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Comparative Study
. 2005 Jun 14;102(24):8472-7.
doi: 10.1073/pnas.0503505102. Epub 2005 Jun 2.

Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration

Vikas B Palhan  1 , Shiming Chen, Guang-Hua Peng, Agneta Tjernberg, Armin M Gamper, Yuxin Fan, Brian T Chait, Albert R La Spada, Robert G Roeder
Affiliations
Comparative Study

Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration

Vikas B Palhan et al. Proc Natl Acad Sci U S A. .

Abstract

Spinocerebellar ataxia type 7 (SCA7) is characterized by cone-rod dystrophy retinal degeneration and is caused by a polyglutamine [poly(Q)] expansion within ataxin-7, a protein of previously unknown function. Here, we report that ataxin-7 is an integral component of the mammalian STAGA (SPT3-TAF9-ADA-GCN5 acetyltransferase) transcription coactivator complex, interacts directly with the GCN5 histone acetyltransferase component of STAGA, and mediates a direct interaction of STAGA with the CRX (cone-rod homeobox) transactivator of photoreceptor genes. Consistent with these results, chromatin immunoprecipitation assays document retinal-specific association of CRX, GCN5, and acetylated histone H3 with CRX _target genes. RNA interference studies also implicate ataxin-7 and GCN5 in CRX-dependent gene activation, and histone deacetylase inhibitors restore the compromised expression of a CRX _target gene in an ataxin-7-deficient background. Significantly, in relation to SCA7, poly(Q)-expanded ataxin-7 gets incorporated into STAGA and, in a dominant-negative manner, inhibits the nucleosomal histone acetylation function of STAGA GCN5 both in vitro and, based on chromatin immunoprecipitation assays, in SCA7 transgenic mice. These results suggest that the normal function of a poly(Q) disease protein may intersect with its pathogenic mechanism, an observation with significant implications for the molecular basis of all poly(Q) disorders and ultimately for their treatment.

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Figures

Fig. 1.
Fig. 1.
Ataxin-7 is an integral subunit of the STAGA complex. (A) SDS/PAGE/silver stain analysis of affinity-purified STAGA from FLAG.HA-SPT3 HeLa cells. The p110 band was identified as ataxin-7 by tandem MS analysis. Asterisks indicate nonspecific bands. (B) Immunoblot of Sephacryl S-300 fractions after chromatography of HeLa S3 NE. (C) SDS/PAGE/silver stain analysis of control (IgG resin) or anti-ataxin-7 affinity-purified STAGA from Y-79 cell NE. (D) Immunoblot of IPs from C with indicated antibodies. Y-79 NE served as positive control. (E) Fluorogram of HAT assay of IPs from C, using free histones as substrate. Recombinant PCAF served as a positive control.
Fig. 2.
Fig. 2.
Poly(Q)-expanded ataxin-7 is incorporated into STAGA and inactivates its HAT function in a dominant-negative manner. (A) Immunoblot of anti-FLAG IPs of NE from control 293T cells (lane 2) or from 293T lines stably expressing FLAG-ataxin-7–24Q (lane 3) or FLAG-ataxin-7–92Q (lane 4). 293T NE served as positive control (lane 1). (B) Fluorogram of HAT assays using an oligonucleosome substrate on IPs from A, a highly concentrated STAGA complex (lane 5), and recombinant p300 (lane 1). (C) Fluorogram of solution HAT assays with an oligonucleosome substrate and purified proteins FLAG-ataxin-7–24Q/-92Q, FLAG-ataxin-7–92Q NT, His6-p300, and affinity-purified PCAF complex added as indicated (top).
Fig. 3.
Fig. 3.
CRX interacts with STAGA through ataxin-7. (A) SDS/PAGE/fluorographic analysis of interactions of 35S-labeled in vitro-translated proteins (LUC, CRX, and CRX-Mutant) with IPs from Fig. 1C. Additions were as indicated (top), and inputs (2%) are shown in lanes 1, 4, and 7. (B) SDS/PAGE/fluorographic analysis of interactions of 35S-labeled in vitro-translated proteins (GCN5-S and GCN5-L) with FLAG-CRX in the presence and absence of Myc-ataxin-7–92Q (Myc-Atx7). Similar results were obtained with Myc-ataxin-7–10Q (data not shown). Additions were as indicated (top). Corresponding empty vector controls are represented by “-.” Inputs (2%) are shown in lanes 7 and 8. (C) ChIP analysis of acetylated histone H3 and GCN5 on CRX-_target genes in mouse retina vs. liver tissues. PCR primers covered the regulatory regions indicated in parentheses. M-opsin, M-cone opsin; LCR, locus control region of M-cone opsin; S-opsin, S-cone opsin; IRBP, interphotoreceptor-binding protein. Input (pre-IP), + and - antibody (Ab), and mock (no DNA) lanes are marked. Similar results were obtained by using an isotype-matched antibody (rabbit IgG, Santa Cruz Biotechnology) as with the no antibody control.
Fig. 4.
Fig. 4.
CRX/NRL-mediated activation of the rhodopsin promoter depends on a functional STAGA complex. (A and B) Immunoblot of whole-cell extracts from HEK293 cells transfected with siRNAs as indicated (top). (C) Effects of ataxin-7, GCN5, and p300 siRNAs on expression of a transfected rhodopsin promoter-driven LUC reporter in HEK293 cells. LUC and nonspecific control (C1) siRNAs served as positive and negative controls, respectively. The activation observed from mock-transfected HEK293 cells was arbitrarily set as 100%. (D) HDAC inhibitor-mediated recovery of ataxin-7 siRNA-compromised rhodopsin-promoter activity in transfected HEK293 cells. Control siRNA (C1) and ataxin-7 siRNA transfected cells (as in C) were treated with suberoylanilide hydroxamic acid and butyrate (HDAC inhibitors) as indicated. Error bars represent mean plus standard deviation in C and D from three independent experiments.
Fig. 5.
Fig. 5.
HAT activity alterations in SCA7 result from a dominant-negative effect on the STAGA complex. ChIP assays on retinas from SCA7–92Q transgenic mice and their nontransgenic WT littermates reveal differences in occupancy of CRX _target and non_target gene regulatory regions. Quantitation of the occupancy differences is provided in Table 1. Brackets group together the enhancer/promoter pair regulating each gene. On the far right, previously reported gene expression alterations for this SCA7 mouse model of retinal degeneration are as indicated (17). SER, S-cone opsin enhancer region; RER, rhodopsin enhancer region; Arr3, cone arrestin 3; IER, IRBP enhancer region. See the legend of Fig. 3C for other definitions. A 100-bp DNA ladder was used as a size marker.
Fig. 6.
Fig. 6.
Mutant ataxin-7 effects on GCN5 function. (A) Retinal sections from 13-week-old SCA7–92Q transgenic mice (92Q), SCA7–24Q (24Q), and nontransgenic littermates (WT) were immunostained with an anti-GCN5 antibody (green). Although confocal images reveal intense immunostaining of photoreceptor nuclei (ONL) and bipolar/interneuron nuclei (INL) in the 92Q mice, no appreciable GCN5 immunostaining is detectable in the 24Q or nontransgenic mice. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (Scale bar, 100 μm.) (B) Models indicating (i) normal function of WT ataxin-7 (Atx7), as an integral GCN5-interacting subunit of STAGA, in facilitating STAGA recruitment and GCN5-mediated histone H3 acetylation through interactions with a promoter-bound transcription factor (TF) such as CRX (Left), and (ii) dominant-negative inhibition of GCN5 function (histone acetylation) within promoter-bound STAGA by poly(Q)-expanded ataxin-7 (Atx7–92Q) after interactions with WT ataxin-7-containing STAGA (Center) or after incorporation into STAGA in place of WT ataxin-7 resulting in reduced incorporation of ADA2b, SPT3, and TAF12 (Right).
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