The THAP domain of THAP1 is a large C2CH module with zinc-dependent sequence-specific DNA-binding activity

Abstract

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-...

Structure-Function Analysis of the THAP Zinc Finger of THAP1, a Large C2CH DNA-binding Module Linked to Rb/E2F Pathways

“…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 typically recognized by classical C2H2 zinc fingers (2,7).…”

“…These data provided the first links in mammals between THAP proteins, cell proliferation, and pRB/E2F cell cycle pathways and complemented genetic data previously obtained in model animal organisms. Indeed, in zebra fish and other fish species, the ortholog of cell cycle transcription factor E2F6, a repressor of E2F-dependent transcription during S phase (11) was found to contain a THAP zinc finger at its N terminus (7). In the nematode Caenorhabditis elegans, five distinct THAP zinc finger proteins (LIN-36, LIN-15B, LIN-15A, HIM-17, and GON-14) (7) were shown to interact genetically with LIN-35/ Rb, the sole C. elegans retinoblastoma homolog (12)(13)(14)(15)(16).…”

“…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 typically recognized by classical C2H2 zinc fingers (2,7). Interestingly, the consensus C2CH signature of the THAP domain was identified in the sequence-specific DNA-binding domain of Drosophila P element transposase, suggesting the THAP zinc finger constitutes a novel example of a DNA-binding domain shared between cellular proteins and transposons from mobile genomic parasites (6,9).…”

Alternative Approaches to Modeling Hereditary Dystonias

“…This technique may also require a priori selection of infusion sites, and as such it is also a biased approach. Unfortunately, some of the most common dystonia genes code for proteins that function in a variety of processes important for cell signaling, stability, and maintenance [48,[77][78][79][80]. In these cases, finding a compound that inhibits a specific function of the protein of interest is likely to be difficult or even impossible.…”

Nitrophorins and nitrobindins: structure and function

“…THAP4 consists of a N-terminal modified zinc finger domain, which has been proposed to bind DNA and of a C-terminus At-Nb-like domain, which could act as a NO sensor (32,113,114). It has been hypothesized that NO-binding to the C-terminal domain modulate the DNA binding activity of the N-terminal region, acting as an NO-dependent transcriptional regulator (31).…”