| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin
Introduction
Results
Isolation of nuclear receptor co-repressor by yeast two-hybrid screening
N-CoR interacts with full-length huntingtin in vitro
Localization of repressor complex proteins in HD brain
Discussion
Materials And Methods
Strains and plasmids
Library screening
Fusion protein expression
Preparation of huntingtin samples
In vitro binding experiments
Immunohistochemistry
Acknowledgements
References
Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin
Received February 1, 1999; Revised and Accepted June 8, 1999
We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system. This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone. The interaction was confirmed using His-tagged Escherichia coli-expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays. The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers. The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases. We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions. The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease.
INTRODUCTION
The association of expanded CAG repeats in the coding regions of genes with a number of inherited diseases has been known for some time (1-9), but the mechanism of toxicity of the polyglutamine tracts translated from these CAG repeats is unknown. Recent work in a number of these diseases and in in vivo and in vitro models has demonstrated the presence of neuronal intranuclear inclusions (NIIs) as a unifying feature of the pathology of expanded polyglutamine tracts, but the nature of their putative toxicity remains unclear (10-17). The inclusions may themselves be directly or indirectly toxic to cells or may be a result of a sequestration of toxic soluble polyglutamine moieties. One of the most intriguing aspects of these diseases is that in each one a specific pattern of neuronal degeneration occurs (18) although the mutated protein is expressed in many cell types (19-22), and one of the most interesting questions is why do only specific neuronal cells die? We and others have been interested in this observed specificity, and have speculated that the interactions of the individual mutated proteins in specific cell types may provide an answer. The proteins carrying the polyglutamine tracts have no similarities apart from these tracts, and are expected to have differing functions. Ataxin 1 (22), atrophin 1 (23), the androgen receptor (24) and ataxin 3 (24,25) are known to localize in the nucleus, but huntingtin is localized in the cytoplasm (27,28), although the inclusions are intranuclear in all of these diseases.
A number of proteins which interact with the N-terminal repeat-carrying region of huntingtin have already been detected, although none of these provide obvious clues to the normal or pathological function of huntingtin. The first such protein isolated, huntingtin-associated protein 1 (HAP1) (29), was found to be a brain-specific protein, although not particularly enriched in areas that degenerate in Huntington's disease (HD) (30). The interaction of huntingtin with HAP1 is polyglutamine length dependent (29) and HAP1 associates with proteins of the neuronal cytoskeleton, adding to earlier evidence that huntingtin may have a role in trafficking proteins along the cytoskeleton (31,32). Burke et al. (33) isolated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an interacting protein by affinity chromatography, but there has been no further evidence that this interaction has a role in pathology. Kalchman et al. (34) found E2-25K, a ubiquitin-conjugating enzyme, to interact with huntingtin; this interaction was not modulated by repeat length, and so its relevance to disease pathology is unclear. This enzyme participates in the conjugation of ubiquitin to cellular proteins, prior to degradation by the ubiquitin-dependent pathway, and so it was thought that interaction with this enzyme might have implications for the catabolism of huntingtin (34); the NIIs in HD brain and in transgenic mice show ubiquitin immunoreactivity (10,11), implying that there is a defect in degradative processing of huntingtin. Huntingtin-interacting protein 1 (HIP1), isolated by the yeast two-hybrid system using N-terminal huntingtin as bait (35,36), shares sequence homology with the yeast protein Sla2p, a protein essential for cytoskeletal function, again linking huntingtin to a cytoskeletal role. This interaction is stronger at shorter repeat lengths so seems unlikely to have a direct role in huntingtin pathology. Cystathionine [beta]-synthase has been shown to interact with huntingtin and, although there is no evidence yet for involvement in pathology, it is a possible candidate for initiating excitotoxic cell death in HD (37). A family of WW domain proteins have been reported to interact with huntingtin (38) through the proline-rich region downstream of the glutamine repeat in a repeat length-dependent fashion; their functions remain unknown. This is also true of the SH3GL3 protein reported by Sittler et al. (39) which interacts with huntingtin exon 1 protein carrying an expanded glutamine repeat and which promotes the formation of insoluble polyglutamine-containing aggregates in vivo.
Some of these interactions are likely to be involved at various stages in the pathology of HD, and we have discovered a further interaction with a protein of known function, the nuclear receptor co-repressor (N-CoR). We used the yeast two-hybrid system (40) with an adult rat brain activation domain library to identify this protein as interacting with the N-terminal region of huntingtin. This protein proved to have 89% homology to the C-terminus of the Mus musculus N-CoR (41), and thus is the rat equivalent. We demonstrate that huntingtin from both rat and human brain interact in vitro with rat and human N-CoR and, further, that N-CoR and other repressor complex proteins which interact with the co-repressor show changed localization in HD brain compared with control brain.
RESULTS
Isolation of nuclear receptor co-repressor by yeast two-hybrid screening
In order to isolate proteins which interact with huntingtin, a combination of Clontech's two-hybrid system 1 and Stratagene's HybriZAP library-making kit was used. Bait plasmids HD17 and HD40 were constructed containing amino acids 1-171 of huntingtin with tracts of 17 or 40 glutamine residues (starting at amino acid number 18) fused in-frame to the DNA-binding domain of GAL4 in vector pGBT9. Yeast strain Y190 carrying HD17 or HD40 was transformed with 40 µg of a rat brain library fused to the GAL4 activation domain (37). Table 1 shows the results of three independent screens, the first screen using HD17 as the bait plasmid, and the others using HD40. All three screens isolated one His+LacZ+ clone, despite screening vastly different numbers of transformants. After re-assaying to confirm the phenotype, the library plasmids were isolated and sequenced. BLAST analysis of the insert from the HD17 screen revealed 89% homology to the M.musculus N-CoR mRNA (GenBank accession no. U35312), and is therefore the rat homologue of this gene (Fig. 1). The 5[prime] end of this insert starts with nucleotide 5875 of the published mouse sequence, giving us residues equivalent to 1921-2453 of the mouse protein, which are homologous with residues 1905-2440 of the human protein (Fig. 1). This library plasmid was named pADNCoR1, and the starting point of its sequence is indicated in Figure 1.
Figure 1. Clustal W alignment of the human, mouse and rat sequences of the C-terminal end of N-CoR, with the yeast two-hybrid library activation domain plasmid start points indicated as shown. Mouse and rat sequences are 96.5% homologous at the amino acid level, and rat and human are 89.6% homologous. Amino acid numbering corresponds to the human N-CoR, GenBank accession no. AF044209. The rat sequence is GenBank accession no. AF124821. The peptide used to produce the polyclonal anti-N-CoR antisera is underlined.
Table 1. Yeast two-hybrid system screens using rat brain cDNA library constructed in HybriZAP vector (Stratagene); all screens were performed in yeast reporter strain Y190
| Bait construct | No. screened | Efficiency/µg | No. of positives |
| HD17 | 84 600 | 2.1 × 103 | 1 |
| HD40 | 27 000 | 675 | 1 |
| HD40 | 3.5 × 106 | 8.7 × 104 | 1 |
The inserts from the two screens with the HD40 expanded repeat bait plasmid were also derived from the N-CoR mRNA. Sequencing of their 5[prime] ends showed them to contain identical inserts, running from nucleotide 6179 of the published mouse sequence, encoding residues equivalent to amino acids 2022-2453. Thus this was a shorter version of the pADNCoR1 construct, and revealed that amino acids 1921-2022 were not essential for the interaction. This plasmid was named pADNCoR2 (Fig. 1).
To determine that the interaction between the 5[prime] end of huntingtin and N-CoR was specific, the pADNCoR1 library plasmid was retransformed into Y190 in combination with pGBT9, HD17, HD50 and pLAMINc. Trp+Leu+ transformants were dotted onto SD-His-Trp-Leu plates and, after growth for 7 days, assayed for lacZ activation. The interaction specifically required N-CoR and the 5[prime] end of huntingtin as shown in Figure 2A, where interaction of htt171 with varying repeat lengths is shown. To analyse whether the polyglutamine repeat region alone could interact with rN-CoR, the library plasmid was co-transformed into Y190 with bait plasmids containing either 20 (pGBTX24) or 56 (pGBTX32) glutamines and the 12 immediately N-terminal amino acids of huntingtin alone. These did not result in a LacZ+ phenotype (Fig. 2A), and so the interaction with huntingtin requires the first six amino acids of the huntingtin N-terminus or protein C-terminal to the polyglutamine tract. Figure 2B shows that the interaction between pHD50 and pADNCoR1 is much stronger than that of pHD17 and pADNCoR1 and stronger than that of the positive control plasmids p53 and pSV40, as judged by the amount of growth on the selective plates and the strength of the lacZ signal.
Figure 2. (A) Interaction of htt1-171 with pADNCoR1 in the yeast two-hybrid system, with 17, 50 and 85 CAG repeats in pHD plasmids as labelled. The negative control reactions are the binding domain vector pGBT9 only with pADNCoR1, and pADNCoR1 with pLaminC, and the positive controls p53 and pSV40. Plasmids pGBTX24 or pGBTX32 including the repeat region plus 12 N-terminal amino acids with 17 or 59 repeats, respectively, do not show any interaction with pADNCoR1. (B) The repeat length dependence of the interaction of pADNCoR1 with pHD17 or 50.
To investigate whether N-CoR interacts with other regions of huntingtin, the pADNCoR1 library plasmid was retransformed into Y190 in combination with JOE1/2, JOE1/3, JOE4/6, JOE5/6, JOE7/8, JOE10/12 and JOE11/12 which cover other areas of the HD cDNA. SBMA bait plasmids pAR24:15 and pAR66:16 and a DRPLA bait plasmid pDR64X with 64 CAG repeats were also co-transformed with the pADNCoR1 activation domain plasmid, to see whether rN-CoR interacts with other triplet repeat disease gene products. Details are as in Boutell et al. (37). A summary of all these results is shown in Table 2.
Table 2. Co-transformation filter assays of various repeat-containing binding domain plasmids with the rN-CoR activation domain plasmid in the yeast two-hybrid system
| Binding domain construct | Activation domain construct | ||
| pADNCoR1 | pADNCoR2 | pSV40 | |
| pGBT9 only | - | - | - |
| HD amino acids 1-171, 17 repeats | + | + | - |
| HD amino acids 1-171, 50 repeats | + | + | - |
| HD amino acids 864-1044 | - | - | - |
| HD amino acids 864-1215 | - | - | - |
| HD amino acids 1279-1695 | - | - | - |
| HD amino acids 1403-1695 | - | - | - |
| HD amino acids 1895-2063 | - | - | - |
| HD amino acids 2741-3144 | - | - | - |
| HD amino acids 2893-3144 | - | - | - |
| HD amino acids 7-16, 20 repeats | - | - | - |
| HD amino acids 7-16, 57 repeats | - | - | - |
| pAR2457 | - | - | - |
| pAR6657 | - | - | - |
| DRPLA amino acids 474-515, 64 repeats | - | - | - |
| pLAMc | - | - | - |
| p53 | - | - | + |
N-CoR interacts with full-length huntingtin in vitro
To confirm the interaction between rN-CoR and huntingtin by an independent method, 6×His-tagged C-terminal rN-CoR (amino acids 1921-2453; C-rN-CoR) and equivalent C-terminal human N-CoR (C-hN-CoR) were produced in Escherichia coli using the pQE vector system and used in pull-down assays with Ni-NTA-agarose. A soluble protein fraction (S1) was prepared from rat or human brain tissue (surgical biopsy tissue from normal human temporal cortex) or from an SK-N-SH cell line and incubated with native recombinant rat or human C-terminal His-NCoR prepared from E.coli and Ni2+-agarose, or with Ni2+-agarose alone in a pull-down assay. Figure 3A shows that full-length rat huntingtin binds specifically to His-tagged rN-CoR in vitro (lanes 1 and 2). Figure 3B shows a similar result for hN-CoR with rat huntingtin (lane 2), although with a weaker result for full-length human huntingtin (lane 3). These results indicate that recombinant C-rN-CoR and full-length rat or human huntingtin do interact in vitro. The addition of 0.5% Triton X-100 to the pull-down assays was found to have no effect on the observed binding.
Figure 3. (A) Pull-down assay showing western blotting using N-675. Lane 1, huntingtin pulled out of a rat brain homogenate using 0.5 ml inputs of rat brain homogenate and soluble C-terminal N-CoR protein from pQENCoR1. Lane 2, an equivalent experiment using 0.25 ml inputs. Lane 3, Ni-NTA-agarose with 0.5 ml of rat brain homogenate and 0.5 ml of sonication buffer. Lane 4, is Ni-NTA-agarose with 0.5 ml of pQENCoR-expressed soluble protein and 0.5 ml of homogenization buffer. (B) Pull-down assay western blot using N-675 showing the extraction of rat huntingtin by human C-terminal N-CoR expressed as a His-tagged protein from pQVW23 (0.5 ml input each) in lane 2 and a similar experiment on human control brain in lane 3. Lane 1, 20 µl of the input rat brain homogenate. Lane 4 shows Ni-NTA-agarose incubated with 0.5 ml of rat brain S1 fraction plus 0.5 ml of sonication buffer input. Lane 5, Ni-NTA-agarose with 0.5 ml of pQVW23 protein and 0.5 ml of homogenization buffer.
Localization of repressor complex proteins in HD brain
Immunohistochemistry with polyclonal antisera to N-CoR shows strikingly different results in HD and control brain (Fig. 4). hN-CoR, normally localized in neuronal nuclei in some populations of large pyramidal neurons of the frontal cortex and giving a less intense signal in cytoplasm (Fig. 4A and C) in control brain, appears to be excluded from the nucleus in the majority of cells of the cortex and caudate in HD brain (Fig. 4B, D and E). This was the case in the affected regions of all HD brains examined, the cortex and caudate; however, in the cerebellum, where cellular morphology is largely unchanged in human HD and inclusions are not observed in our adult onset cases, this change in localization did not occur (data not shown). There is also a noticeable increase in the intensity of immunostaining of the neuronal processes in HD brain compared with control brain (Fig. 4A compared with B). The commercial C-terminal N-CoR antibody AK12 gave similar results (data not shown).
Figure 4. Immunohistochemistry of control and HD brain using anti-N-CoR (1/100 dilution). (A and C) Control temporal cortex, (B and E) HD temporal cortex and (E) HD caudate. (A and B) ×100, (C and E) ×200 and (E) ×400.
hN-CoR interacts with a number of other proteins, including the mammalian homologues of the yeast Sin3 protein, mSin3A and 3B (42,43). Figure 5A and B shows anti-mSin3A immunohistochemistry from control frontal cortex, and Figure 5C and D shows similar immunohistochemistry from HD frontal cortex. It is clear that mSin3A is normally distributed within both the cytoplasm and the nucleus of large pyramidal neurons of the frontal cortex but that in HD cortex mSin3A immunoreactivity is excluded from neuronal nuclei. Figure 4D demonstrates hN-CoR immunoreactivity in the cytoplasm of some of the few neurons remaining in HD caudate and no nuclear staining was observed; the control tissue appears very different morphologically because of the long period of atrophy, and thus direct comparisons are difficult to make but the immunoreactivity looks very similar to that seen in the control cortex (Figs 4A and 5A), in that in some cells it is both cytoplasmic and nuclear and in others is only cytoplasmic (data not shown). Anti-mSin3A shows a distinctly aberrant clumping of immunoreactivity within the cellular cytoplasm compared with control tissue but no localization in the nucleus, apart from an occasional focal inclusion in the nucleus (Fig. 5E). Semi-serial sections from the same series of HD brains have been demonstrated to contain NIIs with ubiquitin and N-terminal huntingtin immunoreactivity (P. Thomas et al., submitted), and counting the NIIs with mSin3 immunoreactivity gives an estimate of 5% of the total NIIs with mSin3 immunoreactivity.
Figure 5. Immunohistochemistry of control and HD brain using anti-mSin3A (AK11, 1/200). (A and B) Control cortex, (C and D) HD cortex and (E) HD caudate. (A and C) ×100, (B and D) ×200 and (E) ×400.
DISCUSSION
Both the yeast two-hybrid system and in vitro studies using His-tagged fusion proteins indicate that C-N-CoR from rat or human brain binds specifically to rat and human huntingtin. Whilst only the N-terminus of huntingtin was used in the yeast two-hybrid screen, the C-rN-CoR and C-hN-CoR proteins with a His tag at their N-termini were capable of specifically attaching full-length huntingtin from a clarified, but non-purified, brain homogenate from fresh rat brain, post-mortem frozen human brain samples (Fig. 3) and neuroblastoma cells (data not shown). The cDNAs initially isolated for rN-CoR contained only the 3[prime] end of this 270 kDa protein (41), and the equivalent section of human N-CoR expressed in E.coli, C-hN-CoR, also interacted with huntingtin. This is not surprising given the high degree of homology of these proteins (Fig. 1).
This interaction is strongly repeat length dependent (Fig. 2B), and is dependent on more than just the polyglutamine repeat, as bait plasmids containing only the polyglutamine region with a few N-terminal amino acids did not interact with the activation domain plasmid carrying an N-CoR insert (Table 2). However, the increased binding of rN-CoR seen at longer repeat lengths indicates that binding affinity is modulated by polyglutamine repeat length. The rat huntingtin pulled out by the in vitro pull-down assay (Fig. 3) has only eight glutamines, so apparently normal huntingtin can interact with N-CoR. Examination of SBMA and DRPLA bait plasmids carrying normal and expanded CAG repeats indicated that these proteins did not interact with rN-CoR. Therefore, it appears that areas of huntingtin outside the polyglutamine repeat tract are important for interaction with N-CoR, and that this interaction is probably specific to huntingtin.
Recent work has established that the repressor complex in which N-CoR functions is common to a number of sequence-specific DNA-binding transcriptional repressors, including the unliganded thyroid hormone (T3R)/retinoic acid (RAR)/retinoid X receptor (RXR) dimers, Mad-Max dimers, RevErb and Dax1 orphan receptors, Pit1 and Ume6 (42-50) as well as others. N-CoR and the homologous silencing mediator of retinoid and thyroid hormone receptor (SMRT) are thought to link the sequence-specific DNA-binding moieties with proteins known to possess histone deacetylase activity (49,51). This link is mediated by interaction between these co-repressors and the mSin3A and mSin3B proteins, mammalian homologues of the yeast Sin3 protein (52,53) which interact with a series of proteins which include the histone deacetylases 1 and 2 (HDAC1 and HDAC2, respectively) (42-48,51). Histone deacetylation is thought to repress transcription by condensation of chromatin, thus preventing access of the basal transcription factors to promotors, and N-CoR also interacts directly with the basal transcription factors TFIIB, TAFII32 and TAFII70 (54).
Mapping of the domains within N-CoR has already revealed that the nuclear receptor interaction domain is located at the C-terminus of the protein (41), and this domain is included in both of the constructs isolated by yeast two-hybrid screening with huntingtin. The two major repression domains responsible for the transcriptional silencing are localized to the N-terminal region of the protein, neither of which are included in our constructs; these are the regions known to carry the Sin3-RPD3 interacting domains (41). So, if huntingtin interacts with the C-terminal domain, in a modular protein it seems feasible that the repression domains would be free to participate in interactions with their usual partners. Because of this confirmed link, we examined the localization of mSin3A as well as N-CoR in HD brain, to see whether either or both proteins were redistributed compared with control brain, and in particular to see whether either protein were being sequestered into NIIs through aberrant interaction with huntingtin.
Figure 4 shows the localization of hN-CoR in normal (Fig. 4A and C) and HD (Fig. 4B, D and E) brain, and Figure 5 of mSin3A in normal (Fig. 5A and B) and HD (Fig. 5C, D and E) brain. In the cortex and caudate, the localization of both proteins is clearly altered in HD brain. The immunostaining in control brain is very specific and indicates that these proteins do not occur everywhere, but are seen in neuronal cells. In some neuronal populations, they are cytoplasmic and in others they occur in both the nucleus and the cytoplasm. Immunoreactivity in normal caudate is quite sparse and specific, particularly localized to the larger neurons and occurring in nucleus and cytoplasm; staining is very similar to that shown in the control cortex (Figs 4A and 5A). The gross morphology of HD caudate is so changed by this stage of the disease (Vonsattel grade 3) that it is difficult to draw definite conclusions about the observed exclusion of these proteins from the nucleus of the surviving neurons, which could be a secondary result of the damage in the caudate (Figs 4E and 5E). In the HD cortex, the same cell populations appear to be immunoreactive for N-CoR and mSin3A as in the control tissue, but the localization of the protein within the cell is different. The most striking observation is that both proteins appear to be excluded from the nucleus in all immunoreactive neurons in HD brain. In HD cortex, ubiquitin antibodies and N-terminal huntingtin antisera reveal the presence of immunoreactive NIIs in the tissue analysed (P. Thomas et al., submitted) and a few of these inclusions are mSin3A positive on immunohistochemistry (Fig. 5E), but huntingtin-immunoreactive inclusions are much more frequent than mSin3A-immunoreactive inclusions. hN-CoR does not appear to be localized to the nuclear inclusions by immunohistochemistry. However, both the antisera used to localize hN-CoR were raised against the far C-terminus of the protein, and, as this is the area that interacts with huntingtin, the relevant epitope of hN-CoR may be masked. Unfortunately, more N-terminal hN-CoR antisera raised have cross-reactions which make them unsuitable for immunohistochemical localizations.
The exclusion of hN-CoR and mSin3A from the nucleus implies that their normal role in binding to sequence-specific receptors is likely to be disrupted and the transcriptional repression associated with the repressors alleviated. In mammalian cells, the magnitude of repression by unliganded receptors and by Mad/Mxi proteins is unknown but, in yeast, Rpd3 deletion strains show 2- to 5-fold defects in both repression and activation of endogenous genes (55). This is a small effect, but a small effect over a long period is the most likely defect in HD as this is generally a disease of adult onset, and the polyglutamine repeat length dependence of the N-CoR interaction makes this a good candidate for involvement in HD pathology. Perhaps the most interesting target of repression is the Mad-Max-binding domain which represses transcription from c-Myc-activated genes; c-Myc is an oncogene which can give rise to uncontrolled cell division by overcoming cell cycle checkpoints, but can also induce apoptosis (56), and may do so when expressed in terminally differentiated neurons. It is possible that huntingtin normally interacts with repressor complex proteins, and has a role in their transport within the cytoplasm (31,33,36,57,58). Transport of inclusions or soluble N-terminal fragments of huntingtin into the nucleus could be mediated by the transport of repressor proteins into the nucleus. The kinetics of association/disassociation of any co-repressor-huntingtin complex is likely to be affected by repeat length, and the disassociation kinetics may be altered such that the repressor complexes remain bound for longer and thus gradually become excluded from the nucleus. This is difficult to assess in in vitro binding assays, where both partners of the interaction are present at artificially high levels, although the yeast system can give an indication of the relative association/dissociation constants as it provides a functional measure of the effects of protein interactions. The question of the functional relevance of this interaction can best be addressed in cell culture and animal models of HD and through assaying transcriptional efficiency in the presence of huntingtin.
MATERIALS AND METHODS
Strains and plasmids
Saccharomyces cerevisiae strain Y190 and bait plasmid pGBT9 were purchased from Clontech. Control plasmids p53, pSV40 and pLAMINc were supplied by Stratagene. Bait plasmids HD17, HD40 and HD50 were generated by inserting 0.5 kb (nucleotides 316-823) of the HD cDNA containing 17, 40 and 50 CAG repeats, respectively, into the SmaI site of pGBT9. Plasmids JOE1/2, JOE1/3, JOE4/6, JOE5/6, JOE7/8, JOE10/12 and JOE11/12 containing other regions of the HD cDNA have been described elsewhere (35). Plasmids containing nucleotides 337-365 of the HD cDNA with either 20 or 57 CAG repeats followed by a stop codon were cloned into EcoRI-BamHI-cut pGBT9. The DRPLA bait plasmids were constructed from pDR64 containing nucleotides 1658-1784 of the DRPLA sequence (35) with 64 CAGs. All inserts were checked for orientation and correct frame by sequencing. Bait plasmids were transformed into yeast Y190 by the lithium acetate method, and plated onto SD-Trp plates. Transformants were also plated onto SD-Trp-His [supplemented with 25 mM 3-aminotriazole(3-AT)] to test for autoactivation of the HIS3 reporter gene.
Library screening
An adult male rat brain cDNA library constructed in the HybriZAP activation domain vector (Stratagene) has been described (35). A single colony of Y190 cells harbouring the HD17 or HD40 bait plasmid was grown overnight in SD-Trp, and transformed with the rat brain library. Transformants were plated on 6 × 150 mm diameter plates containing SD-His-Trp-Leu, supplemented with 25 mM 3-AT to overcome leaky expression of the HIS3 reporter gene in Y190. After incubation at 30°C for 7 days, replica filters were made for each plate, frozen in liquid nitrogen for 10 s and thawed to room temperature to permeabilize the cells. Filters were transferred onto other filters which had been soaked in Z buffer/X-gal solution (16.1 g/l Na2HPO4·7H2O, 5.5 g/l NaH2PO4·H2O, 0.75 g/l KCl, 0.246g/l MgSO4·7H2O, 2.7 ml/l [beta]-mercaptoethanol, 334 mg/l X-gal) and incubated at 30°C for a maximum of 24 h. [beta]-Galactosidase-positive clones were streaked onto SD-His-Trp-Leu and reassayed to confirm a His+LacZ+ phenotype. Positive clones were grown overnight in 2 ml of SD-Leu, and total yeast DNA was prepared. This was used to transform TSS-competent XLI-Blue cells, transformants being selected on LB plates containing carbenicillin. Single colonies were restreaked onto fresh LB plates, and a PCR assay was carried out using primers specific to the activation domain vector (Stratagene). Plasmid DNA was prepared (Qiagen) from clones positive on this PCR assay, and the inserts were sequenced (Sequenase v2.0). To check for the specificity of the interaction, isolated activation domain plasmids were transformed into Y190 in combination with pGBT9, HD17, HD50 and pLAMINc. Transformants were checked for growth on SD-His-Trp-Leu and assayed for [beta]-galactosidase as before.
Fusion protein expression
The N-CoR1 insert was released from the HybriZAP vector by digestion with EcoRI and XhoI, and blunt-end cloned into the SmaI site of pQE31 (Qiagen), creating pQENCoR1. This plasmid was transformed into XLI-Blue, and transformants selected on LB plates containing carbenicillin. Individual colonies were grown at 37°C overnight in 5 ml of LB containing carbenicillin, and this culture was used to inoculate 100 ml of LB. This was grown to an OD600 of 0.7-0.9 before expression was induced for 3 h with 2 mM isopropyl-[beta]-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation at 4000 g for 20 min at 4°C, and stored overnight at -20°C in 10 ml of sonication buffer with protease inhibitors [50 mM NaH2PO4, 300 mM NaCl, pH 7.8, COMPLETE protease inhibitors (Boehringer)]. After thawing in cold water, cells were sonicated and centrifuged at >10 000 g for 20 min, and the supernatant containing soluble proteins removed. The equivalent section of human huntingtin was cloned into BamHI-SalI-digested and dephosphorylated pQE30 using a BamHI-SalI-digested human N-CoR PCR product produced from primers VW01 (5[prime]-CCCGGATCCGATTCTAGTAGCTTATCTTCTCACAG-3[prime]) and VW02 (5[prime]-GCGGTCGACTCATCACTATCCGACAGGNCTCGAC-3[prime]) to give plasmid pQVW23. Expression was induced and soluble fractions generated as above.
Preparation of huntingtin samples
For in vitro binding studies, whole-cell extracts were prepared by homogenizing tissue samples in 5 vol of homogenization buffer with protease inhibitors (50 mM NaH2PO4, 300 mM NaCl, 0.5% Triton X-100, pH 7.8, COMPLETE protease inhibitors), using 20 strokes of a glass homogenizer. Homogenates were centrifuged at 1000 g for 5 min to produce a P1 pellet which was washed by resuspension in 5 vol of homogenization buffer and recentrifuged for 5 min at 1000 g. The two supernatants were combined to give an S1 fraction and stirred on ice for 20 min. The S1 fraction was clarified by centrifugation at 15 000 r.p.m. for 20 min and filtration through a 0.2 µm filter.
In vitro binding experiments
Aliquots of 500 or 250 µl of pQENCoR1 or pQVW23 protein sample were incubated with 500 or 250 µl of rat or human brain S1 fraction (final volume 1 ml) and 50 µl of a 50% slurry of Ni2+-agarose (Qiagen) with shaking at room temperature for 1 h. Bound proteins were collected by centrifugation for 10 s, and the Ni2+-agarose pellet washed with 3 × 1 ml of sonication buffer. Bound proteins were eluted with SDS sample buffer, separated by 4 or 6% SDS-PAGE and transferred onto nitrocellulose membrane. Filters were incubated with a polyclonal antibody raised against a peptide corresponding to the first 17 amino acids of huntingtin (1:1000 dilution), N-675 (35), followed by an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10 000 dilution). Huntingtin signal was detected using the HRPL detection system (National Diagnostics).
Immunohistochemistry
Antibodies to N-CoR were raised in rabbits using five immunizations of 200 µg of peptide JD1 (Fig. 1) attached to a polylysine core, and purchased from Santa Cruz Biotechnology (N-CoR C20) along with mSin3A polyclonal sera (AK11). Polyclonal antiserum N-675 was raised in rabbits as described previously (35). The serum was affinity purified and used at 1/50 in immunohistochemistry. Ubiquitin monoclonal antibody (mAb1510; Chemicon International) was also used at 1:50 dilution. HD and control brain were formalin fixed and paraffin embedded using standard techniques. Six HD and six control brains were used, with the HD brains having expanded repeats in the range 39-45 CAG, as detailed in the figure legends. Sections were processed by an indirect immunohistochemical method using the avidin-biotin complex kit with 3,3-diaminobenzidine (DAB) chromogen (Vectorfasin Elite ABC kits; Vector Laboratories) by standard techniques with minor modifications. Briefly, the sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxidase/methanol for 30 min to block endogenous peroxidase activity followed by two 5 min washes in phosphate-buffered saline (PBS) buffer. Sections were blocked with 10% (60 min) and 1% (5 min) normal serum and then incubated overnight at 4°C with primary antibody diluted as indicated in the figure legends. The sections were washed five times for 5 min in PBS and detected using biotinylated anti-species-specific immunoglobulins (1:200), avidin-biotin complex reagents and antibody reaction, then visualized with DAB (Vector Laboratories). After washing in H2O, the sections were dehydrated through graded alcohols, cleared in xylene and mounted with DPX under glass coverslips. Control sections included the omission of the primary antibody or competition controls with pre-absorbed antibodies treated with excess peptides or antigen.
ACKNOWLEDGEMENTS
The SBMA bait plasmids pAR24:15 and pAR66:16 were a kind gift from Drs K. Fischbeck and D. Merry (University of Pennsylvania, USA) and pDR64 a kind gift from Dr S. Tsuji (Niigata University, Japan). We are grateful to the MRC for a Centre Initiative in Neuropsychiatric Genetics and for a studentship (J.M.B.).
REFERENCES
+Present address: Zeneca Pharmaceuticals, Alderley Park, Cheshire SK10 4TG, UK
§To whom correspondence should be addressed. Tel: +44 1222 745175; Fax: +44 1222 747603; Email: jonesl1{at}cf.ac.uk
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification:
Copyright© Oxford University Press, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M Futter, H Diekmann, E Schoenmakers, O Sadiq, K Chatterjee, and D C Rubinsztein Wild-type but not mutant huntingtin modulates the transcriptional activity of liver X receptors J. Med. Genet., July 1, 2009; 46(7): 438 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Runne, E. Regulier, A. Kuhn, D. Zala, O. Gokce, V. Perrin, B. Sick, P. Aebischer, N. Deglon, and R. Luthi-Carter Dysregulation of Gene Expression in Primary Neuron Models of Huntington's Disease Shows That Polyglutamine-Related Effects on the Striatal Transcriptome May Not Be Dependent on Brain Circuitry J. Neurosci., September 24, 2008; 28(39): 9723 - 9731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kuhn, D. R. Goldstein, A. Hodges, A. D. Strand, T. Sengstag, C. Kooperberg, K. Becanovic, M. A. Pouladi, K. Sathasivam, J.-H. J. Cha, et al. Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage Hum. Mol. Genet., August 1, 2007; 16(15): 1845 - 1861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. O. Evert, J. Araujo, A. M. Vieira-Saecker, R. A. I. de Vos, S. Harendza, T. Klockgether, and U. Wullner Ataxin-3 Represses Transcription via Chromatin Binding, Interaction with Histone Deacetylase 3, and Histone Deacetylation. J. Neurosci., November 1, 2006; 26(44): 11474 - 11486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Bates, M. Victor, A. K. Jones, Y. Shi, and A. C. Hart Differential contributions of Caenorhabditis elegans histone deacetylases to huntingtin polyglutamine toxicity. J. Neurosci., March 8, 2006; 26(10): 2830 - 2838. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kumar, A. E. Gururaj, R. K. Vadlamudi, and S. K. Rayala The Clinical Relevance of Steroid Hormone Receptor Corepressors Clin. Cancer Res., April 15, 2005; 11(8): 2822 - 2831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Lievens, T. Rival, M. Iche, H. Chneiweiss, and S. Birman Expanded polyglutamine peptides disrupt EGF receptor signaling and glutamate transporter expression in Drosophila Hum. Mol. Genet., March 1, 2005; 14(5): 713 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Everett and N. W. Wood Trinucleotide repeats and neurodegenerative disease Brain, November 1, 2004; 127(11): 2385 - 2405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Schilling, A. V. Savonenko, A. Klevytska, J. L. Morton, S. M. Tucker, M. Poirier, A. Gale, N. Chan, V. Gonzales, H. H. Slunt, et al. Nuclear-targeting of mutant huntingtin fragments produces Huntington's disease-like phenotypes in transgenic mice Hum. Mol. Genet., August 1, 2004; 13(15): 1599 - 1610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Tsai, H.-Y. Kao, A. Mitzutani, E. Banayo, H. Rajan, M. McKeown, and R. M. Evans Ataxin 1, a SCA1 neurodegenerative disorder protein, is functionally linked to the silencing mediator of retinoid and thyroid hormone receptors PNAS, March 23, 2004; 101(12): 4047 - 4052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ishihara, N. Yamagishi, Y. Saito, H. Adachi, Y. Kobayashi, G. Sobue, K. Ohtsuka, and T. Hatayama Hsp105{alpha} Suppresses the Aggregation of Truncated Androgen Receptor with Expanded CAG Repeats and Cell Toxicity J. Biol. Chem., June 27, 2003; 278(27): 25143 - 25150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. C. Nucifora Jr., L. M. Ellerby, C. L. Wellington, J. D. Wood, W. J. Herring, A. Sawa, M. R. Hayden, V. L. Dawson, T. M. Dawson, and C. A. Ross Nuclear Localization of a Non-caspase Truncation Product of Atrophin-1, with an Expanded Polyglutamine Repeat, Increases Cellular Toxicity J. Biol. Chem., April 4, 2003; 278(15): 13047 - 13055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Okuda, H. Hattori, S. Takeuchi, J. Shimizu, H. Ueda, J. J. Palvimo, I. Kanazawa, H. Kawano, M. Nakagawa, and H. Okazawa PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype Hum. Mol. Genet., April 1, 2003; 12(7): 711 - 725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gines, I. S. Seong, E. Fossale, E. Ivanova, F. Trettel, J. F. Gusella, V. C. Wheeler, F. Persichetti, and M. E. MacDonald Specific progressive cAMP reduction implicates energy deficit in presymptomatic Huntington's disease knock-in mice Hum. Mol. Genet., March 1, 2003; 12(5): 497 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Li, T. Macfarlan, R. N. Pittman, and D. Chakravarti Ataxin-3 Is a Histone-binding Protein with Two Independent Transcriptional Corepressor Activities J. Biol. Chem., November 15, 2002; 277(47): 45004 - 45012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. R. Singaraja, S. Hadano, M. Metzler, S. Givan, C. L. Wellington, S. Warby, A. Yanai, C.-A. Gutekunst, B. R. Leavitt, H. Yi, et al. HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis Hum. Mol. Genet., November 1, 2002; 11(23): 2815 - 2828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Kazemi-Esfarjani and S. Benzer Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1 Hum. Mol. Genet., October 2, 2002; 11(21): 2657 - 2672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Fossale, V. C. Wheeler, V. Vrbanac, L.-A. Lebel, A. Teed, J. S. Mysore, J. F. Gusella, M. E. MacDonald, and F. Persichetti Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice Hum. Mol. Genet., September 15, 2002; 11(19): 2233 - 2241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kita, J. Carmichael, J. Swartz, S. Muro, A. Wyttenbach, K. Matsubara, D. C. Rubinsztein, and K. Kato Modulation of polyglutamine-induced cell death by genes identified by expression profiling Hum. Mol. Genet., September 15, 2002; 11(19): 2279 - 2287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Bennett, K. E. Huey-Tubman, A. B. Herr, A. P. West Jr., S. A. Ross, and P. J. Bjorkman Inaugural Article: A linear lattice model for polyglutamine in CAG-expansion diseases PNAS, September 3, 2002; 99(18): 11634 - 11639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Luthi-Carter, S. A. Hanson, A. D. Strand, D. A. Bergstrom, W. Chun, N. L. Peters, A. M. Woods, E. Y. Chan, C. Kooperberg, D. Krainc, et al. Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain Hum. Mol. Genet., August 15, 2002; 11(17): 1911 - 1926. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Luthi-Carter, A. D. Strand, S. A. Hanson, C. Kooperberg, G. Schilling, A. R. La Spada, D. E. Merry, A. B. Young, C. A. Ross, D. R. Borchelt, et al. Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects Hum. Mol. Genet., August 15, 2002; 11(17): 1927 - 1937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Y.W. Chan, R. Luthi-Carter, A. Strand, S. M. Solano, S. A. Hanson, M. M. DeJohn, C. Kooperberg, K. O. Chase, M. DiFiglia, A. B. Young, et al. Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease Hum. Mol. Genet., August 15, 2002; 11(17): 1939 - 1951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Lieberman, G. Harmison, A. D. Strand, J. M. Olson, and K. H. Fischbeck Altered transcriptional regulation in cells expressing the expanded polyglutamine androgen receptor Hum. Mol. Genet., August 15, 2002; 11(17): 1967 - 1976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Takahashi, H. Fujigasaki, C. Zander, K. H. El Hachimi, G. Stevanin, A. Durr, A.-S. Lebre, G. Yvert, Y. Trottier, H. d. The, et al. Two populations of neuronal intranuclear inclusions in SCA7 differ in size and promyelocytic leukaemia protein content Brain, July 1, 2002; 125(7): 1534 - 1543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Dunah, H. Jeong, A. Griffin, Y.-M. Kim, D. G. Standaert, S. M. Hersch, M. M. Mouradian, A. B. Young, N. Tanese, and D. Krainc Sp1 and TAFII130 Transcriptional Activity Disrupted in Early Huntington's Disease Science, June 21, 2002; 296(5576): 2238 - 2243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-X. Yu, S.-H. Li, H.-P. Nguyen, and X.-J. Li Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice Hum. Mol. Genet., April 15, 2002; 11(8): 905 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Li, A. L. Cheng, H. Zhou, S. Lam, M. Rao, H. Li, and X.-J. Li Interaction of Huntington Disease Protein with Transcriptional Activator Sp1 Mol. Cell. Biol., March 1, 2002; 22(5): 1277 - 1287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Kegel, A. R. Meloni, Y. Yi, Y. J. Kim, E. Doyle, B. G. Cuiffo, E. Sapp, Y. Wang, Z.-H. Qin, J. D. Chen, et al. Huntingtin Is Present in the Nucleus, Interacts with the Transcriptional Corepressor C-terminal Binding Protein, and Represses Transcription J. Biol. Chem., February 22, 2002; 277(9): 7466 - 7476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Jepsen and M. G. Rosenfeld Biological roles and mechanistic actions of co-repressor complexes J. Cell Sci., February 15, 2002; 115(4): 689 - 698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Khoshnan, J. Ko, and P. H. Patterson Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity PNAS, January 22, 2002; 99(2): 1002 - 1007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Yvert, K. S. Lindenberg, D. Devys, D. Helmlinger, G. B. Landwehrmeyer, and J.-L. Mandel SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types Hum. Mol. Genet., August 1, 2001; 10(16): 1679 - 1692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakamura, S.-Y. Jeong, T. Uchihara, M. Anno, K. Nagashima, T. Nagashima, S.-i. Ikeda, S. Tsuji, and I. Kanazawa SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein Hum. Mol. Genet., July 1, 2001; 10(14): 1441 - 1448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Jana, E. A. Zemskov, G.-h. Wang, and N. Nukina Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release Hum. Mol. Genet., May 1, 2001; 10(10): 1049 - 1059. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Holbert, I. Denghien, T. Kiechle, A. Rosenblatt, C. Wellington, M. R. Hayden, R. L. Margolis, C. A. Ross, J. Dausset, R. J. Ferrante, et al. The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: Neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis PNAS, January 24, 2001; (2001) 41566798. [Abstract] [Full Text]
|
|
|
|
|
|
P. Hilditch-Maguire, F. Trettel, L. A. Passani, A. Auerbach, F. Persichetti, and M. E. MacDonald Huntingtin: an iron-regulated protein essential for normal nuclear and perinuclear organelles Hum. Mol. Genet., November 1, 2000; 9(19): 2789 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Trettel, D. Rigamonti, P. Hilditch-Maguire, V. C. Wheeler, A. H. Sharp, F. Persichetti, E. Cattaneo, and M. E. MacDonald Dominant phenotypes produced by the HD mutation in STHdhQ111 striatal cells Hum. Mol. Genet., November 1, 2000; 9(19): 2799 - 2809. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Wood, F. C. Nucifora Jr., K. Duan, C. Zhang, J. Wang, Y. Kim, G. Schilling, N. Sacchi, J. M. Liu, and C. A. Ross Atrophin-1, the Dentato-Rubral and Pallido-Luysian Atrophy Gene Product, Interacts with Eto/Mtg8 in the Nuclear Matrix and Represses Transcription J. Cell Biol., September 4, 2000; 150(5): 939 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Passani, M. T. Bedford, P. W. Faber, K. M. McGinnis, A. H. Sharp, J. F. Gusella, J.-P. Vonsattel, and M. E. MacDonald Huntingtin's WW domain partners in Huntington's disease post-mortem brain fulfill genetic criteria for direct involvement in Huntington's disease pathogenesis Hum. Mol. Genet., September 1, 2000; 9(14): 2175 - 2182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. McCampbell, J. P. Taylor, A. A. Taye, J. Robitschek, M. Li, J. Walcott, D. Merry, Y. Chai, H. Paulson, G. Sobue, et al. CREB-binding protein sequestration by expanded polyglutamine Hum. Mol. Genet., September 1, 2000; 9(14): 2197 - 2202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Luthi-Carter, A. Strand, N. L. Peters, S. M. Solano, Z. R. Hollingsworth, A. S. Menon, A. S. Frey, B. S. Spektor, E. B. Penney, G. Schilling, et al. Decreased expression of striatal signaling genes in a mouse model of Huntington's disease Hum. Mol. Genet., May 22, 2000; 9(9): 1259 - 1271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Cummings and H. Y. Zoghbi Fourteen and counting: unraveling trinucleotide repeat diseases Hum. Mol. Genet., April 1, 2000; 9(6): 909 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Metzler, C. D. Helgason, I. Dragatsis, T. Zhang, L. Gan, N. Pineault, S. O. Zeitlin, R. K. Humphries, and M. R. Hayden Huntingtin is required for normal hematopoiesis Hum. Mol. Genet., February 12, 2000; 9(3): 387 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. Peters and C. A. Ross Isolation of a 40-kDa Huntingtin-associated Protein J. Biol. Chem., January 26, 2001; 276(5): 3188 - 3194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Hackam, A. S. Yassa, R. Singaraja, M. Metzler, C.-A. Gutekunst, L. Gan, S. Warby, C. L. Wellington, J. Vaillancourt, N. Chen, et al. Huntingtin Interacting Protein 1 Induces Apoptosis via a Novel Caspase-dependent Death Effector Domain J. Biol. Chem., December 22, 2000; 275(52): 41299 - 41308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Khoshnan, J. Ko, and P. H. Patterson Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity PNAS, January 22, 2002; 99(2): 1002 - 1007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Holbert, I. Denghien, T. Kiechle, A. Rosenblatt, C. Wellington, M. R. Hayden, R. L. Margolis, C. A. Ross, J. Dausset, R. J. Ferrante, et al. The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: Neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis PNAS, February 13, 2001; 98(4): 1811 - 1816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Hughes, R. S. Lo, C. Davis, A. D. Strand, C. L. Neal, J. M. Olson, and S. Fields Altered transcription in yeast expressing expanded polyglutamine PNAS, November 6, 2001; 98(23): 13201 - 13206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. McCampbell, A. A. Taye, L. Whitty, E. Penney, J. S. Steffan, and K. H. Fischbeck Histone deacetylase inhibitors reduce polyglutamine toxicity PNAS, December 18, 2001; 98(26): 15179 - 15184. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||