Elsevier

Neurobiology of Disease

Volume 20, Issue 3, December 2005, Pages 646-655
Neurobiology of Disease

A role for both wild-type and expanded ataxin-7 in transcriptional regulation

https://doi.org/10.1016/j.nbd.2005.04.018Get rights and content

Abstract

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease primarily affecting the brainstem, retina and Purkinje cells of the cerebellum. The disease is caused by a polyglutamine expansion in ataxin-7, a protein found in two complexes TFTC and STAGA, involved in transcriptional regulation. Transcriptional dysregulation has been implicated in the pathology of several polyglutamine diseases. In this paper, we analyzed the effect of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP and the Purkinje cell expressed nuclear receptor RORα1. We could show that transcription mediated by both CBP and RORα1 was repressed by expanded ataxin-7. Interestingly, repression of transcription could also be observed with wild-type full-length ataxin-7, not only on CBP- and RORα1-mediated transcription, but also on basal transcription. The repression could be counteracted by inhibition of deacetylation, suggesting that ataxin-7 may act as a repressor of transcription by inhibiting the acetylation activity of TFTC and STAGA.

Introduction

Ataxin-7 is an 892-amino-acid protein found in most CNS and non-CNS tissues (Einum et al., 2001, Jonasson et al., 2002, Lindenberg et al., 2000). The expansion of a polyglutamine domain in the N-terminal region of ataxin-7 has been shown to be the cause of spinocerebellar ataxia type 7 (SCA7), a disease characterized by cerebellar ataxia, dysarthria and visual impairment. The symptoms are primarily due to a selective loss of neurons within the cerebellum, brainstem and retina (David et al., 1997, Konigsmark and Weiner, 1970, Martin et al., 1994). Despite identification of several functional domains, including nuclear localization signals, a motif related to the phosphate-binding sites of arrestins and SH3-binding sites in ataxin-7, little is known about the function of wild-type ataxin-7 (Chen et al., 2004, Kaytor et al., 1999, Lebre et al., 2001, Mushegian et al., 2000). However, recently, a putative ataxin-7 yeast orthologue (SGF73) was identified as a new component of the SAGA complex and subsequently ataxin-7 was found to interact with several components of the mammalian SAGA-like complexes TFTC and STAGA (Helmlinger et al., 2004, Scheel et al., 2003). TFTC and STAGA are large complexes containing several transcription-related factors such as the histone acetyltransferase Gcn5, spt proteins and TBP-associated factors (TAFs) (Brand et al., 1999, Martinez et al., 1998). The complexes have been implicated in several functions including facilitation of transcription by acetylation of histones, recruitment of the preinitiation complex to promoters as well as in splicing and DNA repair (Brand et al., 1999, Brand et al., 2001, Hardy et al., 2002, Martinez et al., 2001, Wieczorek et al., 1998).

Besides SCA7, eight other neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1–3, 6 and 17, DRPLA, SBMA and Huntington's disease (Evert et al., 2000, Koide et al., 1999). The polyglutamine disorders have many features in common, including a negative correlation between repeat length and age of onset, and the formation of nuclear and/or cytoplasmic inclusions containing the mutant protein. The similarities observed in the different disorders have lead to the hypothesis of a common pathological mechanism (Evert et al., 2000). Despite widespread expression of the expanded polyglutamine proteins, the neurons affected in the different disorders are distinct or only partially overlap. The mechanism involved in the disease specificity is not well understood (Evert et al., 2000). One hypothesis suggests that the expanded polyglutamine domain alters the conformation of the protein, resulting in aberrant protein–protein interactions with selectively expressed proteins and thus causing specific neurons to dysfunction and die. Numerous ubiquitously expressed proteins interacting with wild-type and/or expanded forms of polyglutamine proteins have been identified. Many of these proteins carry small glutamine stretches or glutamine-rich domains themselves, which mediate the interaction. Several transcription factors or co-activators such as p53, TAFII130 and CREB-binding protein (CBP) have been found sequestered in inclusions (McCampbell et al., 2000, Nucifora et al., 2001, Zander et al., 2001). Furthermore, it has been shown that these interactions can affect gene transcription. Sp1-TAFII130-mediated transcription is disrupted by expanded huntingtin, and CBP-mediated transcription is affected by both expanded huntingtin and atrophin-1 (Dunah et al., 2002, Nucifora et al., 2001). The observed effect on CBP-mediated transcription has received considerable attention, given the established role for CBP and its target transcription factor, CREB, in neuronal cell survival (Mayr and Montminy, 2001). Interactions with proteins that could explain the selectivity seen between neurons in the different polyglutamine disorders are, however, few in number. The suppression of the cone-rod homeobox protein CRX, a transcription factor predominantly expressed in retinal photoreceptor cells, by expanded ataxin-7 has, however, been suggested to cause the retinal sensitivity in SCA7 (La Spada et al., 2001). To date, no explanation for the specificity of expanded ataxin-7 on cerebellar Purkinje cells and neurons in the inferior olivary nucleus of SCA7 patients has been put forward.

The retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor super family and two of its isoforms, 1 and 4, which contain small polyglutamine domains, are highly expressed in Purkinje cells (Giguere et al., 1994, Matsui et al., 1995, Matysiak-Scholze and Nehls, 1997, Steinmayr et al., 1998). RORα activates transcription of several genes, including the Purkinje cell protein-2 (Pcp-2) and the prosaposin genes, which are highly expressed in Purkinje cells (Matsui, 1997, Sun et al., 2000). Heterozygous RORα knock-out mice have an ataxic phenotype due to progressive loss of cerebellar Purkinje cells, granule cells and neurons in the inferior olivary nuclei. These observations make RORα an interesting candidate target for expanded ataxin-7 in SCA7 pathology (Caston et al., 1995, Hadj-Sahraoui et al., 2001, Zanjani et al., 1992).

In this report, we describe the effects of wild-type and expanded ataxin-7 on transcription mediated by two different co-activators/transcription factors, CBP and RORα1. We observed that both truncated and full-length expanded ataxin-7 repressed CBP- as well as RORα1-mediated transcription. Interestingly, full-length ataxin-7 with a normal glutamine repeat repressed transcription to a similar level as expanded full-length ataxin-7. The negative effect of wild-type full-length ataxin-7 on transcriptional activation could be counteracted by treatment with a deacetylase inhibitor. Our results suggest that ataxin-7 is a negative regulator of transcription, possibly by inhibiting TFTC and/or STAGA functions like histone acetylation and/or recruitment of the preinitiation complex to promoters.

Section snippets

Plasmids

Constructs Tr-Q10 and Tr-Q65 were generated by amplification of codons 2–112 of the SCA7 gene, using the primers SCA7F5: 5′-GGA AAA GAT CTC GGA GCG GG-3′ and SCA7R8: 5′-GAA ACT TTC GTC GAC TTC TGT C-3′, from a human full-length cDNA clone with 10 CAGs (David et al., 1997). The fragment was cloned into pBluescript (Stratagene), and to generate Tr-Q65, the repeat was removed by AatII and NarI digestion, and an expanded polyglutamine domain amplified from genomic DNA from an SCA7 patient using

Expression of ataxin-7 and inclusion formation

To study the effect of wild-type and expanded ataxin-7 on inclusion formation and cell viability, human neuroblastoma cells, SK-N-SH, were transfected with constructs expressing flag and c-myc tagged truncated or full-length ataxin-7 containing 10 or 65 glutamines (Fig. 1A). The correct expression of the constructs was analyzed by Western blotting (Fig. 1B).

The cellular localization of ataxin-7 was determined by immunofluorescence using anti-ataxin-7, anti-flag and anti-c-myc (Fig. 2 and data

Discussion

To date, nine neurodegenerative disorders caused by expanded polyglutamine domains in different proteins are known. The mechanism of polyglutamine pathology is however still largely unknown, but it has been shown that several of the polyglutamine proteins can sequester transcription factors into inclusions and inhibit transcription. Transcriptional repression has therefore been suggested to play a role in the pathology of polyglutamine disease, with repression of cell type-specific

Acknowledgments

We thank Drs. Thomas Grundström and Vincent Giguere for providing DNA constructs. This study was supported by grants from the county council of Västerbotten (‘spjutspets’), the Swedish medical research council and the Swedish foundation for strategic research.

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