Transcriptional regulation of ribonucleotide reductase M1 by the DYT6 dystonia protein THAP1
Andres, Jason Michael
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Dystonia is the third most common neurological disorder after Parkinson's disease and essential tremor, and is characterized by involuntary contractions of agonist and antagonist muscles. DYT6 dystonia, a hereditary form of Primary Torsion Dystonia (PTD), is an autosomal dominant disorder with reduced penetrance, whose causative gene is Thanatos-associated [THAP] domain-containing Apoptosis-associated Protein 1, or THAP 1, that encodes one of twelve human THAP proteins distinguished by their novel DNA-binding domain, the THAP domain. THAP1, a putative transcription factor, is associated with over 50 mutations linked to dystonia; however, relatively little is known about THAP1 target genes or the functional pathways disrupted by its DYT6 mutant forms. It has been suggested that at least some DYT6 mutations may impair DNA binding activity by THAP1, leading to transcriptional dysregulation as a potential pathogenic mechanism underlying the disease phenotype. Recently, a transcriptional target of THAP1 has been identified, the ribonucleotide reductase Ml (RRMJ) gene, which encodes the large subunit of the ribonucleoside-diphosphate reductase enzyme. This enzyme is essential for G liS phase cell-cycle progression and promoting cellular proliferation and has been implicated as a tumor-suppressor gene. However, the regulatory action of THAP1 on this gene was previously unclear, and previous efforts to quantify THAP1-mediated transcriptional regulation have been confounded by secondary artifacts induced by overexpressing THAPl at high levels in cultured cells. In order to further define THAP1's effects on RRMJ, we developed an inducible system to achieve regulated, low level expression of THAPl and performed luciferase reporter assays to quantify RRMJ promoter activity. Inducible plasmid constructs encoding either human wild type THAP1 or turboGFP (tGFP) as a control were transfected separately into HEK-293T cells. After 48 hours, lysates from transfected cells were processed via Western blot analysis to confirm dose-dependent increases in expression of both THAP1 and GFP. This system was then used to probe effects of THAP1 on RRMJ promoter activity in both HEK-293T and human BE(2)C neuroblastoma cells. Both cell types were transfected with a mixture of plasmids consisting of (1) RRMJ promoter-driven firefly luciferase (FLue); (2) Thymidine Kinase (TK) promoter-driven renilla luciferase (RLuc) to normalize for transfection efficiency; and (3) either the inducible THAPl or GFP construct. Lysates from transfected cells were then assayed for both FLue and RLuc activities with appropriate substrates. In both cell types, induction of THAP1 expression by increasing IPTG concentrations resulted in dose-dependent increases in normalized ratios of RRMJFLuc/TK-RLuc activities, whereas cells expressing tGFP failed to exhibit any dose-response relationship. The results of these experiments suggest that THAP1 acts as a positive transcriptional regulator of the RRMJ gene. This study validates a model system for further characterizing THAP1 transcriptional targets and, given RRMJ's known role as a tumor suppressor and cancer drug target, suggests a future avenue of exploration for THAP1 as an anti-cancer drug target.
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