FLI1, friend leukemia insertion 1

FLI1 is an ETS family member located close to ETS1 gene on chromosome 11. FLI1 was originally identified as a proto-oncogene as a site for retroviral integration of Friend virus-induced erytroleukemias.

The FLI1 gene encodes two isoforms of 51 (canonical transcript) and 48 kDa through the use of two alternative and highly conserved in-frame initiation codons, AUG +1 (p51) and AUG +100 (p48) (Fig.9) [134]. Both isoforms differ in their N-terminal end but retain the same functional domains and activity [134]. Two different promoters regulate the transcription of the two splice variants but several transcription factors binding sites, such as sites recognized by CREB, E2A, Oct-3, Sp-1 and c-Myc, are maintained inside both promoter sequence. The promoter encoding for the 48 kDa isoform of FLI1 has very strong transcriptional activity compared to the canonical FLI1 isoform [358].

Moreover, both FLI1 isoforms are cleaved by caspase 3 [359]. In fact, FLI1 has three different and conserved aspartate residues (D20, D155 and D209) involve in the caspase 3 cleavage activity, but maybe the D155 residue is not cleaved in vivo [359]. Three

34

different products are generated by the longer isoform, while only two products arise from the shorter one and this is because the first site of cleavage is inside the N-terminal end specific for the 51 KDa FLI1 isoform [359]. This regulation suggests that FLI1 is a potent anti-apoptotic agent and that its cleavage is functionally important in vivo.

FLI1 functional domains include the pointed domain and a FLI1-specific region (FLS) referred to as the amino terminal transcriptional activation (ATA) domain in the amino terminal half of the protein, an ETS domain and finally a carboxy terminal transcriptional activation (CTA) domain (Fig.9).

The ETS domain is responsible for DNA binding activity having the winged helix structure typical of ETS family members and recognizes the specific sequence ACCGGAAG/AT/C.

The CTA domain is involved in transcriptional activation and protein-protein interaction such as ATA domain but it can simultaneously acts as a transcriptional activator and repressor, while ATA domain is used only for transcriptional activation. The ETS domain contains sequences of secondary structures like helix-loop-helix (H-L-H) and is homologous to the DBD domain of ETS1. Helix-loop-helix structures are also identified in the ATA domain. The FLS and CTA domains contain sequences, which resemble turn-loop-turn (T-L-T) secondary structures. All these structures suggest that FLI1 acts as a transcriptional regulator interacting with others proteins.

FLI1 is well known as an oncogene and is expressed transiently during embryogenesis, while in adult is expressed highly in hematopoietic tissue and endothelial cells with lower levels detect in lung, heart and ovaries [135]. FLI1 is known to take part in vasculogenesis, differentiation of megakaryocytes, promotes cell cycle and inhibits apoptosis [114]. FLI1 is able to down-regulate Rb protein expression leading to the transition of cells through the S phase [136], while its role in apoptotic inhibition correlates with up-regulation of BCL2 expression [137]. FLI1 roles in vasculogenesis and in inhibition of collagen biosynthesis have an impact in systemic sclerosis or scleroderma (SSc), in which reduced levels of FLI1 in endothelial cells may play a critical role in the development of SSc vasculopathy. FLI1 has also been reported to inhibit erythroid differentiation. Together with PU.1, another ETS family member, FLI1 may inhibit erythroid differentiation through functional interference between these ETS family proteins and nuclear hormone receptors [138].

FLI1 gene is rearranged in 95% of Ewing’s sarcoma (ES), a pediatric tumor with neuroectodermal origin [139]. The translocation t(22;11) occurs between the central exons of EWSR1 on chromosome 22 and to the central exons of FLI1 creating a fusion protein acting as a transcriptional activator that recognizes the EBS specific for FLI1. The fusion proteins possess increased transactivation potential in comparison with the wild type FLI1 and this activity is thought to contribute to malignant transformation of the cells

35

[140]. Indeed, the fusion protein, which shows stronger transactivation potential than the former, is often associated with poor prognosis [141]. Overexpression of EWS-FLI1, but not wild type FLI1, transforms NIH/3T3 cells. The expression of EWS-FLI1 leads to a strong up-regulation of the c-myc oncogene [142]. Tumorigenenic role of EWS-FLI1 fusion protein is also promoted by the inhibition of p53 activity. EWS-FLI1 fusion protein, in fact, inhibits the p300-mediated acetylation of p53 at Lys-382 suppressing its transcriptional activity and enhancing MDM2-mediated p53 degradation [143].

Figure 2: FLI1 splice isoforms with functional domains. The full length of FLI1 proteins (p51) consist of 452 amino acids which contain the following domains: ATA: amino-terminal transcriptional activation domain, FLS: FLI1 specific domain, CTA: carboxy-terminal transcriptional activation domain. The functional domains are the same in the shorter FLI1 isoform p48. D20, D155 and D209, aspartate residues.

FLI1 has also an important role in the regulation of autoimmunity. Overexpression of FLI1 protein in transgenic mice results in the development of a lupus-like disease, including hypergammaglobulinemia, splenomegaly, B cell peripheral lymphocytosis, progressive immune complex-mediated renal disease and ultimately premature death from renal failure [144]. Reduced expression of the FLI1 protein in MRL/lpr mice, a murine model of lupus, significantly increases survival and decreases renal disease compared with wild type [145]. In addition, MRL/lpr mice also had decreased total serum Ig. FLI1 expression correlates with patients disease activity, meaning patients with highest disease activity express the highest FLI1 levels and vice versa [146].

In B cells, FLI1 has been reported to coregulate Igα expression with PAX5. Mice with reduced levels of FLI1 have reduced Igα expression and this reduction may contribute to a decreased BCR signaling and concomitant decreased number of follicular B cells and increased number of marginal zone B cells [147]. Reduced mRNA levels of Igα after FLI1 decreased expression is accompanied by a reduction in E2A and ERG1 expression and an increased in Id1 and Id2 expression, all known to be regulators of B cell development.

Proliferation of B cells is reduced although intracellular Ca²⁺ flux in B cells from mice with

36

reduce quantities of FLI1 is similar to that of wild type controls after anti-IgM stimulation.

Finally, immune responses and in vitro class switch recombination are altered in FLI1-deficient mice. Taken together, these studies suggest that FLI1, such as ETS1, plays an important role in the immune system including B cell.