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The THAP Domain of THAP1 Is a Large C2CH Module with Zinc-Dependent Sequence-Specific DNA-Binding Activity

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The THAP Domain of THAP1 Is a Large C2CH Module with Zinc-Dependent Sequence-Specific DNA-Binding Activity The THAP domain of THAP1 is a large C2CH module with zinc-dependent sequence-specific DNA-binding activity Thomas Clouaire*†, Myriam Roussigne*‡, Vincent Ecochard*‡, Catherine Mathe*, Franc¸ois Amalric*, and Jean-Philippe Girard*§ *Laboratoire de Biologie Vasculaire, Equipe Labellise´e ‘‘La Ligue 2003,’’ Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique Unite´Mixte de Recherche 5089, 205 Route de Narbonne, 31077 Toulouse, France; and †Endocube, Prologue Biotech, Rue Pierre et Marie Curie, BP700 31319 Labe`ge Cedex, France Edited by H. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA, and approved March 23, 2005 (received for review September 16, 2004) We have recently described an evolutionarily conserved protein (6), and 9 other human proteins (1). Both THAP1 and DAP4͞ motif, designated the THAP domain, which defines a previously THAP0 appear to function in nuclear apoptotic pathways. THAP1 uncharacterized family of cellular factors (THAP proteins). The interacts and colocalizes within promyelocytic leukemia nuclear THAP domain exhibits similarities to the site-specific DNA-binding bodies with the proapoptotic leucine-zipper protein Par-4 and domain of Drosophila P element transposase, including a putative potentiates both serum withdrawal- and TNF␣-induced apoptosis metal-coordinating C2CH signature (CX2–4CX35–53CX2H). In this ar- (2). DAP4͞THAP0 was initially identified in a screen for genes ticle, we report a comprehensive list of Ϸ100 distinct THAP proteins involved in IFN␥-induced apoptosis in HeLa cells (4) and, more in model animal organisms, including human nuclear proapoptotic recently, as a nuclear partner of MST1 (5), a proapoptotic kinase factors THAP1 and DAP4͞THAP0, transcriptional repressor THAP7, that phosphorylates histone H2B during apoptosis (7). Recently, zebrafish orthologue of cell cycle regulator E2F6, and Caenorhab- THAP7 has been shown to function as a transcriptional repressor ditis elegans chromatin-associated protein HIM-17 and cell-cycle that binds to hypoacetylated histone H4 tails and may also induce regulators LIN-36 and LIN-15B. In addition, we demonstrate the the hypoacetylation of histone H3 by recruiting corepressor NcoR biochemical function of the THAP domain as a zinc-dependent and histone deacetylase HDAC3 to chromatin (6). sequence-specific DNA-binding domain belonging to the zinc- Although orthologous relationships with the human THAP finger superfamily. In vitro binding-site selection allowed us to proteins were not obvious, analysis of the D. melanogaster (1) and identify an 11-nucleotide consensus DNA-binding sequence spe- Caenorhabditis elegans (8) THAP families revealed several inter- cifically recognized by the THAP domain of human THAP1. Muta- esting features. Two of the predicted Drosophila THAP proteins tions of single nucleotide positions in this sequence abrogated were found to contain more than one THAP domain, the double- THAP-domain binding. Experiments with the zinc chelator 1,10-o- THAP protein CG14860 and the multi-THAP protein CG10631, phenanthroline revealed that the THAP domain is a zinc-dependent which is predicted to contain 27 THAP domains occurring as DNA-binding domain. Site-directed mutagenesis of single cysteine internal repeats (1). A third multi-THAP protein, designated or histidine residues supported a role for the C2CH motif in zinc HIM-17, has recently been described in C. elegans and plays a coordination and DNA-binding activity. The four other conserved critical role in chromosome segregation during meiosis by linking residues (P, W, F, and P), which define the THAP consensus chromatin modification and competence for initiation of meiotic sequence, were also found to be required for DNA binding. recombination by double-strand breaks (8). A substantial fraction Together with previous genetic data obtained in C. elegans, our of HIM-17 was found to comprise six internal repeats of the THAP results suggest that cellular THAP proteins may function as zinc- domain (Fig. 1), including two divergent C2CH modules lacking the GENETICS dependent sequence-specific DNA-binding factors with roles in invariant W, F, or P residues. Similar divergent or consensus THAP proliferation, apoptosis, cell cycle, chromosome segregation, chro- domains were also identified in other C. elegans proteins (8), matin modification, and transcriptional regulation. including LIN-36, LIN-15A, and LIN-15B, three proteins initially characterized for their role in vulval development (9–12). Remark- protein motif ͉ zinc finger ͉ Caenorhabditis elegans ͉ cell cycle ably, LIN-36, LIN-15A, LIN-15B, and HIM-17 have all been found to interact genetically with LIN-35͞Rb, the sole C. elegans retino- e have recently described an evolutionarily conserved Ϸ90- blastoma homolog (8, 9, 12, 13). In addition, LIN-36 and LIN-15B Wresidue protein motif, designated the THAP domain, which have been found to function as inhibitors of the G1-to-S-phase defines a previously uncharacterized family of cellular factors, the cell-cycle transition (13), and LIN-36 has also been shown to THAP proteins (1, 2). This motif is characterized by a putative function redundantly with FZR-1, the C. elegans homolog of APC metal-coordinating C2CH module (CX2–4CX35–53CX2H) and four regulator Cdh1, in the global control of cell proliferation (14). additional invariant residues, P26, W36, F58, and P78, in human Although the THAP motif has been well defined and several THAP1 (Fig. 1). The THAP domain was found to be restricted to THAP proteins have been functionally characterized, the bio- animals and is present in both vertebrates (from zebrafish to chemical role of the THAP domain in cellular THAP proteins humans) and invertebrates (e.g., fly and worm) (1). Interestingly, has not yet been described. In this study, using the THAP the THAP-motif signature was identified (1) in the site-specific domain of human THAP1 as a prototype, we show that the DNA-binding domain of Drosophila melanogaster P element trans- THAP domain is a zinc-dependent sequence-specific DNA- posase (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 This paper was submitted directly (Track II) to the PNAS office. and that the THAP proteins may correspond to a previously Abbreviations: RRL, rabbit reticulocyte lysate; SELEX, systematic evolution of ligands by uncharacterized family of cellular DNA-binding proteins (1). exponential enrichment; THABS, THAP-domain-binding sequence. In humans, the THAP family comprises 12 distinct members, ‡M.R. and V.E. contributed equally to this work. including nuclear proapoptotic factor THAP1(2),death-associated §To whom correspondence should be addressed. E-mail: [email protected]. protein DAP4͞THAP0 (4, 5), transcriptional repressor THAP7 © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406882102 PNAS ͉ May 10, 2005 ͉ vol. 102 ͉ no. 19 ͉ 6907–6912 Downloaded by guest on September 27, 2021 Fig. 1. THAP proteins in model animal organisms. (A) Identification of a consensus THAP domain in the zebrafish orthologue of cell-cycle transcription factor E2F6. Shown is CLUSTALW multiple alignment of zebrafish E2F6 with human E2F6 and the THAP domain of human THAP1. Conserved residues are boxed. Black boxes indicate identical residues, whereas boxes shaded in gray show similar amino acids. The consensus THAP motif, defined by the C2CH signature and four other invariant residues (P26, W36, F58, and P78 in human THAP1), is shown above the alignment. (B) Primary structures of C. elegans THAP proteins with known functions. THAP domains are shown in black. Divergent THAP domains containing the C2CH signature are shown in gray. Known protein motifs are indicated. The transcriptional-corepressor function of CtBP has not yet been confirmed in C. elegans. binding module, and we demonstrate that its DNA-binding Systematic Evolution of Ligands by Exponential Enrichment (SELEX) activity absolutely requires the C2CH signature and the four Assay. DNA-binding specificity of the THAP domain from other conserved residues (P, W, F, and P) of the THAP motif. human THAP1 was determined by SELEX, essentially as de- scribed in ref. 15. The following 62-bp oligonucleotide was Materials and Methods synthesized: 5Ј-TGGGCACTATTTATATCAACN25AATGT- Plasmid Constructions. The THAP domain of human THAP1 CGTTGGTGGC CC-3Ј (where N is any nucleotide) along with (amino acids 1–90) was amplified by PCR using as a template primers complementary to each end, F-HindIII 5Ј-ACCG- pGADT7-THAP1 (2) with primers 5Ј-GCGCATATGGTG- CAAGCTTGGGCACTATTTATATCAAC-3Ј and R-XbaI 5Ј- CAGTCCTGCTCCGCCTACGGC-3Ј and 5Ј-GCGCTC- GGTCTAGAGGGCCACCAACGCATT-3Ј. A pool of double- GAGTTTCTTGTCATGTGGCTCAGTACAAAG-3Ј. The stranded 80-bp degenerate oligonucleotides was amplified by PCR product was digested with NdeI and XhoI and cloned PCR using the F-HindIII and R-XbaI primers. Recombinant in-frame with a carboxyl-terminal His tag into plasmid pET-21c THAP domain (Ϸ250 ng) was incubated with Ni-NTA magnetic (Novagen). Similarly, the THAP1 ORF was amplified by PCR beads (Qiagen) in NT2 buffer [20 mM Tris⅐HCl (pH 7.5)͞100 to generate pCDNA3-THAP1 or pCDNA3.1-THAP1 mM NaCl͞0.05% Nonidet P-40] for 30 min at 4°C, and the beads (THAP1-Myc). The THAP1-C5A, THAP1-C10A, THAP1- were washed twice with 500 ␮l of NT2 buffer. The immobilized C54A, THAP1-H57A, THAP1-P26A, THAP1-W36A, THAP domain was incubated with the random pool of oligo- THAP1-F58A, and THAP1-P78A single-point mutants were nucleotides (2–5 ␮g) in 100 ␮l of binding buffer [20 mM Tris⅐HCl obtained by PCR using specific primers containing the corre- (pH 7.50)͞100 mM NaCl͞0.05% Nonidet P-40͞0.5 mM EDTA͞ sponding mutations and cloned as EcoRI–XbaI fragments in 100 ␮g/ml BSA͞20–50 ␮g of poly(dI-dC)] for 10 min at room pCDNA3 expression vector. temperature. The beads were then washed six times with 500 ␮l of NT2 buffer, the protein–DNA complexes were extracted with Protein Expression and Purification.
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