A family with recessive X-linked thrombocytopenia affecting 4 males in 2 generations, characterized by macrothrombocytopenia, profound bleeding, and mild dyserythropoiesis, is described. Microsatellite linkage analysis identified a region of the X chromosome including the GATA-1 gene, which encodes a critical transcription factor involved in erythrocyte and megakaryocyte development. By sequencing the entire coding region of GATA-1, a 2-base mutation was detected that results in a single amino acid substitution (glycine 208 to serine) within a highly conserved portion of the N-terminal zinc finger domain. Restriction fragment length polymorphism confirmed that this novel mutation segregated with the affected males and female carrier. Although not required for DNA binding, Gly208 of GATA-1 is involved in direct interaction with Friend of GATA-1 (FOG), a cofactor required for normal megakaryocytic and erythroid development. These results demonstrate that the GATA-1-FOG interaction is partially disrupted by the mutation and that the greatest effect involves contact with the FOG zinc finger 9. These findings help describe a novel mutation of GATA-1 in humans as a cause of X-linked thrombocytopenia, and they confirm the vital role played by this transcription factor during in vivo megakaryocyte development. (Blood. 2001;98:2681-2688)

Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIb␣ in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIb␣ genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of different transcriptional complexes consisting of cell-specific and ubiquitous transcription factors (24,30). This process is arguably best exemplified in the hematopoietic system, where different transcriptional complexes control the production of distinct cellular lineages from a common hematopoietic stem cell precursor. Among the rarest of the mature hematopoietic cells are megakaryocytes, large polyploid cells that reside in the bone marrow and whose cytoplasmic fragments are extruded into the bloodstream to form platelets.The key transcription factors involved in megakaryocyte differentiation are coming to light (for a review, see Shivdasani [28]). One of these, Friend leukemia integration 1 (Fli-1), is a member of the Ets family of transcription factors. Ets factors encompass a family of over 40 members that are characterized by an 85-amino-acid region of homology termed the Ets domain, which mediates binding to the core Ets recognition element 5Ј-GGA(A/T)-3Ј (36;

The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

Two out of 47 patients with sporadic tetralogy of Fallot (TOF), the most common cyanotic conotruncal heart defect (CTD), showed heterozygous missense mutations of the ZFPM2/FOG2 gene. Knockout mice carrying mutations in the ZFPM2/FOG2 gene have similarly been found to exhibit TOF. While both mutant ZFPM2/FOG2 proteins, E30G (c.88A>G) and S657G (c.1968A>G), retain the ability to bind the partner protein GATA4 and repress GATA4 mediated gene activation, the S657G, but not the E30G, mutation is subtly impaired in this function. ZFPM2/FOG2 gene mutations may contribute to some sporadic cases of TOF.

Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.

CZ415, a potent ATP-competitive mTOR inhibitor with unprecedented selectivity over any other kinase is described. In addition to a comprehensive characterization of its activities in vitro, in vitro ADME, and in vivo pharmacokinetic data are reported. The suitability of this inhibitor for studying in vivo mTOR biology is demonstrated in a mechanistic mouse model monitoring mTOR proximal downstream phosphorylation signaling. Furthermore, the compound reported here is the first ATP-competitive mTOR inhibitor described to show efficacy in a semitherapeutic collagen induced arthritis (CIA) mouse model.

cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term "zinc bundles." Each bundle contains the sequence Cys-X 4 -Cys-X 8 -His-X 3 -Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X 2 -Cys-X 9 -His-X 3 -Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains.The regulation of transcription is coordinated primarily by sequence-specific DNA-binding proteins that recognize elements within the promoters and enhancers of their target genes. Although the mechanisms by which DNA-binding proteins activate or repress transcription are not completely understood, these proteins often work by recruiting coactivators such as cAMP-response element-binding protein-binding protein (CBP) 1 and p300 (1-3). CBP was first isolated as a coactivator that is recruited by the phosphorylated form of cAMP-response element-binding protein (1). It is now clear that CBP is capable of interacting with a wide array of other DNA-bound transcription factors and that it mediates or modulates the transcriptional activity of these proteins (4 -9). The molecular mode of action of CBP is complex. It has been shown that it possesses intrinsic histone acetylase activity and that it is able to recruit P/CAF, another acetyl transferase (10, 11). These results suggest that CBP is involved in the remodeling of chromatin, since it has been shown that the degree of histone acetylation significantly alters chromatin structure (12). Furthermore, CBP can interact directly with the basal transcriptional machinery (13, 14), underlining its central role as a transcriptional adaptor. The intracellular concentration of CBP is thought to be limiting, and it has consequently been proposed that CBP may be instrumental in coordinating different signaling pathways. Thus, for example, the inhibitory effects of nuclear hormone recepto...

Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.