR, one of the principal partners of the RANC, enables us to put forth the assumption that TLS would be related to the way of indication of AR in the hematopoietic cells. TLS, a protein ubiquitairement expressed, intervenes in many molecular mechanisms. Many studies show that it is related to the transciptionnelle machinery as well as with that of the épissage.

We initiated the study of the action of TLS and AR on the transcriptionnelle activity in myeloblastic cells HL-60. The membership of TLS to the complex transcriptionnel of the receivers to the rétinoïque acid is evaluated by the technique of delay on freezing with the two nuclear receivers with the rétinoïdes (RAR and RXR). Moreover, the role of TLS and AR on the épissage was studied by a technique of analysis in vivo, on the alternate épissage into 5 ' of minigene E1A in the hematopoietic cells K562 in absence or in the presence of rétinoïque acid.

Our work made it possible to characterize a role of Co-activator of the transcription by the receivers with the rétinoïdes in the presence of rétinoïque acid in the cells HL-60 and Cos-6, its membership of the RANC and a role of factor of épissage supporting the selection of distal site 5 ' of épissage of minigene E1A in the cells myéloïdes K562. We showed that TLS also acts with the way of indication of the rétinoïdes which accentuate the effects observed of TLS on the épissage.

The rétinoïque acid is thus implied at the same time in the transcriptome and the spliceosome through TLS. Thus, it is the first time that the way of indication of the hormones is implied in the épissage and the transcription through a factor of épissage.

LIST ABBREVIATIONS

ABL : ABeLson

ADN : Acid Desoxyribonucleic

ADNc : Complementary ADN

AF : Activation function

AR : Acid Rétinoïque

ARN : Acid Ribonucleic

ARN polII : ARN ploymérase II

ARNpré-m: ARN pre-messenger

ATRA : Al-trans Retinoic acid

AR 9-cis : Acid Rétinoïque 9-cis

BCR : Breakpoint Cluster Area

CHOP : C/EBP HOmologous Protein

DBD: DNA Binding Domain

DMSO: Dimethyl SulfOxide

DR.: Direct Repeat

E1A: Enhancer 1 Adenovirus

ER: Estrogen Receptor

ERG : ETS-Related Gene

EWS : EWing Sarcomas

GR.: Glucocorticoid Receptor

hnRNP : heterogeneous nuclear

RiboNucleoProtein

LAM: Acute leukemia Myéloïde

LBD : Ligand Binding Domain

NPM: NucleoPhosMin

NuMA: Nuclar Matrix Apparatus

PCR: Polymerization in Chain Reaction

PLZF: Promyelocytic Leukemia Finger Zinc

PML: ProMyelocytic Leukemia

RANC : Retinoic Acid-depend Nuclear Complex

RAR : Retinoic Acid Receptor

RARE : Retinoic Acid Response Element

RGG: Arginine-Glycine-Glycine

RNP-CS : RNP-consensus Sequence

RRM : RNA Recognition Reason

RT: Transfer Transcriptase

RT-PCR: RT followed by a PCR

RXR : Retinoid X Receptor

snRNP: small nuclear RiboNucleoProtein

TAF : TBP Associated Factor

TBP : Binding Protein TOUCHED

TLS : Translocated in LipoSarcomas

TR: Thyroid Receptor

VDR: Vitamin D Receptor

SYNOPSIS

SUMMARY 1

SYNOPSIS 3

I - INTRODUCTION 4

PROJECT DRANK 9

II - MATERIALS AND METHODS 10

A) MATERIALS : 10

a) Cellular lines : 10

c) Inductive agents : 10

d) Plasmides : 10

e) Oligonucléotides : 11

B) METHODS 11

a) Cellular cultures 11

b) Plasmidic preparation of the ADN 11

c) Transfections cellular 11

d) Epissage in vivo 12

e) Test of transactivation 13

f) Technique of Blot Western 13

G) Technique of the delay on freezing 14

III RESULTS 15

1. TLS is an Co-activator of the RANC 15

2. TLS makes party of the RANC 17

3. Analyze in vivo activity of TLS and rétinoïque acid on the alternate épissage of them 5 ' of E1A 19

IV - DISCUSSION 23

V - REFERENCES 28

I - INTRODUCTION

One of the principal topics of studies of our laboratory relates to the study of the hématopoïèse and leukemias, with an aim of diagnosis, forecast and therapeutic targeting. Thus, the comprehension of the molecular mechanisms which control the differentiation of the cells myéloïdes is an essential element.

The hématopoïèse is an active process, highly controlled, of proliferation and differentiation of the precursors and progéniteurs hematopoietic. Within the active compartment of the cells stocks, a cellular program of differentiation is set up. Initially, the cell stock loses its capacity of car-renewal, then gradually its capacity of proliferation. The cell engaged in this process will be directed towards one of the ways of hematopoietic differentiation (granulo-monocytaire, mégacaryocytaire, érythroïde or lymphocytary).

The extracellular signals, intra- or, which put in cycle the cell stock and which secondarily control the first stages of its determinism are little known. Two models are currently proposed to give an account of this phenomenon. First is known as « stochastic model » in which these stages are done in absence of any external signal, in an intrinsic way to the genetic program. The second model is known as « deterministic model », where the cell receives outside, in an extrinsic way, a signal which will start the genetic program of differentiation. Whatever the model considered, these mechanisms lead in an ultimate way to activation of factors of transcription which activate genes responsible for cellular differentiation (Felsenfeld and coll, 1996). They is probably combinative factors of transcription expressed by the cell which will lead to the preferential expression of specific genes of line. In other words, it is the genic expression which influences differentiation.

It was shown that the rétinoïdes stimulated in a preferential way the granulopoïèse (To endow and coll, 1982 ; Gratas and coll, 1993). The rétinoïdes act while being fixed on specific receivers which are the nuclear receivers with the rétinoïdes. The nuclear receivers (Figure 1) belong to a family of factors of transcription which control the form of genes according to the fixing of a ligand. The members of the superfamille of the nuclear receivers include the receivers with the hormones stéroïdiennes, such as the receivers with the glucocorticoïdes (GR.) and the estrogens (ER), of the receivers for hormones not stéroïdiennes like the receiver with the thyroid hormones (TR), the receiver with the Vitamin D3 (VDR), the receivers with the rétinoïdes, and also of the receivers for various metabolites lipidic like the fatty acids and the prostaglandins (Green and Chambon, 1986).

The superfamille of the nuclear receivers also includes/understands a great family of receivers known as orphan when the ligand was not identified yet or does not exist.

A

B

C

E/F

AF-1

AF-2

Activation

indépendante du ligand

DBD

Dimérisation

LBD

Activation dépendante du ligand

Dimérisation

D

Figure 1. General structure of the nuclear receivers

Two classes of receivers to the rétinoïdes were identified : receivers with the rétinoïque acid (RAR) and receivers with the rétinoïde X (RXR). Three types of RARs (, and) coded by distinct genes localized on different chromosomes are present at the Man, the Mouse, then in other species (Giguère and coll, 1987 ; Petkovich and coll, 1987 ; Krust and coll, 1989 ; Zelent and coll, 1989). RARs are able to fix two metabolites credits of Vitamin A, the all-trans rétinoïque acid (ATRA) and the rétinoïque acid 9-cis (Allegretto and coll, 1993 ; Allenby and coll, 1993). The comparison of the proteinic sequences of different RARs and other members from the superfamille of the nuclear receivers made it possible to subdivide them in various fields having of the distinct functions. These fields are more or less preserved of a receiver at the other and one species at the other (Kastner and coll, 1994). The second class of receivers to the rétinoïdes, RXRs, binds specifically and only the rétinoïque acid 9-cis. The genes coding for the three types of RXRs (, and) were clones in the Mouse and the Man. In the field of activation of the nuclear receivers, one finds two sequences carrying of the functions activatrices : AF-1 (field A/B), independent of the ligand and AF-2 (field E/F), depend on the ligand. These two fields (but mainly AF-2) can be used for recruitment of the Co-activator and Co-répresseurs (Kastner and coll, 1994). The structure of RARs is divided into six functional areas of A with F whereas RXRs do not have an area F. the various types of RXRs present a strong identity of sequence in their areas C and E, but strongly differ on the level from their areas A/B and D. This difference is thus specific of a given type of RXR (Chambon, 1996).

The nuclear receivers exert their action by various mechanisms. They can activate or repress target genes by setting directly on specific sequences of ADN called brief replies in the shape of homo-dimer (example of the receivers stéroïdiens and RXRs) or of hétéro-dimers (example of RARs, the VDR and TRs) with RXRs like partners. Or by fixing other classes of factors of transcription (Chambon, 1996). Certain nuclear receivers such as the TR and the RAR, can repress target genes in absence or in the presence of the ligand. These effects are related to the interaction of the nuclear receivers with intermediate protein classes of which the function is either to activate (Co-activators), or to repress (Co-répresseurs) the transcription. The complex transcriptionnel of the nuclear receivers with AR is called RANC. analyze areas promotrices of target genes of the rétinoïque acid allowed to describe brief replies, the RARE one (Retinoic Acid Response Element). These brief replies consist of two hexa-nucleotidic sequences preserved PuG (C/T) TCA laid out in repetitions direct (DR.), palindromes or reversed and separated by a number of nucleotides varying from 1 to 5 (Giguère, 1994). Thus, a spacing of 1 nucleotides (DR1) between the two reasons consensus AGGTCA defines a brief reply of homo-dimer RXR-RXR, a spacing of 5 nucleotides (DR5) between the two reasons leads to RARE.

The deterioration of RAR causes disturbances on the level of the mechanisms of replanning of chromatin. These deteriorations are due to genes of fusion implying gene RAR at the time of chromosomal translocations responsible for acute leukemia promyélocytaire (LAM3 according to classification FAB). It is about the first human malignant pathology answering therapeutic differentiating (for review to see Chomienne and coll, 1996 ; Fenaux and coll, 1994). It thus shows a single characteristic compared to the other sub-types of acute leukemias (LAM, anomaly of the myélopoïèse).

In an interesting way, it was revealed that in 60% of the LAM, TLS, a protein ubiquitaire, was very strongly expressed (Aman and coll, 1996). In addition, of the direct interactions between TLS and the receiver with rétinoïdes RXR were shown (Power and coll, 1998). These two observations us incited to study the possible bonds between TLS and the ways of indication to the rétinoïdes in a normal and deteriorated context hematopoietic.

Gene TLS, indeed, was initially identified in a malignant tumor starting from the translocation T (12; 16) like gene coding for the N-final part of TLS/CHOP, a oncoprotéine of fusion which is expressed invariably associated the liposarcome myxoïde human (Crozat and coll, 1993 ; Rabbits and coll, 1993). Other chromosomal translocations (at the origin of sarcomes human and leukemias), amalgamate either TLS or a similar gene, EWS with a great number of factors of transcription (Zinszner and coll, 1994). The common point of these various oncoprotéines of fusion is the presence of the N-terminal field of TLS or EWS. This field plays an essential part in the transformation confirmed by experiments of transformation using of the lines of mouse (Ichikawa and coll, 1999) or of the hematopoietic normal cells (Pereira and coll, 1998).

Many experiments made it possible to highlight certain roles of TLS, in particular the realization of two lines of mouse nullizigotes for TLS. The mice homozygotes carrying an induced change of TLS are sterile with an important increase in chromosomal axes not paired or mésappariés in the premeiotic spermatocytes. These results show a role of TLS in the pairing of the homologous ADN and the recombination. The analysis of these Mice indicates that TLS is essential for the survival of the new-born baby, influences the lymphocytary development, has a role in the proliferative response of the lymphocytes B to stimuli mitogenes, and is necessary for the maintenance of genomic stability (Hicks and coll, 2000). The capacity of TLS to be bound to the ADN is amongst other things induced by its phosphorylation by the PKCII which is allowed by the activity tyrosin kinase of BCR/ABL. These results suggest that TLS plays a part of regulator in the leucémogénèse caused by BCR/ABL, supporting independence towards the growth promoters and preventing differentiation, by modulating the expression of receivers of cytokines (Perroti and coll, 1998) confirming that its surexpression in the LAM can be one of the causes of the disorders observed.

TLS also interacts with the ARN polII by its N-terminal field and is implied in the formation of complex TFIID. It could thus act like a regulator of the basal transcription taking part in the recognition of promoter transcriptionnel and the initiation of transcription (Yang and coll, 2000). Moreover, TLS interacts with RXR and TR by their field N-terminal (Powers and coll, 1998). Factors of épissage also interact with TLS, implying it in this mechanism. TLS interacts with Spi-1/PU.1 a protein ETS able to control the transcription and maturation ARN in the cells myéloïdes (Hallier and coll, 1998). Three proteins SR, SC35, TASR-1 and TASR-2 interact with TLS by its C-terminal field.

Figure 2. TLS is a hnRNP and belonged to the family of EWS and hTAFII68.

The study of TLS made it possible to specify its structure (Figure 2). TLS belongs to a family of proteins including EWS (Delattre and coll, 1992) and TAFII68 (Bertolotti and coll, 1996). The N-terminal field of proteins of this family is rich in glutamine, serine and tyrosin, which are the amino acids found in the fields of activation of the transcription. In oncogenic dreams, the addition of the N-terminal field of TLS to the regulators transcriptionnels CHOP, FLI-1 or ERG-1 generates proteins whose transcriptionnelle activity differs from that of these respective components. Moreover, the N-terminal field of TLS present in these dreams can have negative effect dominating over the function of TLS (coming from the germinal line) as suggests the recent identification of determinants oncogenic in the N-terminal field of TLS. The C-terminal field contains several reasons : a sequence consensus of ribonucleoprotein (RRM : or RNP-CS), of the repetitions arginine-glycine-glycine (RGG 2/3), being the signature of proteins of connection to ARN (Burd and coll, 1994) and a zinc C2C2 finger. TLS in vivo binds the ARN or in vitro (Zinszner and coll, 1997b). In vitro, ARNs selected by TLS share a reason GGUG. TLS recognizes a ARN containing this reason in a cellular extract. Each field of connection to the ARN (three boxes RGG and the reason for recognition of the ARN) contribute to the specificity of interaction TLS-ARN (Lerga and coll, 2001). TLS is also called WERE or hPOMp75. This last denomination was given when it was shown that TLS lieny with the simple and double ADN bits. TLS supports the hybridization of bits complementary to ADN and the incorporation of a oligonucléotide of ADN simple bit in a super propeller of ADN to form one « D-loop », suggest that TLS is implied in the homologous recombination (Baetchtold and coll, 1999).

Figure 3. Reaction of cross esterification in the mechanism of the épissage of the ARN pre-Mr.

At Eucaryotes superiors, the genic expression implies the transcription, it « 5 ' capping », the épissage of the pre-m ARN, it « 3 ' processing », the export of ARNm and possibly translation in cytoplasm (Misteli and coll, 2000). The épissage (derivation of a nautical term which means to put end to end the ends of two ropes) consists of a reaction of cross esterification eliminating certain sequences from a pre-m ARN (will introns them) and of leaving other sequences (let us exons them). Sequences consensus were identified with the junctions exons/will introns, they are defined like the sites of épissage donor into 5 ' of the intron (dinucléotide GU), and acceptor, at end 3 ' of the intron (dinucléotide AG) (Breatnach and Chambon, 1981). Functions of the snRNA U1, U2, U4, U5, and U6 in the épissage are related to their inclusion within complexes made up of snRNA and many other proteins, called the snRNP. The fitting of the five snRNP with about fifty proteins on the matrix of the pre-m ARN constitutes a dynamic complex ribonucleoproteic, it « spliceosome ». The proteins SR take part in this complex, they are essential factors of épissage characterized by a reason rich in dipeptide serine-arginine having a role in the definition of exons and the selection of the alternative sites of épissage (Valcarcel and coll, 1996). Lastly, the capacity to select various combinations of let us exons at the time of the épissage, in order to generate proteins of distinct functions. This alternative phenomenon of épissage is essential to the development of many organizations. Approximately 60% of genes would be expressed according to such a mechanism (Croft and coll, 2000).

TLS belongs to the hnRNP family. And it was identified like the hnRNP p2 in a proteinic complex assembled on the pre-m ARN of the adénovirus. TLS committed in a complex with the hnRNPs A1 and C1/C2 and is associated several snRNP of « spliceosome ». TLS is complexed with transcribed ARN polII in extracts of irradiated cells of HeLa (Perroti and coll, 1998). Moreover, an inhibiter of the ARN polII induces the relocalization of TLS of the core with the cytoplasm, depend on the presence of its C-terminal field. All these proptiétés are characteristic of the hnRNPs. TLS was found associated the dinucléotide AG during the stage of recognition of site 3 ' of épissage and takes part in the selection of alternative site 5 ' of épissage in the pre-m ARN of minigene E1A. TLS can recruit by its field C-terminal, two regulators of épissage of the family of the proteins SR. Thus, TLS is one of factor of épissage.Lorsqu' it is surexprimée in cells érythroïdes, TLS preferably induces the use of distal site 5 ' of épissage, during the maturation of the pre-m ARN of E1A (Hallier and coll, 1998). This preference is counterbalanced by Spi-1, suggesting that TLS can belong to a protein network implied in the regulation of maturation ARN.

Several pathologies related to deteriorations of the épissage were characterized. Certain affections like the hereditary amyotrophies spinales or the neurodégénératives diseases related to the protein tau, represent true problems of public health and give place to profitable co-operations between hospital and university laboratories (Philips and Cooper, 2000). For example of pathologies implying the alternate épissage, one finds Spi-1/PU.1 in the erythroleucemy of Friend and CD44 in certain transformations tumoral (Stickeler and coll, 1999).

The role of TLS in the transcriptionnelle regulation is not yet clearly elucidated. TLS increases the transactivation directed by NFB induced by physiological stimuli such as the TNF, IL-1 and the surexpression of a kinase inducing NFB. TLS increases the activity of the promoter NFB-dependant on an intercellular gene of adhesion and gene on the IFN. These results suggest that TLS acts like an Co-activator of NFB (Uranishi and coll, 2001).

PROJECT DRANK

The fact that TLS is surexprimée in 60% of the LAM and that it interacts with RXR, one of the principal partners of the RANC, enables us to put forth the assumption that TLS would be related to the way of indication of AR in the hematopoietic cells. We propose to study two functions of TLS which could be brought into play in the LAM, as an Co-activator of the transcription and its role of factor of épissage. The action of TLS (in the presence of AR) as Co-activator of the transcription is tested in two cellular models, HL-60 and Cos-6, by the proportioning of the expression of a gene targets under the dependence of a promoter fixing the RANC. The membership of TLS to the RANC is observed by the setting in the presence of the RANC (RAR, RXR, DR5 with or without AR) and of TLS. The effect TLS and of AR on the épissage are analyzed qualitatively by obtaining of alternative profile of épissage of minigene E1A and the quantitative study of the produced isoformes.

II - MATERIALS AND METHODS

A) MATERIALS :

a) Cellular lines :

Three cellular lines were used :

- Line HL-60 was obtained starting from cells of a patient reached of a myeloblastic acute leukemia of type 2 according to classification FAB (French-American-British). Cells HL-60 have the characteristic to be Bi-potential, i.e. to be different either towards the way granulocytaire, or towards the way monocytaire, according to whether the ATRA or the VD3 is used (Breitman and coll, 1980).

- The K562 line was obtained starting from cells of a patient reached of a chronic leukemia myéloïde. The cellular population was identified like undifferentiated and of granulocytic series.

- The Cos-6 line comes from a kidney of Monkey.

b) Antibody :

Several antibodies were used : the monoclonal antibody of anti-myc Mouse (anti myc-TLS, IgG1, Kappa, Rock Molecular Biochemicals), the antibody polyclonal of Anti-RXR Rabbit (RXR 444, gift of Cecile Rochette-Egly and Pierre Chambon), the antibody polyclonal of Anti-RAR Rabbit  (RAR 115, gift of Cecile Rochette-Egly and Pierre Chambon) and the antibody polyclonal of Anti-TLS Rabbit (gift of Francoise Moreau-Gachelin).

c) Inductive agents :

The ATRA (Hoffmann the Rock) east dissolves in the DMSO with a concentration of 10-2 M, preserved at- 80°C safe from the light, diluted in RPMI to obtain a concentration of 10-4 M, stored with- 20°C and used in the culture media to the final concentration of 10-6 Mr. AR  9-cis carried out and is in the same way used with the final concentration of 10-6 Mr.

d) Plasmides :

- The plasmide Tkgal is a plasmide in which is clone ADNc of - galactosidase under the control of a délété fragment of promoter TK of the virus of the Herpes (gift of H. of Tea).

- The RARE plasmide is a plasmide pXp2, in which ADNc of the luciférase is under the control of the promoter of gene RAR2 (inserted promoter upstream, gift of H. of Tea).

- The vector of expression pcs3-MT (Myc-Tag) contains the promoter/enhancer cytomegalovirus simien IE94, promoter SP6 and six copies of the épitope Myc 9e10 (gift of Francoise Moreau-Gachelin).

- pcs3-MT-TLS codes for a protein containing the épitope Myc and the amino acids of TLS of 1-526 (gift of Francoise Moreau-Gachelin).

- pcs3-MT-E1A was generated by insertion of the EcoRI-XbaI fragment of E1A of the plasmide Sp4 in the EcoRI-XbaI site of the vector pcs3-MT (gift of Francoise Moreau-Gachelin).

- The pSG5-hRAR and the pSG5-mRXR are plasmides of expression containing ADNc of nuclear receivers RAR and RXR (gift of Cecile Rochette-Egly and Pierre Chambon).

- The MVC-Luc is a plasmide puC18 of 6 KB, without «polylinker», in which ADNc of the gene luciférase under the control of the promoter of the cytomegalovirus is clone, which is a strong promoter (gift of H. of Tea).

e) Oligonucléotides :

In the technique of delayed-action on freezing :

DR5: 5 ' - GATCAGGGTTCACCGAAAGTTCACTCGCATATATTAG-3' (Eurogentec).

In the technique of RT : start RT: 5 ' - GGAGAGCTTGGGCGACC-3 '(Genset).

In the technique of PCR: start PCR-E1A: 5 ' - ATTATCTGCCACGGAGGTGT-3': (Genset).

B) METHODS

a) Cellular cultures

The cells of lines HL-60 and K562 are sown with the concentration of 3x105 cells per ml and the Cos-6 cells with the concentration of 4x105 cells per ml.

The cells HL-60 and K562 are cultivated in RPMI 1640, added with fetal calf serum (SVF, décomplémenté by heat (20 min. with 56°C)) respectively with 15 and 10%, of glutamine 2 mm and Penicillin/Streptomycine with 100 ug/ml. The cells are maintained in exponential phase of growth by a dilution, twice by weeks.

The adherent cells Cos-6 are cultivated in DMEM, added with 6% with SVF, glutamine 2 mm and Penicillin/Streptomycine with 100 ug/ml. The cells are maintained in exponential phase of growth by a dilution, twice per weeks, after a washing with the medium without serum and an separation by twice an addition of 1 ml of trypsin during 5 min. with 37°C.

b) Plasmidic preparation of the ADN

The ADN plasmidic is obtained by culture of bacteria transformed beforehand by the plasmides of interest, in medium LB plus ampicilline 1X. The ADN plasmidic is prepared according to the protocol of plasmidic purification (Plasmid Purification, Qiagen). It undergoes then a precipitation with cesium chloride, this stage increases the purity of the ADN but is not obligatory. The quality of this ADN is analyzed by spectrophotometry to evaluate the quantity and migration on freezing of agarose 1% to evaluate quality.

c) Transfections cellular

1) Transitory Transfection of Cos-6 cells by calcium phosphate

Twenty four hours before the transfection, the cells are put in wells of culture 6 cm in diameter at a concentration of 600000 in 5 ml of medium. The next day, the medium is replaced by 4 ml of fresh medium. A solution of 1 ml containing 5 to 30 ug ADN plasmidic in 450 ul of H2 O, 50 ul of CaCl2 (2,5 M) and 500 ul of HBS 2X (NaCl 280 mm, KCl 10 mm, Na2HPO4 12 mm, Glucose 12 mm, Hépès 50 mm) beforehand incubated 30 min. at ambient temperature is added on the cells. They are given in the drying oven 12 hours, then washed with the PBS 1X. Fresh medium is added and as well as the ligand, possibly. The cells are again incubated 12 midnight for a proportioning of the luciférase at 2 days for a proteinic extraction with 37°C. Before the extraction of proteins, the cells are washed with the PBS 1X.

2) Transitory Transfection of cells HL-60 by electroporation

The cells passed the day before to the usual concentration corresponding to the cellular type and the tanks with electroporation are cooled beforehand with 4°C. Each tank is used for 30x106 cells. Three hours before the transfection, the cells are put at 1x106 per ml then incubated with 37°C. Then they are centrifuged, washed and gathered with 4°C in a volume of OPTIMEM-1 equivalent to 10 ml for 100x106 cells. They are then included in a volume containing 100 ul OPTIMEM-1 by tank. After placehaving placed 100 ul in each tank of them, the mixture is incubated during 10 min. with 4°C. The cells are added in the tanks, with a plasmidic solution containing 100 ul OPTIMEM-1 by tank. The electroporation is carried out in a electroporator of which parameters are: C= 960 uF and V=250 V, they are then put to incubate at ambient temperature during 30min. The cells are recovered with 1 ml of complete medium heated beforehand with 37°C. The cells are incubated with 37°C during three hours, then given in culture.

3) Transitory Transfection of K562 cells by the lipofectamine

The ADN plasmidic is prepared in a volume of 100 ul per well with 5% of lipofectamine more and supplemented by 95 ul of OPTIMEM-1 which is left incubated during 15 min. a solution of 12,5 ul of lipofectamine and of 87,5 ul of OPTIMEM-1 by well is prepared. The two solutions are mixed in sterile polypropylene tubes, by adding the plasmidic solution drop by drop. This mixture is incubated one hour at ambient temperature. The cells are conditioned with the number of 2x106 per well in 0,8 ml of OPTIMEM-1 (the concentration initial of the cells in their medium was 500000 per ml) and incubated one hour with 37°C. The 200 ul prepared are deposited drop by drop in each well. Then the cells are incubated two hours with 37°C and finally 3,5 ml of medium are added and the ligand éventuellement.d

) Epissage in vivo

The cells are collected 24 hours after the transfection. Co-transfection with the plasmide pCMV-luciférase allows the standardization of the RT according to the differences observed in the effectiveness of transfection. A aliquotée fraction of these cells is used to measure the activity luciférase and the remainder is used to carry out a total extraction of ARNs. ARNs totals are prepared starting from the cells by using the protocol of purification of ARN and according to the instructions of the supplier (RNeasy, Qiagen) and treated twice by Dnase I (Qiagen). The quality of ARNs obtained is observed by migration on a gel of agarose denaturing agent. The isoformes of ARN E1A are analyzed by RT-PCR as described previously (Hallier and coll, 1998). One microgram of ARN retro-is transcribed by SuperscriptII transcriptase reverse of the virus of the murine leukemia of Moloney (Gibco-BRL) in the presence of 50 uM of dNTP and 2 picomoles of preceding 3 ' RT E1A in a solution of 8 ul. Approximately a tenth of ADNc is used for the reaction of amplification (PCR) carried out with Taq polymerase ADN (Perkin Elmer) in the presence of the starter 5 ' E1A marked at its end 5 ' by T4 polynucléotide kinase (Boehringer) and of the ATP P32 (Amersham) like previously described (Hallier and coll, 1998). The minimum of cycles PCR is used (18 to 22 cycles) in order to detect the signal in a window of adequate detection. The reactions of control of the RT-PCR contain a matrix ARN not having undergone an opposite transcription. The products of the RT-PCR of E1A are put to migrate on a urea/polyacrylamide gel 6% denaturing agent, autoradiographies and quantified with a Biorad system. The form of proteins coded by the plasmides is checked by the technique of the Blot Western carried out on samples of transfectées cells from which come ARNs totals.

e) Test of transactivation

The transfection day before, the Cos-6 cells are put out of well, in 5ml of DMEM-6% SVF décomplémenté at a rate of 0,6x106 per well. The next day, the medium is changed for 4 ml of fresh medium. In each well, is added a beforehand incubated solution of 1 ml with ambient temperature including/understanding a plasmidic solution (TKGal, Rare-Luciférase,...), 450 water ug Bi-distilled in addition to 50 ul of CaCl2 (2,5 M) and 500 ul of HBS 2X (NaCl 280 mm, KCl 10 mm, Na2HPO4 1,2 mm, Glucose 12 mm, Hépès 50 mm).

Cells HL-60 are électroporées with an equivalent plasmidic solution.

After a 12 hours incubation to 37oC, the cells are washed 2 times at the PBS 1X. A plug of lysis (200 ul) is added in each well and the cells are separated using a scraper. The cells are recovered in tubes Eppendorff, are vortexées and centrifuged 2 min. with 12000g and 4oC. The activity of the luciférase (according to the protocol of Luciferase Assay System, Promega, Madison, EUA) in the lysate is measured in a luminometer and is expressed in arbitrary unit. The activity of - Galactosidase (according to the protocol of BM Chemiluminescence ELISA Substrate - Gall, Boehringer Mannheim, Mannheim, the Federal Republic of Germany) is also measured in order to standardize measurements obtained of the luciférase activity.

f) Technique of Blot Western

Total proteinic extracts are carried out. The cells are washed twice with the PBS 1X, the base is included in 300ul plug of extraction (Hepès 20 mm pH 8, NaCl 450 mm, EDTA 0,4 mm and glycerol 25%) containing inhibiters of proteases (pepstatine, leupeptine, aprotinine). For the lysis of the cells, three successive baths in nitrogen and with 37°C are carried out. After centrifugation (15min. with 14000 rpm), the supernatant corresponding to the total extract is taken, made up in aliquot fractions and is frozen with - 80°C.

After a denaturation of 10 min. with 100oC, the protein samples are deposited on a denaturing gel of acrylamide 12%. The migration is done in 2 stages, first of all to 80 V during 15 min. then with 125 V during one hour. The proteins are then transferred on a membrane from nitrocellulose by électrotransfert to 600 my during 60 min. cold. To use the anti-RAR and anti-RXR antibodies, the membrane is washed twice with PBS 1X then saturated with milk 5% during 2 hours at ambient temperature. During all the night, the membrane is incubated with 4oC with the anti-RAR primary education antibody diluted with the 1/100è and anti-RXR diluted with the 1/100è. Washed 5 times 10 min. with PBS 1X, the membrane Re-are saturated with milk (2,5%) during 10 min. then incubated with the secondary antibody during 30 min. (Protein A diluted to the 1/10000è). Then, 5 washings are carried out at the PBS 1X containing 0,01% of Tween.

For the anti-myc antibody, the membrane is saturated with PBS Tween 20 to 0,1% during 2 hours at ambient temperature. Several washings with the PBS Tween are made during two min. the membrane is incubated with 4oC all the night with the anti-myc primary education antibody diluted to the 1/10000è. Washed several times during 5 min. with 500 ml of PBS Tween, the membrane is incubated with the secondary antibody during one hour (anti-kappa of mouse diluted to the 1/1500è). Then the membranes are washed with 1l PBS Tween during at least three times 15min.

Lastly, the membrane is put in the presence of a solution of detection (ECL, Amersham, Piscataway, NJ, EUA) during one minute. Radiography is made by exposing a film to variable times (Hyperfilm ECL, Amersham, Piscataway, NJ, EUA).

G) Technique of the delay on freezing

The probe DR5, which corresponds to the RARE brief reply of the promoter of RAR, is marked at its end 5 ' while incubating during 60 min. with 37oC: 30 ng of the probe, the plug kinase 10X, 3 L of ATPP32, 1 L of T4 polynucléotide kinase (5 units). After having purified the probe marked on a column (centri.spin-40, hydrated beforehand with 0,5 ml of plug YOU 1X during 30 min. and centrifuged 2 min. to 700 G at ambient temperature) by centrifuging it 2 min. to 700 G at ambient temperature, the control of the specific activity is carried out thanks to a scintillation counter. The probe doubles bit is elaborate while adding to the marked probe 60 ng of the complementary bit, the plug kinase 10X and 150 mm of NaCl. The solution is denatured with 100oC then gradually cooled with 37oC.

The proteinic extracts and the antibodies are incubated 20 min. on the ice with a solution containing 2 ul plug Darnell (KCl 400 mm, Hepès 200 mm, MgCl2 10 mm, EGTA 1 mm, DTT 5 mm, Ficoll 4%, pH 7,5), 1 ul of poly dI-cd. (1 ug/ul) and 40000 cpm of the radiomarquée probe. The extracts are then deposited on a polyacrylamide gel 4% and migrated to 200 V during 82 min. freezing is dried in neutral during 120 min with 80°C. An autoradiography is carried out with an amplifying screen in the presence of a film Kodak X-Omat AR with- 800C during 12 hours.

III RESULTS

1. TLS is an Co-activator of the RANC

Firstly, we analyzed the effect of a transitory surexpression of TLS on the expression of a gene rapporteur (luciférase) under the control of the RARß2 promoter (Figure 4A). We used as cellular model hematopoietic cells HL-60. These cells are électroporées in the presence of Tkßgal, to evaluate the effectiveness of transfection and to standardize the results, of ßRARE-Luc, used like gene rapporteur to evaluate the transcriptionnelle activity and of various quantities of vector of expression of TLS, as indicated. pCS3, a plasmide control is used in such way that all the transfections have the same total quantity of plasmide of expression (Figure 4). The cells are collected 24 H after and the luciférase activity is measured. The values represent the averages of the doublets carried out. Similar results were obtained in several independent experiments.

Expressed results corresponding to the increase in the activity of the luciférase compared to a negative witness (cells transfectées with pCS3 only, in absence of ligand).

At the time of the transfection in the presence of AR alone (1 '), an increase in the luciférase activity is observed. Co-transfection by pcs3-MT-TLS alone and Rare-Luc (2 and 3) does not have an effect on induction of the luciférase activity, except in the presence of AR (ATRA 10-6 M) (2 ' and 3 ', multiplied by 25, in the presence of 5 ug and 10 uG of TLS (Figure 4B). These results suggest that TLS increases the transcriptionnelle activity induced by the endogenous receivers in cells HL-60.

In order to determine that Co-activation by TLS acts by and thanks to the RANC, we used Cos-6 cells having few receivers to the endogenous rétinoïdes which we transfectées with the plasmides expression of RAR and RXR. The Cos-6 cells are thus Co-transfectées with calcium chloride with the plasmides TKgal and RARE, as previously described.

Figure 4 . TLS increases the transcriptionnelle activity of the nuclear receivers to the rétinoïque acid in cells HL-60. With, descriptive diagram of the construction used in the test of transactivation. B, the transcriptionnelle activation of RARE A place with AR alone or TLS in a way dependant on AR in cells HL-60.

In Figure 5, controls (1 and 4) indicate the basal transactivation of the gene rapporteur. At the time of the transfection in the presence of AR alone (1 ' and 4 ', multiplied by 40) or with TLS (2 ', 3 ', 5 ', 6 ' and 7 '), an induction is observed. Induction is optimal with 2 ug of TLS (3 ' and 5 ', multiplied by 85). TLS is thus an Co-activator of the complex transcriptionnel receivers RAR and RXR, depend on AR. With larger quantities, 5 ug and 10 ug of TLS (6 ' and 7 '), a reduction in induction in the presence of AR, lower at the level reached in absence of TLS, are observed. This reduction could be due for a purpose of « quenching » or with the effect TLS which, in great quantities, would prevent RXR from binding to the ADN.

The whole of these results indicate that protein TLS is an Co-activator of the transcription by the receivers with AR. TLS thus seems implied in the way of indication of AR.

Figure 5 A and B. TLS act like Co-activator of the RANC, way proportions dependant. The two experiments are carried out independently.

2. TLS makes party of the RANC

The effect TLS on the transcription passing by the way of indication of AR suggests that TLS can belong to the RANC, including/understanding the nuclear receivers. In order to determine the membership of TLS to the RANC, the experiments of delay on freezing were realized.

Proteinic extracts of Cos-6 transfectés by calcium chloride with the plasmides of expression of RAR, RXR and TLS are used in the experiments of delay on freezing.

These total proteinic extracts of Cos-6 are préalablements checked by Western Blot after immunoblotting with specific antibodies, of which the antione for labelled protein TLS (Figure 6).

Figure 6: Analyze by Western Blot of the proteinic expression of Cos-6 transfectées

The technique of delay on freezing is carried out with the total proteinic extracts tested previously in presence or absence of TLS (Figure 7).

RXR RAR

DR5

Figure 7. descriptive diagram of the construction used in the delay on freezing and of the fixing of the RANC

The signal in 1 corresponds to the fixing of RAR and RXR on the DR5. The complex observed is thus consisted of the nuclear receivers and is called RANC. Signal 2 indicates that RXR in fact part. The intensity and the delay of bands 3 and 4 indicate that TLS belonged to the RANC. From 1 ul (band 4), plus the quantity of TLS is increased, plus the complex seems destabilized (band 5 and 6). The maximum quantity of TLS for its participation in the RANC is 1 ul total proteinic extracts. Beyond this confirmation and by realizing experiments of controls of the phenomena of competition or allostery, TLS could interfere with the complex, being able to prevent RXR from binding to the ADN.

In conclusion, these preliminary results strongly suggest that TLS would increase the target gene transcription of AR in a way dependant on the ligand in the cells myéloïdes and that this would be related to a direct participation of TLS in the complex transcriptionnel RANC. The experiments in the Cos-6 cells show that this regulation by TLS and AR could be a general phenomenon.

Figure 8. Delay on freezing carried out with RAR and RXR, in presence or absence of TLS.

The effect of the amount of TLS on the RANC is given.

3. Analyze in vivo activity of TLS and rétinoïque acid on the alternate épissage of them 5 ' of E1A

In the light of the results obtained, it is clear that TLS acts on the way of indication to the rétinoïdes at least by its transcriptionnelle activity.

Since TLS can play a part of factor of épissage by supporting the selection of distal site 5 ' of épissage of the pre-m ARN of minigene E1A in the cells érythroïdes murine IW1-32, we studied the role of TLS on the épissage of the pre-m ARN of minigene E1A in a human model hematopoietic, the line myéloïde K562.

ARN pre-m E1A contains sites 5 ' of épissage which lead to the formation of several isoformes principal of ARN (of which 13S, 12S and 9S). These isoformes can be highlighted and be quantified by PCR using selected starters spécifiquements (Figure 9).

Figure 9. Diagrammatic representation of the alternate épissage of transcribed E1A. the isoformes of the pre-m ARN of E1A and the corresponding size of the products of RT-PCR are shown. The starters for the analysis in RT-PCR are indicated by arrows.

In order to study the possible role of TLS or AR on the épissage into 5 ' of minigene E1A, we transitorily have Co-transfecté the K562 cells by the lipofectamine with the plamides of expression of E1A, pCS3 (to standardize the quantities of plasmide used), of TLS (the quantities used are indicated) and of MVC-Luc (to measure the effectiveness of the transfection). When the luciférase is expressed, ARNs are extracted, then rétrotranscrits. Then a PCR is carried out. The detection of the products of PCR is made after separation on a gel Page-urea by autoradiography (Figure 10). Lastly, the quantitative analysis of the results is obtained by the system Molecular Analyst de Biorad (Figure 11).

After ensurehaving ensured itself of the effectiveness of the transfection, it is advisable to obtain the adequate experimental conditions to secure of the detection of all the isoformes. It is a question of varying either the quantity of products of RT used for the PCR, or the number of cycles of the PCR, or the exposure of autoradiography. Each experiment is made in doublet and is reproduced to be ensured of the reproducibility of the results obtained. The study is based on the choice of site 5 ' of épissage of the minigene E1A which one expressed as a percentage deduces from the expression of the isoformes 13S, 12S and 9S. It is, moreover, requirement to visualize a band corresponding to the not spliced form of E1A so that the results are interpretable.

By comparing the results without AR (1) with the results with AR (2), it is possible to affirm, subject to additional controls (between 40 and 60% of 9S) that it would not seem that AR alone has a significant effect on the épissage of E1A in K562.

On the other hand, the results in the presence of TLS are reproducible and confirm that in the K562 cells, protein TLS allows a preferential épissage isoforme 9S the detriment of the isoformes 13S and 12S. This phenomenon would be increased in the presence of AR. The results (3) show that TLS alone supports the formation of the isoforme 9S (75% of 9S), whereas when AR is used with TLS, the isoforme 9S is increased considerably (90% of 9S).

These results indicate that TLS acts on the selection of distal site 5 ' of épissage, supporting the formation of the isoforme 9S minigene E1A, phenomenon accentuated by AR. Thus, these results suggest that TLS acts on the épissage in the human hematopoietic cells and that AR intervenes on the épissage through TLS.

In conclusion, these results would show for the first time that AR would be implied directly in control post-transcriptionnel through protein TLS.

Figure 10. The alternate épissage in vivo of minigene E1A in the K562 cells.

The intensity of the bands corresponding to different the isoformes from E1A for each profile is quantified by densitometry. The results are expressed in percentage of the isoformes 13S, 12S and 9S. the studies are made in doublets. The RT- represents the negative control of the RT, it was carried out without enryme RT and no ADN is revealed. ARNs extracted and used are thus free from ADN.

Figure 11. Quantification of the isoformes of the pre-m ARN of E1A by a software Molecular Analyst de Biorad. The percentage of the isoformes 13S, 12S and 9S is represented.

IV- DISCUSSION

These research tasks relate to the study of the role of TLS in the ways of indication of the rétinoïque acid. TLS is a bifunctional protein in the mechanism of action of AR on the level of the transcriptionnelle regulation and the post-transcriptionnelle regulation by AR. Indeed, of the experiments of transactivation and delay on freezing highlight that TLS takes part in the complex transcriptionnel of hétérodimère RXR-RAR, called RANC allowing to increase the transactivation dependant on AR on the target gene promoters. Initially, of the techniques of transitory transfection in Cos-6 cells and myeloblastic cells HL-60 show that TLS is able to increase the transcriptionnelle activity of the natural promoter of RAR sensitive to the action of the rétinoïque acid in the presence of rétinoïque acid. It had been previously highlighted that TLS had a transcriptionnelle activity per. Indeed, the N-final part of TLS implied in protein of fusion TLS/ERG is regarded as pre-necessary for the factor of transcription ERG deteriorated in its potential leucemogene by increasing its transcriptionnelle activity and/or by changing its specificity on the level of genes target (Zinszner and coll, 1994 ; Ichikawa and coll, 1999). Moreover, it was highlighted that the N-final part of TLS amalgamated with the field of connection of the ADN of Gal4 has a strong transcriptionnelle activity (Uranishi and coll, 2001).

In order to determine if TLS belonged to the RANC, of the experiments of delay on freezing were carried out while using like probes oligonucleotidic DR5. For this purpose, extracts of Cos-6 cells transfectées by RAR, RXR or TLS were used. The results show that TLS belonged to RANC. Another study carried out in vitro shows that this interaction is direct and independent of the presence of the rétinoïque acid and seems specific to the receivers with rétinoïque acid RXR, the glucocorticoïdes, with the estrogens, and with the thyroid hormone (Powers and coll, 1998). This direct interaction is independent of the presence of the ligand. This observation is due to the fact that the complex is formed between the field of connection of the ADN (field C) and the area D of the nuclear receiver with the N-final part of TLS (Powers and coll, 1998). The area C, made up of 66 amino acids, is preserved among all the members of the superfamille of the nuclear receivers. It contains two structures of ^« finger with zinc ^» and corresponds to the field of connection of the nuclear receiver with the ADN. The area D, also called zone hinge, is between the field of connection to the ADN and the area E. It is subdivided in three under-areas (D1, D2 and D3) of which the N-final D1 area, the most preserved, contains many basic amino acids which would correspond to a signal of nuclear localization. The central area D2 is more variable. It is interesting to note that the interaction of TLS with the field of connection to the ADN of RXR does not deteriorate the connection of the nuclear receiver on its specific brief reply.

The function Co-activatrice of TLS on the level of the complex transcriptionnel of the receivers to the rétinoïque acid could seem paradoxical in the fact that the interaction of TLS with RXR implies its field of connection of the ADN. However, this interaction is not destabilizer of the complex protein/ADN. Quite to the contrary, since it is established that this interaction protein/protein is stabilizing complex RXR-ADN (Powers and coll, 1998). Moreover, this interaction does not prevent the change of conformation of the nuclear receivers. Thus, TLS by its action would stabilize the complex Co-activator of the receivers to the rétinoïque acid. The principal complexes Co-activators of the nuclear receivers include/understand the complex Brg (SWI/SNF), Co-integrators CBP and p300, the family of the proteins p160, the protein p/CIF and the complexes TRAP/DRIP/ARC.

TLS was already implied in the complex transcriptionnel of another factor of transcription, NFB. Thus, TLS increases the transactivation dependant on NFB induced by physiological stimuli such as TNF and IL-1 (Uranishi and coll, 2001). TLS thus acts as an Co-activator of various factors of transcriptions.

Lastly, TLS shares structural characteristics with hTAFII68. These proteins were found associated complexes TFIID (Bertolotti and coll, 1997 and 1998) and are implied in activation transcriptionnelle (Prasad and coll, 1994 ; Zinszner and coll, 1994 ; Bertolotti and coll, 1999 ; Ichikawa and coll, 1999). TLS interacts in vivo with TFIID (Uranishi and coll, 2001). Moreover, TLS is associated the ARN polII via its field N-terminal (Yang and coll, 2000). The whole of these interactions could make it possible TLS to establish the molecular link between factors of transcription and the complex of initiation of the transcription.

The whole of these studies implies in an undeniable way TLS in the transcriptionnelle regulation on the level of the ways of indication of the rétinoïdes.

The second part of the research project relates to the possible bond between the ways of indication of the rétinoïdes and the post-transcriptionnelle regulation through the modulation of the alternate épissage. Indeed, the fact that TLS, factor of épissage, is implied in the ways of indication of AR through its direct role in the transcriptionnelle coactivation dependant on the RANC encourages to study the role of the rétinoïque acid and its receivers in the épissage.

The experimental results show that TLS acts in vivo on the selection of sites 5 ' of épissage alternate of ARN pre-m E1A in the hematopoietic cells K562. The selection of distal site 5 ' of E1A corresponding to the formation of the isoforme 9S is favoured with the detriment of that of the proximaux sites 12S and 13S.

Certain data in vivo switch us on the role of TLS in the regulation of the alternate épissage. TLS is associated RNPs by in vivo forming a complex with the hnRNP A1, a factor of épissage able to support the selection of distal site 5 ' of épissage during an alternate épissage of any pre-m ARN (Uranishi and coll, 2001). It was previously shown that TLS acted on the selection of the distal site of E1A in the erythroblastic cells of Mouse IW1-32 (Hallier and coll, 1998). The experiments carried out on the K562 cells transfectées by E1A make it possible to highlight an increase in the rate of isoforme 9S in the presence of AR and TLS. The functional interference between TLS and the rétinoïque acid can be due to molecular interferences, i.e. physical interactions protein/protein. This assumption can be based on the direct interaction shown by preceding work. Consequently, these results identify the rétinoïque acid as an actor implied in the regulation of the épissage. It is the first time that it is highlighted the action of a hormone on the épissage. This work makes it possible to define a new level of regulation of the ways of indication of the rétinoïdes.

The fact that TLS interacts with RXR, TR and GR. make it possible reasonably to think that this phenomenon is found on a more general level.

The épissage of the ARN, critical stage of the form of genes, is regarded more and more as an event Co-transcriptionnel. Experimental obviousnesses state from now on that stages of transcription, it « capping » and the polyadenylation, are closely related to the ARN polII through its association with the factors implied in these mechanisms molecular (Cho and coll, 1997 ; Hirose and coll, 1998). The proteins of regulation of the épissage, the proteins SR, are associated the ARN polII. However, the means by which this association is carried out were not identified. The Co-activator transcriptionnel p52 is able to interact with the protein SR, ASF/SF2. p52 thus acts as an adapter in order to coordinate the transcription and the épissage (Ge and coll, 1998). TLS also interacts with the ARN polII and the proteins SR (Yang and coll, 2000). TLS could thus act as a recruteuse molecule of the factors of regulation of the épissage SR towards the ARN polII, thus coupling the transcription with the épissage. TLS could also act directly on the épissage by its interaction with ARN (Lerga and coll, 2001).

TLS contains three fields potentially implied in the connection with the ARN, RGG1, RRM and RGG2-3. The mechanism implied in the recognition of the ARN by TLS is still unknown. The secondary structure of the ARN represents a significant part of the interactions protein/ARN. The selected sequences are fixed at TLS with affinities of about 250 Nm with 600 Nm, Kd of 250 Nm corresponding to the complex ggugARN/TLS (Lerga and coll, 2001). Consequently, TLS seems to be a protein having a weak affinity for its sequence ARN. Thus, it is not excluded that Kd of in vitro given complex TLS/ggugARN is far away from Kd from a complex ARN/protein on the level of the spliceosome. However, a weak affinity of a factor of épissage for its target sequence ARN could represent a condition compatible with the assembly of « spliceosome » which is held through the exchange and replacement of a great number of proteins on the level of the substrate, the ARN pre-Mr.

The functions of TLS in the regulation of the transcription and the épissage make it possible to consider the consequences of a deterioration of TLS in certain pathologies. In the leukaemic cells myéloïdes having the translocation T (16; 21) and in the cells of liposarcome in the translocation T (12; 16), only one allele are stopped whereas the other allele is intact (Crozat and coll, 1993 ; Rabbitts and coll, 1993 ; Yamamoto and coll, 1997). These observations suggest a role of protein of generated fusion of dominant type negative. The protein of fusion TLS/ERG, observed in human leukemia myéloïde T (12;16), in vivo pertuberait the épissage of E1A observed in cells HeLa (Yang and coll, 2000). It should be noted that TLS was not considered able to act on E1A in these cells, which seems erroneous starting from other observations carried out (F. Moreau-Gachelin, personal communication). Nevertheless, TLS is able to inhibit the function of the TASR on the épissage of ARN pre-m E1A (Yang and coll, 2000). Moreover, it is interesting to note that the épissage CD44 is deteriorated by the presence of protein of fusion TLS/ERG in stable clones generated starting from cells of the line K562 (Yang and coll, 2000). Gene CD44 codes for a molecule of adhesion made up of ten exons constitutive and ten let us exons variable. Various combinations formed by let us exons variable is at the origin of a large variety of isofomes of épissage of CD44 which differ on the level from their extracellular field. The abnormal épissage of ARN pre-m CD44 was found in various solid tumors and of leukemias (Cooper and coll, 1995). Moreover, It was suggested that the variations in proteinic rate of SR according to various stages' of the development of the breast cancer can be directly related to the variations of various the isoformes of CD44 (Stickeler and coll, 1999). Two mechanisms are planned to explain the negative effect dominating of TLS/ERG over the épissage of CD44. The first considers that TLS clinging to the specific isoformes CD44, TLS/ERG would block this way of regulation, at the origin of a premature degradation of the pre-m ARN of CD44 not spliced completely. In the second mechanism, if one of the ways of regulation is blocked and that another way of regulation of the alternate épissage exists, the inhibition of the first way by TLS/ERG could encourage the épissage CD44 through another way of regulation, thus increasing the risk to generate an aberrant épissage.

If TLS is expressed in way ubiquitaire, it is interesting to note differences in expression in the normal and pathological hematopoietic cells. The cells hematopoietic stocks purified of blood of cord have lower rates of TLS compared with the cells myéloïdes. In addition, an important expression of TLS was highlighted in cells of LAM (Millets and coll, 2000). It is interesting to note that TLS was also identified by its rate of expression decreased in cells HL-60 treated by the rétinoïque acid during one hour. Moreover, the expression of TLS is strongly decreased in cells HL-60 during the granulopoïèse induced by the DMSO or the ATRA. There is a specificity with the granulopoïèse since differentiation monocytaire induced by the TPA or the D3 vitamin does not act on the expression of TLS (Millets and coll, 2000). This reduction in expression is associated the fall of the cellular proliferation in the various LAM. The whole of these observations suggests that the expression of TLS is a key regulator of the myélopoïèse where a strong rate of expression of TLS supports the cellular proliferation with the detriment of differentiation.

The prospects for this work are articulated on two axes. The first relates to the fundamental molecular mechanisms which imply TLS in the ways of indication of the rétinoïque acid. We plan to study TLS in the regulation of the épissage constitutive and the alternate épissage into 3 ' of épissage. The effect of AR on the épissage of E1A will be also studied through the roles of RAR and RXR. It is also a question of determining with precision the association of TLS in the complex transcriptionnel of AR. Thus, of the experiments of identification of the direct interactions between TLS and RAR will be carried out knowing that they were highlighted with RXR (GST sweater down and Co-immunoprécipitation). We want to study the coordination of the épissage and the transcription to know if these mechanisms are dependant or independent one of the other (a construction joining together genes E1A and the luciférase under the dependence of a transferred promoter sensitive to AR, not allowing the RANC to fix itself to increase the transcription). The biological role of TLS will be determined by its transitory invalidation (RNAi). The second axis consists in confirming the bond between TLS and pathology. It is a question of including/understanding the consequences of the deterioration of the expression of TLS and its targeting within the framework of a new therapeutic and diagnostic futurology.

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