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1742 Humań Molecular Genetics. 1999. Vol. S. No. 9

STAT5a^_;‘ and/or STAT5b^_/“ knockout mice have pheno-lypes mainly aiiributable to deficiencies in prolactin and growth hormone functions (14). STAT5a"^/_b"^/" double mutant mice have a profound deficicncy in peripheral T ccii prolifera-tion together with a reduced number of cytokine colony-forming unit responses of bonę marrow cells (14). Moreover. STAT5a^-/" and STAT5b^_/~ mice exhibit a defect in natural killer celi proliferation and function (21). STAT5s reguiate expression of many genes (8.21.27) coding for milk proteins <p-casein and p-lactoglobuiin) and for proteins involved in haemato-poiesis and the immune response [oncostatin M. c-myc. cytokine-inducible SH2 domain coniaining protein (CIS), !L-2Ra. IL-2R(3. perforin and pim-1). Undergrowth hormone stimulation. STAT5b controis liver gene expression in a sex-specific man-ner (28).

We report on the molecular characterization of the STAT5n RARA <zene fusion at both the cDNA and the aenomic levels together with immunolocalization studies of the chimeric protein in the patient leukaemic cells. We discuss the potential implication of the STAT5b component of the fusion protein in leukaemogenesis in the present APL-L.

RESULTS

Fusion of STAT5b to RARA

In order to isolate the gene fused to RARA. a 5' rapid amplifica-tion of cDNA ends (5'-RACE) was perfonned on total RNA> e.\tracted from the patient bonę marrow cells (Fig. lat. A Blastn anaiysis of a 5-RACE product-denved secjucnce showed an in-frame fusion between STATób and RARA genes (Fig. 1 at. The resulting protein is composed of 1038 amino acids: a schemaiic representation of the anticipated structure of the STAT5b-RARA protein is shown in Figurę Ib. The putative STAT5b-RARA protein contains all known functional domains of RARA except for the N-terminal tr^/i5-activating function. Apan from all R.ARA functional domains. the fusion protein contains the Statób coiled-coil region (29). DNA-binding domain and a tmneated SH2 domain. Tvr699 essential for STATób activation and the C-terminal transactivation domain with Ser731 invołved in regula-tion of STAT5b tran.scriptional activiiy are absem from the chimeric STAT5I>-RARA protein. The reciprocal RARA-STAT5b mRNA was not detected after RT-PCR on patient bonę marrow total RNAs (data not shown). Tlie STAT5b rearrangemeni was confimied at the genomie level by Southern biot anaiysis with STAT5b cDNA (Fig. 2).

Characterization of the fusion at the genomie leve! was done with a PAC which contains all the STAT5b coding region and with a breakpoint spanning phage clone (Fig. 3). Sequcncc anaiysis showed a 3 bp mseriion at the joining sile; the 2.4 kb chimeric intron is mainly composed of STAT5b-deri\ed intronic segucncc. No homologics were detected with highly rcpcatcd long (LINES) and short interspersed elemenis (SINES). The breakpoint in RARA gene intron 2 is located in the so-called ‘cxtreniely resirieted region' (ERRi ol 50 bp which has bcen identified as an illcgilimalc rccombination hotspot involved in PML-RARA rearrangements i30.3b. .Vloreovcr. we found short idcniical sireiches of DNA between STAT5b and RARA at the joining points in the present case (Fiu. 3». rc.scmblino those obscrved in PML-RARA fu^.on.'.

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132). The model of the u!5:I7> translocation propo>ed

Yoshida et a!. (32) is therefore likely to be relevam to this STAT5b-RARA gene fusion. The reciprocal chimeric RARA-STAT5b intron. estimated to be slightlv larsrer than 17 kb long [the normal RARA intron 2 is 17 kb long (33)], was not detected by PCR with two different oligonucleotide pairs on patient genomie DNA (data not shown). This suggests a smali interstitial deletion ranainsz from STATSb intron n to RARA intron 2 as the most probable mechanism for formarion of the STAT5b-RARA gene fusion. Duplication of the 17q21.3—q23 region observed after comparative genomie hybridization (CGH) anaiysis (see Materials and Methods) is probably a sec-ondary event in leukaemogenesis.

The STATSb gene inaps to 17q21.1-q21.2 close to the RARA loeus

STAT5a and STAT5b were both mapped at l?qll.2 (10) by FISH: however. the metapha.se chromosome resolution did not allow precise localization of STAT5b in the I7q proximal region. Our FISH anaiysis with PAC 196pl7 on normal pro-meraphases seems to inćicate that the STATób and RARA genes are very close to each other in 17q2l.l-q21.2 and that the phvsical distance between the two genes is most likely <3 Mb <34: Fig. 4).

Localization of the STAT5b-RARA protein in leukaemic cells

Immunolocalization studies on patient bonę marrow ccils show that the STAT5b-RARA protein is mainly iocalized in the nucleus but also to a lesser extent in the c\ toplasm and displays a microspeckled paltem, whereas the normal STAT5b protein shows a normal diffuse cyioplasmic localization (Fig. 5).

DISCUSSION

In this study. we identified STAT5b as the flfth gene fused to RARA in APL and APL-L leukaemias. PML. PLZF. NPM. .NuMA and STAT5b (in its phosphorylated form) are nuclear proteins. Howevcr. for the First time. the protein derived from the RARA gene partner can be Iocalized in the cytosol (in its latem form). To datę. all RARA partners contain an N-terminal protein-protein interaction domain; the STATSb coiled-coil domain is located between amino acids 232 and 321 (29).

RARA is essential for proper myeloid celi differentiation in response to retinoic acid (RA). As for other APLs and APL-Ls. the chromosome 17 breakpoint in the present ca.se is located in intron 2 of the RARA gene. This suggests that an ubnormul receptor with almost all functional domains of the nativc RARA is the

hailmark for inhibition of myeloid celi differentiation. The arrest

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of maturation of myeloid ccils in APL-L reponed in this study is possihb due lo an abnormal RARA protein.

The wcll-characterized PML-RARA and PLZF-RARA onco-genic proteins mediate leukaemogenesis through reeruitment of histone dcaeetylase leading to aberram chromatin acctylation and transcriptional repression of RA-regulated genes (35-38). An N-( nR box has heen implicated in the ability of RARA to internet u uh co-rcpressors (35). Moreover. in PML-RARA-associatcd APL>. ddocali/ation of the nomiai PML protein in hlast cells pro-motev survival ol myeloid prccursors. as PML has reccntly hecn shown to be involved in apoptoiic pathuays <39.40). Patients with the PML-RARA. NPM-RARA and NuMa-RARA cene fusion>

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