The role of LANP and ataxin 1 in E4F-mediated transcriptional repression

Abstract

The leucine-rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1--a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)--we tested whether ataxin 1 can alter the E4F-LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP-E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1.

Figure 1

Delimiting the interaction between E4F and LANP. (A) Interaction of full-length bait LANP with prey E4F constructs (full-length E4F (E4F 1–773) or deletion constructs as indicated, with zinc-finger motifs shown as black boxes). (B) Full-length (FL), amino-terminal (ΔN) and carboxy-terminal (ΔC) terminal deletion constructs of LANP were tested as interacting baits against full-length E4F as prey. Interaction was scored on the basis of relative growth on auxotrophic (4−) selection media (−Trp/−Leu/−His/−Ade); the presence of both bait and prey plasmids is recorded by growth on nonselective (2−) media (−Trp/−Leu). LANP, leucine-rich acidic nuclear protein; LRR, leucine-rich-repeat domain.

Figure 2

LANP and E4F interact in mammalian cells. (A–E) Confocal immunofluorescence of HeLa cells showing that endogenous LANP and E4F colocalize. Stained for (A) E4F, (B) LANP, (C) DAPI (nuclear), and (D) A and B merged. (E) Fluorescence intensity profile over a random cross-section of the merged image in (D) shows a correlation of intensities of fluorophores used to visualize E4F and LANP. (F,G) Co-precipitation of S-tagged E4F and Myc-tagged LANP in HeLa cells. Western blots (WB) showing the proteins in the immunoprecipitates. (H) In N2A cells, endogenous LANP is precipitated with S-beads when S-tagged E4F is expressed. (I) Chromatin immunoprecipitation (IP) showing that LANP–Myc and S-E4F occupy E4 promoter. (J) Real-time PCR to quantify the amount of pGL-E4 immunoprecipitated with LANP. DAPI, 4,6-diamidino-2-phenylindole; LANP, leucine-rich acidic nuclear protein.

Figure 3

LANP and E4F synergistically repress the E4 promoter. The effects of E4F and LANP on (A) pGL-E4 and (B) pGL-E4 mut promoter activity in CV-1 cells. The left side of each panel shows the activity expressed from pGL-E4 alone (black bars) or with increasing amounts (25, 50, 100, 150 ng) of E4F or LANP expression plasmids (colour shaded). The right side of each panel shows the effect of coexpression of E4F and LANP on pGL-E4 activity. The amount of E4F (E) and/or LANP (L) expression plasmid in each transfection is indicated by colour. The asterisk indicates synergistic repression. LANP, leucine-rich acidic nuclear protein.

Figure 4

LANP is required for E4F-mediated repression. (A) N2A cells were transiently transfected with E4F, pGL-E4 and siRNA targeting LANP (or control siRNA). Expressed in terms of percentage inhibition, it shows that E4F causes significantly less repression when LANP is depleted (P<0.05 for siRNA#1 and siRNA#3). The inset shows the data presented in the form of relative luciferase activity. Depletion of LANP by two distinct siRNA targeting LANP (#SP1 and #3) relieved exogenous E4F repression (lanes 3 and 4 compared with lane 2; P<0.05), and also relieved baseline repression of the reporter by targeting LANP complexed to endogenous E4F (lanes 5 and 6 compared with lane 1; P<0.05). The asterisks indicate statistical significance P<0.05. (B) Western bloting (WB) shows the level of overexpression of E4F (approximately 30-fold) and the depletion of LANP (greater than 60%). (C) Reducing LANP decreases E4F binding to the E4 promoter. Chromatin immunoprecipitation (IP) with S-beads was carried out on HeLa cells transfected with siRNA targeting LANP (or control siRNA) and S-E4F. Western blots show the level of E4F overexpression and LANP depletion. (D) Quantitative PCR of (C). LANP, leucine-rich acidic nuclear protein; RLU, relative luciferase units; siRNA, short interfering RNA.

Figure 5

Ataxin 1 relieves the transcriptional repression induced by the LANP–E4F complex. (A) Ataxin 1 relieves E4F-induced inhibition. N2A cells were transfected with the indicated constructs. E4F-induced repression relieved by ataxin 1 (compare histograms 3 and 2; P<0.05) could be restored by the addition of exogenous LANP (compare histograms 4 and 3; P<0.05). Ataxin 1 and LANP alone are shown as controls in histograms 5 and 6. (B) The amount of LANP bound to E4F is reduced in the presence of mutant ataxin 1. Co-precipitation of LANP with S-beads was carried out in HeLa cells transfected with the indicated constructs. (C) Chromatin immunoprecipitation (IP) with a Myc antibody that precipitates Myc-tagged LANP. (D) Quantitative PCR of (C). LANP, leucine-rich acidic nuclear protein; WB, western blot.