The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington's disease

Abstract

The transcription factor REST silences neuronal gene expression in non-neuronal cells. In neurons, the protein is sequestered in the cytoplasm in part through binding to huntingtin. Polyglutamine expansions in huntingtin, which causes Huntington's disease (HD), abrogates REST-huntingtin binding. Consequently, REST translocates to the nucleus, occupies RE1 repressor sequences and decreases neuronal gene expression. In this work, we found that levels of several microRNAs (miRNAs) with upstream RE1 sites are decreased in HD patient cortices relative to healthy controls. Interestingly, one of these, the bifunctional brain enriched miR-9/miR-9*, targets two components of the REST complex: miR-9 targets REST and miR-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.

Figure 1.

Several REST-regulated miRNA levels change with HD progression, and miR-9/9* targets a silencing complex. A, Longitudinal expression profiles of REST-regulated miRNAs in HD and healthy control cortex (BA4) HD grade 1, 2, and 3/4 as measured by QPCR. Mature miRNA levels in HD brain relative to controls (*p < 0.05, **p < 0.01). B, C, miR-9/9* reduces expression of reporter proteins containing the 3′ UTR of REST or CoREST. B, HEK 293 cells were cotransfected with a vector expressing miR-9/9* (mU6 miR-9/9*) and REST 3′ UTR luciferase reporters. Relative luciferase activity was measured 24 h later. Increasing doses of mU6 miR-9/9* relative to target caused a dose-dependent decrease in relative luciferase. Control vectors containing a single site with perfect complementarity to mature miR-9 (miR-9-PT) or miR-9* (miR-9*-PT) were included as positive controls (n = 3). C, Experiments as in (B) with CoREST 3′ UTR luciferase reporters (n = 3). D, E, Transfection of HEK 293 cells with mU6 miR-9/9* reduces both REST and CoREST protein expression levels 24 h later. D, Representative Western blots and densitometry for (E) REST (n = 5) and CoREST (n = 5). Histograms show mean ±SEM after normalization to β-actin levels. *p < 0.05, **p < 0.01.

Figure 2.

miR-9 targets the 3′ UTR of REST. A, Cotransfection of HEK 293 cells with luciferase reporters containing the 3′ UTR of REST in the presence of increasing concentrations (0.1, 1.0, 5.0, or 15.0 nm) of artificial pre-miR-9 reduces relative luciferase activity (n = 4). B, C, Knockdown of REST following transfection of HEK 293 cells with pre-miR-9 (30 nm). A representative Western blot (B) and the densitometry from (n = 4) independent experiments (C) after normalization to β-actin. Data are mean ±SEM; *p < 0.05, **p < 0.01. D, A representative Western blot showing that target site protectors (TSP) designed to block the predicted miR-9 binding site of REST (TSP +) rescues mU6 miR-9/9*-mediated knockdown of REST. HEK 293 cells were cotransfected with mU6 miR-9/9* and TSP+ or TSP negative control (Neg) and REST levels measured in cell lysates 24 h later. Densitometry from n = 4 independent experiments indicate increased REST in TSP+ (0.97 ± 0.065)-treated versus TSP Neg (0.54 ± 0.212)-treated cells.

Figure 3.

miR-9* targets the 3′ UTR of CoREST. A, Cotransfection of HEK 293 cells with luciferase reporters containing the 3′ UTR of CoREST in the presence of increasing concentrations (0.1, 1.0, 5.0, or 15.0 nm) of artificial pre-miR-9* reduces relative luciferase activity (n = 5). B, C, CoREST levels are reduced following transfection of HEK 293 cells with pre-miR-9* (30 nm). A representative Western blot (B) and densitometry (n = 4) (C) after normalized β-actin levels. Data are mean ± SEM; *p < 0.05, **p < 0.01. D, TSP designed the predicted miR-9* binding site of CoREST (TSP +) rescues pre-miR-9* mediated knockdown (B). HEK 293 cells were cotransfected with pre-miR-9* (15 nm) and TSP+ or TSP Neg and CoREST levels measured in cell lysates by Western blot 24 h later. Densitometry from n = 4 independent experiments indicate increased CoREST in TSP+ (1.02 ± 0.05)-treated versus TSP Neg (0.565 ± 0.08)-treated cells.