RESUMO
Thanatos-associated protein domain containing, apoptosis-associated protein 1 (THAP1), the gene mutated in DYT6 dystonia, encodes a transcription factor. While the N-terminal THAP domain allows for specific DNA-binding, the functional relevance of the other regions is largely unknown. The C-terminus contains a 4-amino-acid-spanning host cell factor 1 (HCFC1)-binding domain (HBM) that mediates the interaction with HCFC1. Interestingly, three mutations affecting the HBM (p.N136S, p.N136K, p.Y137C) have been reported in dystonia patients. We investigated the consequences of these mutations on the interaction of THAP1 with HCFC1 and demonstrated that all three mutations abolished HCFC1-THAP1 complex formation. Notably, HCFC1 co-localization was found in >90% of the almost 3,500 chromatin regions loaded with THAP1 in publicly available genome-wide ChIP data. By siRNA-mediated depletion of HCFC1, we detected an increase of THAP1 expression, indicating a co-repressor activity of HCFC1 for THAP1. Quantitative ChIP on selected promoters revealed that none of the mutations significantly decreased the DNA-binding ability of THAP1 while HCFC1 binding was highly reduced. Our findings indicate a THAP1-mediated recruitment of HCFC1 to THAP1 target sites. Of note, dystonia-causing mutations within the HBM in THAP1 abolished this interaction. Thus, we demonstrate disrupted THAP1-HCFC1 complex formation as another mechanism of dystonia-causing mutations leading to transcriptional dysregulation.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação a DNA/genética , Distonia/genética , Fator C1 de Célula Hospedeira/genética , Proteínas Nucleares/genética , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Distúrbios Distônicos/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Fator C1 de Célula Hospedeira/metabolismo , Fator Proteico 1 do Hospedeiro , Humanos , Mutação/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
Mutations in the THAP1 gene encoding the transcription factor THAP1 have been shown to cause DYT6 dystonia. THAP1 contains a highly conserved THAP zinc finger at its N-terminal region which allows specific binding to its target sequences as well as a coiled-coil domain (amino acids 139-190) towards its C-terminus postulated as a protein-protein-binding motif. While several DYT6-causing mutations within the THAP domain were shown to decrease THAP1 activity in transcriptional regulation and DNA-binding, the role of mutations within the coiled-coil domain is rather unknown. Therefore, assigning a function to this domain may enable functional testing of mutations in this region. Notably, THAP1 and other THAP proteins form homodimers; however, the responsible domain has not been elucidated in detail. We show that the region of amino acids 139-185 is involved in formation of THAP1 homodimers by using yeast-two-hybrid, GST pull-down, and cross-linking assays. Surprisingly, all nine reported DYT6-causing missense mutations within this region had no effect on dimerization of THAP1 in GST pull-down and formaldehyde cross-linking assays. In conclusion, we demonstrated that a region of 47 amino acids is involved in THAP1 homodimerization but mutations in this region seem not to impair this mechanism.
