Huntingtin regulates RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting LIM domain protein (RILP) and dynactin p150 Glued - PubMed (original) (raw)

Huntingtin regulates RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting LIM domain protein (RILP) and dynactin p150 Glued

Masahito Shimojo. J Biol Chem. 2008.

Abstract

Huntingtin has been reported to regulate the nuclear translocation of the transcriptional repressor RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). The REST/NRSF-interacting LIM domain protein (RILP) has also been shown to regulate REST/NRSF nuclear translocation. Therefore, we were prompted to address the question of how two distinct proteins could have the same function. We initially used a yeast two-hybrid screen to look for an interaction between huntingtin and RILP. This screen identified dynactin p150(Glued) as an interacting protein. Coimmunoprecipitation of proteins in vitro expressed in a reticulocyte lysate system showed an interaction between REST/NRSF and RILP as well as between RILP and dynactin p150(Glued). Coimmunoprecipitation analysis further showed a complex containing RILP, dynactin p150(Glued), and huntingtin. Huntingtin did not interact directly with either REST/NRSF or RILP, but did interact with dynactin p150(Glued). The N-terminal fragment of wild-type huntingtin did not affect the interaction between dynactin p150(Glued) and RILP; however, mutant huntingtin weakened this interaction. We further show that HAP1 (huntingtin-associated protein-1) prevents this complex from translocating REST/NRSF to the nucleus. Thus, this study suggests that REST/NRSF, dynactin p150(Glued), huntingtin, HAP1, and RILP form a complex involved in the translocation of REST/NRSF into the nucleus and that HAP1 controls REST/NRSF cellular localization in neurons.

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Figures

FIGURE 1.

FIGURE 1.

Pulldown assays for analyzing the interaction of RILP and dynactin p150Glued. His-tagged RILP and FLAG-tagged dynactin p150Glued were coexpressed in the TNT coupled reticulocyte lysate system. The lysate was applied to a Ni-NTA-agarose column, and the various fractions were subjected to SDS-PAGE, followed by Western blot analysis using anti-FLAG (upper panels) or anti-His (lower panels) antibody. S, starting lysate; P, passthrough; E, eluate.

FIGURE 2.

FIGURE 2.

Coimmunoprecipitation of RILP with dynactin p150Glued and huntingtin from HeLa cells. HeLa cell lysates were prepared in lysis buffer and subjected to immunoprecipitation with anti-RILP, anti-dynactin p150Glued (p150), anti-huntingtin (Htt), or nonimmune antiserum, respectively. Nonimmune antibodies were from goat and rabbit. Aliquots of immunoprecipitates were subjected to Western blot analysis with anti-RILP, anti-dynactin p150Glued, or anti-huntingtin antibodies. INPUT is one-tenth of what was applied to immunoprecipitation.

FIGURE 3.

FIGURE 3.

Analysis of the direct interaction of huntingtin and REST/NRSF. Proteins were coexpressed in the TNT coupled reticulocyte lysate system. Lysates were subjected to immunoprecipitation (IP) with anti-RILP (α_-RILP_), anti-dynactin p150Glued (α_-p150_), anti-huntingtin (α_-Htt_), or anti-NRSF (α_-NRSF_) antibodies. Aliquots were subjected to Western blot analysis (5% SDS-polyacrylamide gel) with the indicated antibodies. + and -, presence and absence, respectively, of each protein in the lysate; S, starting lysate for immunoprecipitation; wt, wild-type; mut, mutant. The input was one-fifth of what was applied to immunoprecipitation.

FIGURE 4.

FIGURE 4.

RILP interacts with huntingtin through dynactin p150Glued. Each protein was coexpressed and subjected to immunoprecipitation (IP) using the appropriate antibodies as shown in Fig. 3. Immunoprecipitation in the absence (a) or presence (b) of dynactin p150Glued was carried out with anti-RILP (α_-RILP_), anti-dynactin p150Glued (α_-p150_), or anti-huntingtin (α_-Htt_) antibodies. Aliquots of immunoprecipitates were subjected to Western blot analysis with anti-RILP, anti-p150, or anti-Htt antibodies. + and -, presence and absence, respectively, of each protein in the lysate; S, starting lysate for immunoprecipitation;wild, wild-type; mut, mutant. The input was one-fifth of what was applied to immunoprecipitation.

FIGURE 5.

FIGURE 5.

Effect of HAP1 on the interaction between RILP and huntingtin. a, RILP coimmunoprecipitated with dynactin p150Glued, HAP1, and huntingtin in NT2 cells. NT2 cell lysates were prepared in lysis buffer and subjected to immunoprecipitation (IP) with anti-RILP, anti-dynactin p150Glued (p150), anti-huntingtin (Htt), anti-HAP1, or nonimmune antiserum. Nonimmune antibodies were from goat and rabbit. Aliquots of immunoprecipitates were subjected to Western blot analysis with antibodies. INPUT is one-tenth what was applied to immunoprecipitation. b, proteins were coexpressed and subjected to immunoprecipitation using anti-huntingtin or anti-RILP antibodies as described in the legend to Fig. 3. Immunoprecipitation was carried out with anti-RILP (α_-RILP_) or anti-huntingtin (α_-Htt_) antibodies. Aliquots of immunoprecipitates were subjected to Western blot analysis with anti-RILP or anti-Htt antibodies. + and -, presence and absence, respectively, of each protein in the lysate; S, starting lysate for immunoprecipitation. The input was one-fifth of what was applied to immunoprecipitation.c, the interaction between REST/NRSF and HAP1 or RILP and HAP1 was analyzed. Immunoprecipitation was carried out with anti-HAP1 (α_-HAP_), anti-RILP, or anti-NRSF (α_-NRSF_) antibodies. Aliquots of immunoprecipitates were subjected to Western blot analysis with anti-RILP, anti-NRSF, or anti-HAP1 antibodies.

FIGURE 6.

FIGURE 6.

Coimmunoprecipitation of complexes from mouse striatal cells expressing wild-type or mutant huntingtin. Cell lysates were prepared in lysis buffer and subjected to immunoprecipitation with anti-RILP, anti-dynactin p150Glued (p150), anti-huntingtin (Htt), or nonimmune antiserum. Nonimmune antibodies were from goat and rabbit. Aliquots of immunoprecipitates were subjected to Western blot analysis with anti-RILP (α_-RILP_), anti-dynactin p150Glued (α_-p150_), or anti-huntingtin (α_-Htt_) antibodies.INPUT is one-tenth of what was applied to immunoprecipitation.

FIGURE 7.

FIGURE 7.

Effect of HAP1 on the expression of reporter gene constructs containing an RE1/NRSE element in HeLa and NT2 cells. Reporter gene assays were performed using the 5′-noncoding region of the human choline acetyltransferase gene driving the luciferase reporter gene. Reporter constructs were pXP2-EX, which contains a cholinergic gene locus fragment with an active RE1/NRSE, and pXP2-EXmut, which contains a mutant inactive RE1/NRSE. Constructs were transiently transfected into the indicated cell lines as described under “Experimental Procedures.” Cells were transfected with HAP1 expression plasmid (+HAP1) or siRNA for HAP1. For the control, vehicle pcDNA3.1 plasmid or a scrambled siRNA was also transfected. After 24 h, reporter gene constructs with β-galactosidase plasmid were transfected with Effectene reagent. After transfection, cells were harvested, and extracts were prepared for assays of luciferase activity.a, luciferase activity is reported as the relative reporter activity normalized to β-galactosidase activity. Data are the means ± S.E. (n = 6). b, each lysate expressing HAP1 was subjected to SDS-PAGE, followed by Western blot analysis using anti-HAP1 or anti-tubulin antibodies.

FIGURE 8.

FIGURE 8.

Effect of HAP1 expression on REST/NRSF localization. HeLa and NT2 cells were grown on a coverslip and transfected with HAP1 siRNA or HAP1 expression vector. After 24 h of culture, REST/NRSF was transfected, followed by culturing for an additional 24 h. The cells were then fixed with dry ice/cold methanol and incubated with anti-REST/NRSF (12C11-1) and anti-HAP1 antibodies followed by fluorescently labeled IgG. Red fluorescence is shown as REST/NRSF; green fluorescence is due to RILP; and_blue_ fluorescence is due to nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Upper panels, cells transfected with REST/NRSF as well as with a non-related sequence control siRNA; middle panels, cells treated with HAP1 following transfection with REST/NRSF; lower panels, cells transfected with HAP1 siRNA followed transfecting with REST/NRSF. The subcellular distribution of REST was examined by confocal microscopy as described under “Experimental Procedures.” The relative staining intensities yielded the following percentages of nuclear REST in HeLa cells: 91.24 ± 3.17% (+REST), 20.19 ± 4.42% (+REST and HAP1), and 91.86 ± 1.70% (+REST and HAP1 siRNA). The percentages of nuclear REST in NT2 cells were as follows: 9.73 ± 2.09% (+REST), 9.71 ± 2.20% (+REST and HAP1), and 81.73 ± 2.92% (+REST and HAP1 siRNA).

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