The DYT6 dystonia causative protein THAP1 is responsible for proteasome activity via PSMB5 transcriptional regulation

Abstract

The proteasome plays a pivotal role in protein degradation, and its impairment is associated with various pathological conditions, including neurodegenerative diseases. It is well understood that Nrf1 coordinates the induction of all proteasome genes in response to proteasome dysfunction. However, the molecular mechanism regulating the basal expression of the proteasome remains unclear. Here we identify the transcription factor THAP1, the causative gene of DYT6 dystonia, as a regulator of proteasome activity through a genome-wide genetic screen. We demonstrated that THAP1 directly regulates the expression of the PSMB5 gene, which encodes the central protease subunit β5. Depletion of THAP1 disrupts proteasome assembly, leading to reduced proteasome activity and the accumulation of ubiquitinated proteins. These findings uncover a regulatory mechanism for the proteasome and suggest a potential role for proteasome dysfunction in the pathogenesis of dystonia.

Fig. 1. Identification of THAP1 as a regulator of the proteasome through a genome-wide screen.

a The dots represent the RRA scores of genes in the CRISPR screen. The blue dots and red dot represent proteasome subunits and THAP1, respectively. b, d Flow cytometry analysis of HEK293T (b) and U2OS cells (d) stably expressing mCherry-P2A-ZsGreen-mODC transfected with sgControl (negative control), sgPSMA6 (positive control), and sgTHAP1. The numbers in the upper right quadrant of (b) and (d) represent the percentage of the total population. c, e Mean fluorescence of ZsGreen-positive cells in the upper right quadrants of (b) and (d), respectively. Data represent the mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction and one-way ANOVA Tukey with multiple comparisons test. f Proteasome chymotrypsin-, trypsin-, and caspase-like activities of HEK293T cells transfected with sgControl and sgTHAP1. The activity was measured in the absence of SDS for measuring the 26S activity. g HEK293T cells were transfected with the indicated sgRNA and subjected to immunoblot analysis with antibodies against the indicated proteins. h, i The chymotrypsin-, trypsin-, and caspase-like activities (h) and immunoblot analysis (i) of the THAP1 knockout cells in which sgRNA-resistant THAP1 cDNA was added back. The 26S proteasome activities were measured in the absence of SDS. Data represent the mean ± SEM (n = 3 from three biological replicates). The significance were calculated using one-way ANOVA Tukey with multiple comparisons test, with exact P-values as follows: Chymotrypsin-like relative peptidase activity: ****P < 0.0001 (P_{sgControl vs sgTHAP1 #1}); ****P < 0.0001 (P_{sgControl vs sgTHAP1 #1+THAP1}); ****P < 0.0001 (P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1}), Trypsin-like relative peptidase activity; **P < 0.001 (P_{sgControl vs sgTHAP1 #1} = 0.0002); n.s., not significant, (P_{sgControl vs sgTHAP1 #1+THAP1} = 0.6161); **P < 0.01 (P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1} = 0.0016), Caspase relative peptidase activity: ****P < 0.0001 (P_{sgControl vs sgTHAP1 #1});n.s., not significant,(P_{sgControl vs sgTHAP1 #1+THAP1} = 0.2211); ****P < 0.0001(P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1}). Data represent the mean ± SEM (n = 3 from three biological replicates). n. s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 2. THAP1 is essential for CP assembly.

a Immunoblot analysis of lysates from HEK293T cells using antibodies against the indicated proteins. b Schematic diagram of the assembly process of the proteasome CP. c, d HEK293T cells were transfected with the indicated sgRNA and selected by puromycin for 6 days. The cell extracts were fractionated by glycerol gradient centrifugation, and an equal amount of each even-numbered fraction was used for measuring proteasome chymotrypsin-like activity in the presence of 0.025% SDS. c and for immunoblot analysis using antibodies against the indicated subunits. Fractions 8–12, 16–18, and 22–26 correspond to fractions containing CP assembly intermediates, assembled 20S CP, and the 26S proteasome, respectively (d). All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 3. THAP1 regulates the expression of PSMB5.

a mRNA expression levels of proteasome β subunit genes in HEK293T cells transfected with the indicated sgRNAs. All data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. b mRNA expression level of the β5 subunit gene in THAP1 knockout cell with or without the addition of sgRNA-resistant THAP1 cDNA. All data are presented as mean ± SEM (n = 3 from three biological replicates). Statistical significance is indicated by one-way ANOVA Tukey with multiple comparisons test, with exact P-values as follows: **P < 0.01(P_{sgControl vs sgTHAP1 #1+THAP1} = 0.0097); ***P < 0.001 (P_{sgControl vs sgTHAP1 #2} = 0.0002); n.s., not significant. (P_{sgControl vs sgTHAP1 #2+THAP1} = 0.4859); **P < 0.01(P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1} = 0.006); **P < 0.01 (P_{sgTHAP1 #2 vs sgTHAP1 #2+THAP1} = 0.0017); n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. c Volcano plot of RNA-seq results of THAP1 knockout HEK293T cells, compared to control HEK293T cells. HEK293T cells were transfected with a control sgRNA and a THAP1-targeting sgRNA, selected by puromycin for 3 days, and subjected to RNA extraction. PSMB5 and other proteasome subunits are shown in red and blue, respectively. The significance were calculated using an unpaired two-tailed Student’s t-test correction. d Gene Ontology analysis for biological process of genes downregulated by THAP1 knockout with the changes of fold change > 0.2 and a P-value < 0.01 of (c). The significance were calculated using Modified Fisher Exact test and adjusted by Bonferroni, Benjamini, and FDR. All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 4. Overexpression of β5 restores proteasome dysfunction caused by THAP1 knockout.

a The HEK293T cells were transfected with the indicated sgRNA targeting THAP1 and subsequently transfected with each cDNA encoding proteasome β subunits β1–7. After five days, the cells were lysed and subjected to proteasome chymotrypsin-like activity assay. b The lysates from (a) were fractionated by glycerol gradient centrifugation, and an equal amount of each even-numbered fraction was subjected to a proteasome chymotrypsin-like activity assay. c, e The HEK293T cells stably overexpressing β5-FLAG were transfected with sgRNA targeting THAP1. Cell lysates were subjected to proteasome chymotrypsin-like activity assay (c) and immunoblot analysis using antibodies against the indicated proteins (e). d, f The lysates from (c) were fractionated by glycerol gradient centrifugation, and equal amounts of each even-numbered fraction were subjected to proteasome chymotrypsin-like activity assay. (d) and immunoblot analysis using antibodies against the indicated proteins (f). Proteasome activity assays were performed in the presence of 0.025% SDS for measuring both the 20S and 26S activities. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction and one-way ANOVA Tukey with multiple comparisons test. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 5. THAP1 binds to the upstream sequence of the PSMB5 gene.

a Analysis of publicly available THAP1 ChIP-seq data with a data height of 120 and data range of 0-2.5. THAP1-binding sites within each proteasome subunit of the CP and RP are shown. b Close-up view of THAP1-binding sites on the PSMB5 gene. Three putative THAP1 binding sites around the transcription start site (TSS) of the PSMB5 gene were aligned with the THAP1 binding motif sequence and depicted. The constructs and the results of the luciferase assay for promoter activity are shown. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. *P < 0.05. No adjustments were made for multiple comparisons. c Relative luciferase activity in HEK293T luciferase reporter cells transfected with the indicated sgRNA. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. **P < 0.01. No adjustments were made for multiple comparisons. d ChIP-qPCR analysis of THAP1 wild-type and C54Y mutant constructs. Cell lysates from HEK293T cells transfected with cDNAs encoding FLAG-tagged THAP1 wild-type or C54Y mutant cDNA were subjected to ChIP-qPCR to quantify binding to the promoter region of the PSMB5 gene. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using two-way ANOVA Tukey with multiple comparisons test. n.s., not significant; **P < 0.01. e, f Chymotrypsin-like activity (e) and immunoblot analysis (f) in THAP1 knockout cells in which sgRNA-resistant THAP1 wild-type, C54Y mutant, and THAP binding-domain deletion were added back. The 20S proteasome activity was measured in the presence of 0.025% SDS. Statistical significance is indicated (e) by one-way ANOVA Tukey with multiple comparisons test, with exact P-values as follows: P < 0.0001 (P_{sgControl vs sgTHAP1 #1}); n.s., not significant (P_{sgControl vs sgTHAP1 #1+THAP1} = 0.1851); p < 0.0001 (P_{sgControl vs sgTHAP1 #1+THAP1 C54Y}); P < 0.0001(P_{sgControl vs sgTHAP1 #1+THAP1 ΔTHAP domain}); n.s., not significant (P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1 C54Y} = 0.369); n.s., not significant (P_{sgTHAP1 #1 vs sgTHAP1 #1+THAP1 ΔTHAP domain} = 0.9778). Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 6. THAP1 regulate proteasome function in neurons and mice embryos.

a mRNA expression levels of proteasome β5 subunit gene in SH-SY5Y cells transfected with the indicated sgRNAs. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. **P < 0.01. b Proteasome chymotrypsin-like activity of SH-SY5Y cells transfected with indicated sgRNA. The activity was measured in the presence of 0.025% SDS. Data are presented as mean ± SEM (n = 3 from three biological replicates). The significance were calculated using an unpaired two-tailed Student’s t-test with Welch’s correction. *P < 0.05; **P < 0.01. c mRNA expression levels of proteasome β5 subunit gene in the Thap1^+/+, Thap1^C54Y/+ and Thap1^C54Y/C54Y embryos at E10.5. Data represent the mean ± SEM (n = 4 for Thap1^+/+ n = 5 for Thap1^C54Y/+ and n = 6 for Thap1^C54Y/C54Y from three biological replicates). d Volcano plot of RNA-seq results of Thap1^C54Y/C54Y embryos at E10.5, compared to Thap1^+/+ embryos at E10.5. PSMB5 is highlighted in red and other proteasome subunits are colored in blue. Data represent the mean ± SEM (n = 3 for Thap1^+/+ and n = 3 for Thap1^C54Y/C54Y) The significance were calculated using an unpaired two-tailed Student’s t-test correction. e Proteasome chymotrypsin-like activity of Thap1^C54Y/C54Y embryos at E10.5. The activity was measured in the presence of 0.025% SDS. f The Thap1^+/+, Thap1^C54Y/+ and Thap1^C54Y/C54Y embryos at E10.5 were subjected to immunoblot analysis with antibodies against the indicated proteins. Data represent the mean ± SEM (n = 3 from three biological replicates). Significance was calculated using an unpaired two-tailed Student’s t-test with Welch’s correction and one-way ANOVA Tukey with multiple comparisons test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All experiments were performed at least three biologically independent times with similar results. Source data are provided as a Source Data file.

Fig. 7. The model of proteasome regulation by THAP1.

THAP1 is responsible for constitutive proteasome activity by regulating the transcription of the PSMB5 gene. A deficiency in THAP1 activity causes a reduction in PSMB5 expression, an insufficient production of the β5 subunit, a defect in the assembly of the proteasome CP, and a decline in proteasome activity, which leads to the accumulation of the ubiquitinated proteins and disruption of cellular homeostasis. These results suggest the involvement of proteasome dysfunction due to THAP1 mutations in the development of DYT6 dystonia, but direct evidence is not yet available.