AF10-dependent transcription is enhanced by its interaction with FLRG.

BACKGROUND INFORMATION: FLRG (follistatin-related gene) is a secreted glycoprotein which is very similar to follistatin. As observed for follistatin, FLRG is involved in the regulation of various biological processes through its binding to members of the TGFbeta (transforming growth factor beta) superfamily, activin, BMPs (bone morphogenetic proteins) and myostatin. Unlike follistatin, FLRG has been found to be both secreted and localized within the nucleus of many FLRG-producing cells, suggesting the existence of specific intracellular functions of the protein. RESULTS: In order to analyse the function of the nuclear form of FLRG, we performed a yeast two-hybrid screen, in which we identified AF10 [ALL1 (acute lymphoblastic leukaemia) fused gene from chromosome 10], a translocation partner of the MLL (mixed-lineage leukaemia) oncogene in human leukaemia, as a FLRG-interacting protein. This interaction was confirmed by far-Western-blot analysis and co-immunoprecipitation with transfected COS-7 cells. The N-terminal region of AF10, including the PHD (plant homeodomain), is sufficient to mediate this interaction, and has been shown to be involved in AF10 homo-oligomerization. By immunoprecipitation experiments, we showed that FLRG enhances the homo-oligomerization of AF10. Functional studies demonstrated that FLRG enhances the transactivation properties of the AF10 protein fused to Gal4 DNA-binding domains in transient transfection assays. CONCLUSIONS: Our present study provides novel insights into the function of the nuclear form of the FLRG protein, which is revealed as a novel regulator of transcription. The nuclear isoform of FLRG lacks an intrinsic transactivation domain, but enhances AF10-mediated transcription, probably through promoting the homo-oligomerization of AF10, thus facilitating the recruitment of co-activators.

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin.

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

AF4p12, a human homologue to the furry gene of Drosophila, as a novel MLL fusion partner.

More than 35 different partner genes with the mixed lineage leukemia (MLL) gene have been cloned from leukemia cells with translocations involving chromosome 11 band q23. In this study, we report on a novel fusion partner of the MLL gene, AF4p12, which we have identified as the human homologue to the furry gene of Drosophila. AF4p12, highly conserved in evolution, encodes a large protein of 3,105 amino acids. The expression of AF4p12 has been preferentially detected in colon, placenta, and brain tissues and in tumor cells of lymphoid origin. We show that the t(4;11)(p12;q23) translocation results in the creation of a chimeric RNA encoding a putative fusion protein containing 1,362 amino acids from the NH2-terminal part of MLL and 712 amino acids from the COOH-terminal part of AF4p12. FLT3 and HOXA9 genes are overexpressed in this leukemia. We found that the COOH-terminal part of AF4p12 fused to MLL contains a leucine zipper motif and exhibits transcriptional activation properties when fused to Gal4 DNA-binding domains in transient transfection assays. The AF4p12 fragment fused to MLL may contribute to the oncogenic activation of MLL, possibly through specific recruitment of the transcriptional machinery.

FLRG, a new ADAM12-associated protein, modulates osteoclast differentiation.

BACKGROUND INFORMATION: FLRG (follistatin-related gene) is a secreted glycoprotein that is highly homologous with follistatin. These proteins are involved in the regulation of various biological effects mediated by their binding to TGF-beta (transforming growth factor-beta) superfamily members, activin A and bone morphogenetic proteins. To characterize further the function of FLRG, we used a yeast two-hybrid screen to look for other possible functional partners. RESULTS: We report a direct interaction between the cysteine-rich domain of FLRG and ADAM12 (a disintegrin and metalloprotease 12). ADAMs are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Several studies have reported that ADAM12 protein, as well as activin A, are important regulators of osteoclast differentiation. We observed that the expressions of ADAM12 and activin A are modulated during osteoclast formation, whereas the FLRG expression seemed to remain quite constant. We showed that the FLRG protein inhibits osteoclast differentiation from murine primary spleen cells and macrophage RAW264.7 cells cultured in the presence of RANK-L (receptor activator of nuclear factor kappaB ligand) and M-CSF (macrophage colony-stimulating factor). Addition of FLRG protein to precursors significantly reduces the number of osteoclasts, as well as the average number of nuclei in each osteoclast. CONCLUSIONS: Our study indicates that the FLRG protein may contribute to bone formation by inhibiting osteoclast differentiation.