Yin Yang 1 is a repressor of glutamate transporter EAAT2, and it mediates manganese-induced decrease of EAAT2 expression in astrocytes

Abstract

Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.

FIG 1

YY1 is a negative regulator of EAAT2, and it mediates Mn-induced repression of EAAT2. (A) Astrocytes were cotransfected overnight with 0.5 μg of EAAT2 luciferase plasmid and 0.1 μg of either the pcDNA control vector or YY1, followed by a luciferase assay to determine EAAT2 promoter activity as described in Materials and Methods. (B) The YY1 consensus site (+34) in the EAAT2 promoter was mutated by site-directed mutagenesis, and the promoter activity of the YY1 mutant (YY1m) of EAAT2 was compared with that of the wild-type EAAT2 by luciferase assay. (C) Astrocytes were transfected with YY1 siRNA or a scrambled control siRNA (scRNA) for 48 h, followed by a luciferase assay (C₁). The YY1 mRNA levels from qPCR (C₂) and YY1 protein levels from Western blotting (C₃) were measured to determine the efficiency of YY1 siRNA knockdown. (D) Astrocytes were treated with Mn (250 μM) for the indicated time periods, and EAAT2 promoter activity was measured by luciferase assay. (E) After overnight transfection with the wild-type or YY1 mutant EAAT2 promoter vector, astrocytes were treated with Mn (250 μM) for 6 h, followed by a luciferase assay. #, P < 0.05; ##, P < 0.01; ###, P < 0.001; *, P < 0.05; **, P < 0.01 (ANOVA followed by Tukey's post hoc test; n = 3). WT, wild type.

FIG 2

Mn increases YY1 expression. (A) Confocal image showing YY1 and GFAP expression in rat primary astrocytes. (B) After overnight transfection with the EAAT2 promoter vector, astrocytes were treated with Mn (0, 125, and 250 μM) for 6 h, followed by a luciferase assay. (C and D) Astrocytes were treated with Mn (with 250 μM for up to 3 h or for 3 h with up to 250 μM), followed by measurement of YY1 mRNA levels by qPCR (C) and conventional reverse transcription-PCR (D) using GAPDH as a control. (E) After treatment with Mn (250 μM), astrocytes were lysed, and YY1 protein levels were measured in whole-astrocyte lysates (top panel) or nuclear extract (bottom panel) by Western blotting. Equal amounts (30 μg) of cell lysates or nuclear extracts were loaded using β-actin and histone H3 as internal controls. *, P < 0.05; **, P < 0.01 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 3

Mn treatment releases TNF-α in astrocytes. (A) Astrocytes were treated with Mn (250 μM) for 6 h, and the release of TNF-α was determined by ELISA. (B) After overnight transfection with EAAT2 promoter vector, astrocytes were treated with 15 ng/ml of TNF-α for 6 h, followed by a luciferase assay. (C) After overnight transfection with 0.5 μg of YY1 promoter, astrocytes were treated with Mn (250 μM) or TNF-α (15 ng/ml) for 6 h, followed by a luciferase assay. (D and E) Astrocytes were treated with TNF-α (15 ng/ml) for the indicated periods of time and YY1 mRNA (D) and protein (E) levels were measured using quantitative PCR and Western blotting, respectively. **, P < 0.01; ***, P < 0.001; #, P < 0.05 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 4

Mn recruits YY1 to the EAAT2 promoter. (A) EMSA was performed in nuclear extracts prepared from astrocytes treated with Mn (250 μM for 6 h) as described in Materials and Methods. The arrowhead shows the DNA-protein complex. (B) DAPA was performed with nuclear extracts prepared from astrocytes treated with Mn (250 μM for 6 h), and the YY1 consensus sequence-bound protein was subjected to Western blotting to probe YY1. As an input control (C), 10 μg of nuclear extract was used. (C) Astrocytes were treated with Mn (250 μM) for the indicated time periods, followed by a ChIP assay to determine YY1 binding to its consensus site in the EAAT2 promoter in vivo. (D) The PCR products were also quantified. **, P < 0.01; ***, P < 0.001 (Student's t test; n = 2).

FIG 5

NF-κB regulates YY1 activation. (A) There is one critical NF-κB consensus site (−170) in the YY1 promoter. Astrocytes were transfected overnight with a wild-type (B) or an NF-κB mutant (C) YY1 luciferase plasmid (0.5 μg) and 0.1 μg of the control vector pRC-RSV or p65, and promoter activity was determined by luciferase assay. (D) Astrocytes were transfected with 0.5 μg of either the wild-type or NF-κB mutant (−170) YY1 promoter, followed by a luciferase assay. (E) After overnight transfection with an NF-κB mutant of the YY1 promoter vector, astrocytes were treated with Mn (250 μM) and TNF-α (15 ng/ml) for 6 h, followed by a luciferase assay. ##, P < 0.01; **, P < 0.01 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 6

YY1 interacts with NF-κB p65, overriding the p65 effects. (A) Astrocytes were cotransfected overnight with the EAAT2 promoter vector and either YY1, p65, or both, followed by a luciferase assay. (B) Astrocytes were treated with Mn (250 μM) for the indicated time periods, followed by nuclear extract preparation and co-IP for YY1 and p65 as described in Materials and Methods. ###, P < 0.001; ***, P < 0.001 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 7

HDACs serve as corepressors of YY1. (A and B) Astrocytes were cotransfected overnight with the EAAT2 promoter vector and either YY1, HDACs, or both expression vectors, followed by a luciferase assay. (C) Astrocytes were treated with Mn (250 μM) for the indicated times, and nuclear extracts were prepared, followed by co-IP for YY1 and HDAC1. C, control; IB, immunoblotting. #, P < 0.05; ##, P < 0.01 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 8

HDAC inhibits the stimulatory p65 effects on EAAT2 promoter activity. (A and B) Astrocytes were cotransfected overnight with either or both p65 and HDAC1 or HDAC4 expression vectors or their empty vectors (control) along with the EAAT2 promoter vector, followed by a luciferase assay. (C) The co-IP of HDAC1 and p65 was carried out using the nuclear extracts prepared from control and Mn-treated cells. (D) The co-IP of HDAC1 and p65 was performed using nuclear extracts which were prepared from astrocytes transfected with scrambled control (sc siRNA) and YY1 siRNAs for 48 h. Nuclear extracts were also blotted for YY1 to confirm the efficiency of knockdown with YY1 siRNA. Histone H3 was used as a loading control. #, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 9

HDAC inhibitors increase and reverse Mn-induced repression of EAAT2 promoter activity in astrocytes. Astrocytes were exposed to vehicle (controls) or the following HDACi: romidepsin (FK228; 10 nM) and trichostatin A (TSA; 200 nM) (A) or sodium butyrate (NaB; 1 mM), SAHA (vorinostat; 1 μM), and valproic acid (VPA; 4 mM) (B) for 24 h. Mn, where indicated, was added at hour 18 during this incubation. #, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA followed by Tukey's post hoc test; n = 3).

FIG 10

Proposed mechanism for Mn-induced repression of EAAT2. TNF-α is released by Mn, which activates the NF-κB pathway, followed by YY1 activation. The upregulation of YY1 represses EAAT2 using HDACs as corepressors. YY1 also physically interacts with NF-κB, inhibiting its positive regulation on EAAT2.