TLS, une protéine du spliceosome, est impliquée dans le mécanisme d'action de l'acide rétinoà¯que à travers les régulations post-transcriptionnelle et transcriptionnelle

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.