We have recently described an evolutionarily conserved protein motif, designated the THAP domain, which defines a previously uncharacterized family of cellular factors (THAP proteins). The THAP domain exhibits similarities to the site-specific DNA-binding domain of Drosophila P element transposase, including a putative metal-coordinating C2CH signature (CX 2-4CX35-53CX2H). In this article, we report a comprehensive list of Ϸ100 distinct THAP proteins in model animal organisms, including human nuclear proapoptotic factors THAP1 and DAP4͞THAP0, transcriptional repressor THAP7, zebrafish orthologue of cell cycle regulator E2F6, and Caenorhabditis elegans chromatin-associated protein HIM-17 and cell-cycle regulators LIN-36 and LIN-15B. In addition, we demonstrate the biochemical function of the THAP domain as a zinc-dependent sequence-specific DNA-binding domain belonging to the zincfinger superfamily. In vitro binding-site selection allowed us to identify an 11-nucleotide consensus DNA-binding sequence specifically recognized by the THAP domain of human THAP1. Mutations of single nucleotide positions in this sequence abrogated THAP-domain binding. Experiments with the zinc chelator 1,10-ophenanthroline revealed that the THAP domain is a zinc-dependent DNA-binding domain. Site-directed mutagenesis of single cysteine or histidine residues supported a role for the C2CH motif in zinc coordination and DNA-binding activity. The four other conserved residues (P, W, F, and P), which define the THAP consensus sequence, were also found to be required for DNA binding. Together with previous genetic data obtained in C. elegans, our results suggest that cellular THAP proteins may function as zincdependent sequence-specific DNA-binding factors with roles in proliferation, apoptosis, cell cycle, chromosome segregation, chromatin modification, and transcriptional regulation.protein motif ͉ zinc finger ͉ Caenorhabditis elegans ͉ cell cycle W e have recently described an evolutionarily conserved Ϸ90-residue protein motif, designated the THAP domain, which defines a previously uncharacterized family of cellular factors, the THAP proteins (1, 2). This motif is characterized by a putative metal-coordinating C2CH module (CX 2-4 CX 35-53 CX 2 H) and four additional invariant residues, P26, W36, F58, and P78, in human THAP1 (Fig. 1). The THAP domain was found to be restricted to animals and is present in both vertebrates (from zebrafish to humans) and invertebrates (e.g., fly and worm) (1). Interestingly, the THAP-motif signature was identified (1) in the site-specific DNA-binding domain of Drosophila melanogaster P element transposase (3). This finding suggested that the THAP domain may constitute an example of a DNA-binding domain shared between cellular proteins and transposases from mobile genomic parasites and that the THAP proteins may correspond to a previously uncharacterized family of cellular DNA-binding proteins (1).In humans, the THAP family comprises 12 distinct members, including nuclear proapoptotic factor THAP1 (2), death-...
We recently cloned a novel human nuclear factor (designated THAP1) from postcapillary venule endothelial cells (ECs) that contains a DNA-binding THAP domain, shared with zebrafish E2F6 and several Caenorhabditis elegans proteins interacting genetically with retinoblastoma gene product (pRB). Here, we show that THAP1 is a physiologic regulator of EC proliferation and cell-cycle progression, 2 essential processes for angiogenesis. Retroviral-mediated gene transfer of THAP1 into primary human ECs inhibited proliferation, and large-scale expression profiling with microarrays revealed that THAP1-mediated growth inhibition is due to coordinated repression of pRB/E2F cell-cycle target genes. Silencing of endogenous THAP1 through RNA interference similarly inhibited EC proliferation and G1/S cell-cycle progression, and resulted in down-regulation of several pRB/E2F cellcycle target genes, including RRM1, a gene required for S-phase DNA synthesis. Chromatin immunoprecipitation assays in proliferating ECs showed that endogenous THAP1 associates in vivo with a consensus THAP1-binding site found in the RRM1 promoter, indicating that RRM1 is a direct transcriptional target of THAP1. The similar phenotypes observed after THAP1 overexpression and silencing suggest that an optimal range of THAP1 expression is essential for EC proliferation. Together, these data provide the first links in mammals among THAP proteins, cell proliferation, and pRB/ E2F cell-cycle pathways. (Blood. 2007; 109:584-594)
THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/ E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structurefunction relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of ϳ80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel -sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.Zinc finger proteins represent the most abundant class of DNA-binding proteins in the human genome. Zinc fingers have been defined as small, functional, independently folded domains that require coordination of a zinc atom to stabilize their structure (1). The zinc finger superfamily includes the C2H2-type zinc finger, a compact ϳ30-amino acid DNA-binding module repeated in multiple copies in the protein structure (2, 3), the C4-type zinc finger found in the GATA family of transcription factors (4), and the zinc-coordinating DNA-binding domain of nuclear hormone receptors (5). We recently described an atypical zinc finger motif, characterized by a large C2CH module (Cys-X 2-4 -Cys-X 35-53 -Cys-X 2 -His) with a spacing of up to 53 amino acids between the zinc-coordinating C2 and CH residues (6). This motif, designated THAP domain or THAP zinc finger, defines a previously uncharacterized large family of cellular factors with more than 100 distinct members in the animal kingdom (6, 7). We showed that the THAP domain of THAP1, the prototype of the THAP family (8), possesses zinc-dependent sequence-specific DNA binding activity and recognizes a consensus DNA target sequence of 11 nucleotides (THABS, for the THAP1 binding sequence) (7), considerably larger than the 3-4 nucleotides motif typ...
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