Zinc fingers as protein recognition motifs: Structural basis for the GATA-1/Friend of GATA interaction

Abstract

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1͞FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.transcription ͉ factor ͉ gene expression T he erythroid transcription factor GATA-1 has become a paradigm for understanding the control of gene expression by sequence-specific DNA-binding proteins. Numerous studies (reviewed in refs. 1 and 2) have revealed that the function of GATA-1 depends on its ability to bind both DNA and other proteins such as the DNA-binding proteins PU.1, Fli-1 Sp1, and erythroid Krüppel-like factor, as well as coregulators such as LIM-only 2 protein, cyclic AMP response element-binding protein-binding protein, and the zinc-finger protein friend of GATA (FOG). It is thought that different partnerships involving GATA-1 occur at different DNA elements, in different cell types, and at distinct stages of development, and that these regulatory networks contribute to the ability of GATA-1 to direct the complex patterns of gene expression required for the differentiation of blood cells.GATA-family proteins contain two highly conserved trebleclef zinc fingers (ZnFs) domains (3): the N-terminal (NF) and the C-terminal (CF) fingers. These domains mediate both DNA and protein interactions, but despite high sequence similarity to each other (Ͼ50% identity), have quite distinct functions. The CF appears to be the main determinant of DNA-binding and recognizes sequences of the form T͞AGATAA͞G, whereas the NF can participate in binding to tandem T͞AGATAA͞G sites (ref. 4 and references therein) and can independently bind GATC motifs (5). The protein-binding specificities of the two ZnFs also differ; most notably, the NF alone is able to recruit FOG, an Ϸ1,000-aa protein (6) that contains five classic CysCys-His-His (C 2 H 2 ) and four related Cys-Cys-His-Cys (C 2 HC) ZnFs (Fig. 1A).FOG is essential for the normal development of both erythrocytes and megakaryocytes (7) and the interaction of FOG with GATA-1 is indispensable for these events (8). Of the nine ZnFs in FOG (Fig. 1A), four are not found in the tandem arrays that are generally associated with DNA-binding activity. Intriguingly, each of these four isolated domains can independently bind NF, consistent with the emerging view that single fingers can mediate protein-protein interactions (9, 10).Here, we describe the solution structure of...

Tumor necrosis factor alpha-mediated inhibition of erythropoiesis involves GATA-1/GATA-2 balance impairment and PU.1 over-expression

“…Page 18 of 38 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 -18 -and inhibiting its binding to the DNA of erythro-specific genes [46,49,57]. We show in this study that TNF induced increased expression of the myeloid and lymphoid transcription factor, PU.1 [58,59].…”

“…Moreover, Ikonomi et al reported that GATA-2 over-expression induced the arrest of erythroid differentiation in K562 cells [53]. However, it has also been reported that over-expression of GATA1 in erythroid cells, both in vitro, and in -18 -and inhibiting its binding to the DNA of erythro-specific genes [46,49,57]. We show in this study that TNF induced increased expression of the myeloid and lymphoid transcription factor, PU.1 [58,59].…”

“…PU.1 and GATA-1 are described as physically interacting with each other, which results in the inhibition of GATA-1 transactivation and, therefore, the down-regulation of erythroid specific genes [49]. To assess the possible capacity of TNF to affect GATA-1 activity via PU.1 over-expression, the interaction between GATA-1 and PU.1 was studied by immunoprecipitation assay in HSPCs that had undergone EPO-mediated differentiation in the presence or absence of TNF.…”

CTCF terminal segments are unstructured

“…37 Moreover, the classical zinc fingers of CTCF bind DNA, 1,38 but these domains in other transcription factors also bind proteins. 39,40 We now know that CTCF predominantly consists of zinc fingers and unstructured polypeptides, so we surmise that perhaps the entire protein functions as a scaffold or adaptor. Extreme speculation could even envisage recruitment regulated by post-translational modifications similar to that observed with histone tails or the RNA polymerase II C-terminal extension.…”

Zinc fingers are known as domains for binding DNA and RNA. Do they also mediate protein-protein interactions?

“…It is the N-terminal ZnF from GATA-1 that mediates the interaction with FOG-1 described above, and in fact this domain is able to bind simultaneously to FOG-1 and to DNA, thus illustrating the versatility of these domains (7,18).…”