Loss Of Ataxin-3 Induces Transcriptional Dysfunction Via Repressing The Expression Of HDAC3 And NCoR

Partâ… . Loss of ataxin-3 induces transcriptional dysfunctionObjective:To investigate the difference of gene expression between ataxin-3 KO MEF cells and ataxin-3 WT MEF cells.Methods:RNA was isolated from ataxin-3 WT and ataxin-3 KO MEF cells, and Microarray was applied to analyse the gene expression. RT-PCR was performed to confirm Microarray data.Results:In comparison with ataxin-3 WT control, loss of ataxin-3 induced the upregulation of 260 genes and downregulation of 150 genes. These genes were categorized based on different biological process, and several pathways including immune response pathway and cell adhesion were altered due to loss of ataxin-3.Conclusion:Loss of ataxin-3 induces transcriptional dysfunction in MEF cells. Partâ…¡. Promoter regions of EphA3 and their transcriptional activities in 293T and MEF cell linesObjective:To construct reporter vectors containing EphA3 promoter region in different lengths and test their transcriptional activities in 293T and MEF cells.Methods:Genomic DNA from Balb/C mice was used as a template to synthesize various PCR products in different lengths, and then the PCR products cloned into pGL3-Basic vector to develop a series of reporter constructs. These constructs were cotransfected into 293T or MEF cells with pRL-CMV vector, and luciferase activities were measured.Results:All constructs were verified by restriction enzyme digestion and sequencing. The basic promoter region of EphA3 was from -279bp to transcription start site (+110bp), and the promoter activities of EphA3 in 293T and MEF cells were similar.Conclusions:EphA3 promoter region in different lengths are successfully cloned into reporter vector and the basic promoter region is confirmed. Partâ…¢. Loss of ataxin-3 leads to transcriptional dysfunction of EphA3 gene via repressing the expression of HDAC3 and NCoRSpinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease and one of the nine known polyglutamine (PolyQ) neurodegenerative diseases. An unstable CAG repeats in the corresponding protein ataxin-3 leads to SCA3. Growing evidences indicate that ataxin-3 is a deubiquitinating enzyme (DUB) linked to the ubiquitin-proteasome pathway. Another line of evidence indicates ataxin-3 is also involved in transcriptional regulation, and expanded ataxin-3 leads to transcriptional dysfunction. However, how ataxin-3 regulates transcription is still a mystery. Here, we chose EphA3 as a target to investigate the molecular mechanism of endogenous ataxin-3 in transcriptional regulation. We firstly found that EphA3 gene transcription was upregulated without ataxin-3, then we generated reporter constructs containing EphA3 promoter region in different lengths and tested their activities in ataxin-3 WT and ataxin-3 KO MEF cells. Dual luciferase assays demonstrated that the promoter activities of EphA3 were significantly upregulated without ataxin-3. Transient transfection of human ataxin-3 into ataxin-3 KO MEF cells repressed the promoter activities of EphA3 gene. To reveal whether ataxin-3 could bind to the promoter region of EphA3 directly, chromatin immunoprecipitaion (ChIP) was applied. Although no any data supported that ataxin-3 could bind to the promoter region of EphA3 gene, ataxin-3 altered histone acetylation and loss of ataxin-3 induced the hyperacetylation of histone H3 and H4 in the promoter region of EphA3 via repressing the expression of HDAC3 and NCoR. Consistently, HDAC broad inhibitor TSA and HDAC3 specific inhibitor Apicidin induced the upregulation of EphA3 gene transcription in ataxin-3 WT cells. Taken together, these results demonstrate that loss of ataxin-3 induces hyperacetylation of histones H3 and H4 via repressing the expression of HDAC3 and NCoR, and provide the direct evidence that loss of ataxin-3 induces transcriptional dysfunction. Loss-of-function may contribute to transcriptional dysregulation in SCA3 pathogenesis.