The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.
Background: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.
The nuclear receptor COUP-TFII was recently shown to bind to the promoter of the ⑀-and ␥-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult ⑀ and ␥ expression. We show that, on the ⑀-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5 or the 3 COUP-TFII site but not to both sites simultaneously. The non-␣-globin genes are clustered in several species within a relatively small chromosomal region. The expression of these genes is precisely regulated both spatially and quantitatively during embryonic, fetal, and postnatal development to match the expression of ␣-globin genes, resulting in a perfectly balanced ␣/non-␣ synthetic ratio (1-3).In man, the predominant non-␣-globin chain during the embryonic period is ⑀-globin, which around the third month of gestation (embryonic-fetal switch) is replaced by ␥-globin (encoded by two non-allelic genes, G ␥-and A ␥-globin) and finally, around birth, by -globin (fetal-adult switch). DNA sequences regulating globin gene expression have been extensively investigated; in addition to the upstream locus control region, essential for the correct activity of all the genes in the cluster (4 -8), several promoter, enhancer, and other non-conventional elements have been described (9, 10).Despite the detailed knowledge of the DNA elements regulating globin expression, the mechanisms underlying the transition from embryonic (⑀) to fetal (␥) and then to adult () gene expression remain largely unclarified. In particular, no transcription factor has been detected whose activity during development varies in a way consistent with the changes observed in globin gene expression at the various stages; even erythroid Krü ppel-like protein, although necessary for -globin (11-14) but not ⑀-and ␥-globin expression, is present and active during the embryonic and early fetal stages, when -globin is not yet expressed (13).Some clues to the nature of the DNA sequences controlling the temporal expression of ␥-globin genes have come from inherited conditions usually observed in heterozygous individuals and known as hereditary persistence of fetal hemoglobin (HPFH) 1 (1-3). Such individuals present, postnatally, moderate or high levels of fetal hemoglobin (␣ 2 ␥ 2 ). Some HPFHs are caused by point mutations in either the G ␥ or A ␥-globin gene; the mutated gene is selectively overexpressed in adults. Six different mutations causing HPFH cluster around the double CCAAT box region and affect the binding of several proteins (3,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Among them, all of the four different HPFH mutations studied so far greatly diminish or abolish the binding of a protein called NF-E3 (23).These results suggested that NF-E3 might be a ␥-globin repressor and that the inability of the ␥-globin promoter to bind it ...
Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of beta-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.
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