Oligomerization of THAP9 Transposase via Amino-Terminal Domains.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

Comparative analysis of ChIP-exo peak-callers: impact of data quality, read duplication and binding subtypes.

BACKGROUND: ChIP (Chromatin immunoprecipitation)-exo has emerged as an important and versatile improvement over conventional ChIP-seq as it reduces the level of noise, maps the transcription factor (TF) binding location in a very precise manner, upto single base-pair resolution, and enables binding mode prediction. Availability of numerous peak-callers for analyzing ChIP-exo reads has motivated the need to assess their performance and report which tool executes reasonably well for the task. RESULTS: This study has focussed on comparing peak-callers that report direct binding events with those that report indirect binding events. The effect of strandedness of reads and duplication of data on the performance of peak-callers has been investigated. The number of peaks reported by each peak-caller is compared followed by a comparison of the annotated motifs present in the reported peaks. The significance of peaks is assessed based on the presence of a motif in top peaks. Indirect binding tools have been compared on the basis of their ability to identify annotated motifs and predict mode of protein-DNA interaction. CONCLUSION: By studying the output of the peak-callers investigated in this study, it is concluded that the tools that use self-learning algorithms, i.e. the tools that estimate all the essential parameters from the aligned reads, perform better than the algorithms which require formation of peak-pairs. The latest tools that account for indirect binding of TFs appear to be an upgrade over the available tools, as they are able to reveal valuable information about the mode of binding in addition to direct binding. Furthermore, the quality of ChIP-exo reads have important consequences on the output of data analysis.

Classification of the human THAP protein family identifies an evolutionarily conserved coiled coil region.

BACKGROUND: The THAP (Thanatos Associated Proteins) protein family in humans is implicated in various important cellular processes like epigenetic regulation, maintenance of pluripotency, transposition and disorders like cancers and hemophilia. The human THAP protein family which consists of twelve members of different lengths has a well characterized amino terminal, zinc-coordinating, DNA-binding domain called the THAP domain. However, the carboxy terminus of most THAP proteins is yet to be structurally characterized. A coiled coil region is known to help in protein oligomerization in THAP1 and THAP11. It is not known if other human THAP proteins oligomerize. We have used bioinformatic tools to explore the possibility of dimerization of THAP proteins via a coiled coil region. RESULTS: Classification of human THAP protein into three size based groups led to the identification of an evolutionarily conserved alpha helical region, downstream of the amino terminal THAP domain. Secondary structure predictions, alpha helical wheel plots and protein models demonstrated the strong possibility of coiled coil formation in this conserved, leucine rich region of all THAP proteins except THAP10. CONCLUSIONS: The identification of a predicted oligomerization region in the human THAP protein family opens new directions to investigate the members of this protein family.

Correction to: Classification of the human THAP protein family identifies an evolutionarily conserved coiled coil region.

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Bioinformatic analysis of THAP9 transposase homolog: conserved regions, novel motifs.

THAP9 is a transposable element-derived gene that encodes the THAP9 protein, which is homologous to the Drosophila P-element transposase (DmTNP) and can cut and paste DNA. However, the exact functional role of THAP9 is unknown. Here, we perform structure prediction, evolutionary analysis and extensive in silico characterization of THAP9, including predicting domains and putative post-translational modification sites. Comparison of the AlphaFold-predicted structure of THAP9 with the DmTNP CryoEM structure, provided insights about the C2CH motif and other DNA binding residues, RNase H-like catalytic domain and insertion domain of the THAP9 protein. We also predicted previously unreported mammalian-specific post-translational modification sites that may play a role in the subcellular localization of THAP9. Furthermore, we observed that there are distinct organism class-specific conservation patterns of key functional residues in certain THAP9 domains.

Pan-Cancer Analysis Reveals the Prognostic Potential of the THAP9/THAP9-AS1 Sense-Antisense Gene Pair in Human Cancers.

Human THAP9, which encodes a domesticated transposase of unknown function, and lncRNA THAP9-AS1 (THAP9-antisense1) are arranged head-to-head on opposite DNA strands, forming a sense and antisense gene pair. We predict that there is a bidirectional promoter that potentially regulates the expression of THAP9 and THAP9-AS1. Although both THAP9 and THAP9-AS1 are reported to be involved in various cancers, their correlative roles on each other's expression has not been explored. We analyzed the expression levels, prognosis, and predicted biological functions of the two genes across different cancer datasets (TCGA, GTEx). We observed that although the expression levels of the two genes, THAP9 and THAP9-AS1, varied in different tumors, the expression of the gene pair was strongly correlated with patient prognosis; higher expression of the gene pair was usually linked to poor overall and disease-free survival. Thus, THAP9 and THAP9-AS1 may serve as potential clinical biomarkers of tumor prognosis. Further, we performed a gene co-expression analysis (using WGCNA) followed by a differential gene correlation analysis (DGCA) across 22 cancers to identify genes that share the expression pattern of THAP9 and THAP9-AS1. Interestingly, in both normal and cancer samples, THAP9 and THAP9-AS1 often co-express; moreover, their expression is positively correlated in each cancer type, suggesting the coordinated regulation of this H2H gene pair.

THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain.

The catalytic domain of most 'cut and paste' DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp-DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

P Transposable Elements in Drosophila and other Eukaryotic Organisms.

P transposable elements were discovered in Drosophila as the causative agents of a syndrome of genetic traits called hybrid dysgenesis. Hybrid dysgenesis exhibits a unique pattern of maternal inheritance linked to the germline-specific small RNA piwi-interacting (piRNA) pathway. The use of P transposable elements as vectors for gene transfer and as genetic tools revolutionized the field of Drosophila molecular genetics. P element transposons have served as a useful model to investigate mechanisms of cut-and-paste transposition in eukaryotes. Biochemical studies have revealed new and unexpected insights into how eukaryotic DNA-based transposons are mobilized. For example, the P element transposase makes unusual 17nt-3' extended double-strand DNA breaks at the transposon termini and uses guanosine triphosphate (GTP) as a cofactor to promote synapsis of the two transposon ends early in the transposition pathway. The N-terminal DNA binding domain of the P element transposase, called a THAP domain, contains a C2CH zinc-coordinating motif and is the founding member of a large family of animal-specific site-specific DNA binding proteins. Over the past decade genome sequencing efforts have revealed the presence of P element-like transposable elements or P element transposase-like genes (called THAP9) in many eukaryotic genomes, including vertebrates, such as primates including humans, zebrafish and Xenopus, as well as the human parasite Trichomonas vaginalis, the sea squirt Ciona, sea urchin and hydra. Surprisingly, the human and zebrafish P element transposase-related THAP9 genes promote transposition of the Drosophila P element transposon DNA in human and Drosophila cells, indicating that the THAP9 genes encode active P element "transposase" proteins.

The human THAP9 gene encodes an active P-element DNA transposase.

The human genome contains ~50 genes that were derived from transposable elements or transposons, and many are now integral components of cellular gene expression programs. The human THAP9 gene is related to the Drosophila P-element transposase. Here, we show that human THAP9 can mobilize Drosophila P-elements in both Drosophila and human cells. Chimeric proteins formed between the Drosophila P-element transposase N-terminal THAP DNA binding domain and the C-terminal regions of human THAP9 can also mobilize Drosophila P elements. Our results indicate that human THAP9 is an active DNA transposase that, although "domesticated," still retains the catalytic activity to mobilize P transposable elements across species.

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins.

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins.

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.