MTA1-mediated transcriptional repression of BRCA1 tumor suppressor gene

Abstract

Metastasis-associated tumor antigen 1 (MTA1), a component of the nucleosome remodeling and deacetylating (NuRD) complex is routinely upregulated in several cancers. In the present study, we investigated the potential role of MTA1 in BRCA1 transcriptional repression and subsequent chromosomal instability. MTA1-NuRD complex was found to negatively regulate BRCA1 transcription by physically associating with an atypical estrogen-responsive element (ERE) on the BRCA1 promoter. Moreover, MTA1 and HDAC complex recruited to the ERE of BRCA1 promoter in an ER alpha-dependent manner. Accordingly, BRCA1 protein levels were enhanced by silencing of either MTA1 expression or by treatment with the specific histone deacetylase inhibitor trichostatin A. MTA1's strong repressive effects on BRCA1 expression was supported by our observation that cells stably overexpressing MTA1 showed centrosome amplification which has been long implicated as a phenotype for BRCA1 repression. Accordingly, overexpression of BRCA1 in cells stably over expressing MTA1 resulted in restoration of normal centrosome numbers. Together, these findings strongly implicate MTA1 in the transcriptional repression of BRCA1 leading to abnormal centrosome number and chromosomal instability.

Figure 1. Inverse relationship between expression of MTA1 and BRCA1

A, RT-PCR analysis showing decreased BRCA1 and PARP1 expression in MTA1-overexpressing MCF7 clones (first and second panels, right lane) when compared with control cells. Actin expression was used as an internal control for both RNA samples (third panel). B, Northern blot analysis showing a drastic decrease in BRCA1 mRNA levels in MCF7/MTA1 cells when compared with MCF7/pcDNA (control) cells (first panel, second lane), thus implicating MTA1 over expression in the repression of BRCA1 gene transcription. C, Significant decreases in the protein levels of BRCA1 (left) and PARP1 (right) in the presence of MTA1 over expression (first panel, second lane). Actin or Vinculin expression was used as an internal control. D, Increase in BRCA1 levels as a result of MTA1 knockdown with small interfering RNA (siRNA) (first panel, right lane). A decreased level of MTA1 expression (second panel, right lane) shows effective knockdown of MTA1 by MTA1 siRNA. Actin expression was used as an internal control (third panel). Expression levels were quantified using the ImageQuant version 5.1 program.

Figure 2. MTA1-mediated repression of BRCA1 promoter activity

A, An atypical ERE located on the human BRCA1 gene promoter and through which estrogen regulates transcription of the BRCA1 gene. B, Luciferase assay in cells transfected with a pGL3-BRCA1 promoter (1-2696) reporter construct containing one atypical ERE. Luciferase activity was assayed 48 h after transfection. Values were normalized to β-galactosidase activity (n=3). BRCA1 promoter activity was reduced approximately 50% in MCF7, ZR75 and HC11 cells over expressing MTA1. C, Luciferase assay of HSY (ERα-negative) or HeLa cells (ERα-, ERβ-negative) showing no change in BRCA1 promoter activity in the presence of MTA1 over expression, thus implicating ERα in MTA1-mediated repression of BRCA1.

Figure 3. MTA1-mediated repression of the BRCA1 promoter via its atypical ERE site

A, ChIP assay in MCF7/pcDNA and MCF7/MTA1 clones with a T7-tag specific antibody. Results demonstrate the physical association of MTA1 with BRCA1 promoter chromatin and not with the PhosphoFructokinase M promoter chromatin. B, Double or sequential ChIP in MCF7/pcDNA and MCF7/MTA1 clones. Results demonstrate the recruitment of both MTA1 and HDAC2 to the BRCA1 promoter, thus implying recruitment of the MTA1-containing NuRD complex to that chromatin region. C, Decreased MTA1-induced repression of BRCA1 at both the protein (first panel, right lane) and mRNA levels (third panel, right lane) in MCF7 cells treated with the specific histone deacetylase (HDAC) inhibitor trichostatin A (TSA; 300 nmol/L per milliliter of culture medium). Actin expression was used as an internal control. Functional assay of pGL3 BRCA1-luciferase activity demonstrating the effect of TSA treatment on MTA1-mediated repression of BRCA1 expression in MCF7 cells. In brief, MCF7 pcDNA and MCF7 MTA1 over expressing cells were treated with TSA (D) or ZR75 cells were transiently transfected with a pcDNA or T7-tagged MTA1 expression construct (1μg) and then treated with TSA (E). Western blot for T7 in TCA precipitated luciferase samples shows efficient transfection (above the bar chart). The TSA treatment relieved MTA1-induced repression of BRCA1, thus implying that HDACs were involved in such repression.

Figure 4. ERα-mediated recruitment of the MTA1-NuRD complex to the BRCA1 promoter

A, Single Chip with ERα antibody and IgG control shows recruitment of ERα on BRCA1 promoter. Double ChIP assays with ERα antibody and anti-T7-tag antibody proved that ERα was involved in recruiting MTA1 to the BRCA1 promoter. B, ChIP assay with MTA1-specific antibody showed that MTA1 was recruited to the BRCA1 promoter only in ER-positive (MCF7) cells and not in ER-negative (HeLa) cells, thus implicating ERα in such recruitment. Western blot showing endogenous MTA1 levels in MCF7 and Hela cells. Actin was used as an internal control. C, ChIP assay with MTA1-specific antibody of MCF7 cells treated with anti-estrogen ICI-182780 for 1h before E2 treatment. Results demonstrated that MTA1 was not recruited to the BRCA1 promoter in the treated cells. D, functional assay of pGL3 BRCA1-luciferase activity demonstrating relief of MTA1-mediated repression of BRCA1 expression in MCF7 cells over expressing MTA1 on ICI treatment, further implicating ERα as a mediator of MTA1’s recruitment to the BRCA1 promoter.

Figure 5. Co-operation between MTA1 and ERα in regulation of BRCA1 repression

pGL3 BRCA1-luciferase activity demonstrating relief of MTA1-mediated repression of BRCA1 expression in MTA1 over expressing MCF7 cells A, and in HC11 cells B, on ERα knockdown with small interfering RNA (siRNA). A decreased level of ERα expression (Top panel, right lane) shows effective knockdown of ERα by ERα siRNA. Equal MTA1 levels in both contol siRNA and ERα knockdown samples (second panel) indicate specificity of ERα siRNA. Actin expression was used as an internal control (third panel). C, Functional assay of pGL3 BRCA1-luciferase activity demonstrating relief of BRCA1 repression in ERα transfected Hela cells on MTA1 knockdown with siRNA. A decreased level of MTA1 expression (Top panel, right lane) shows effective knockdown of MTA1. Actin expression was used as an internal control (second panel). D, Luciferase activity demonstrating that mutated ERE on BRCA1 promoter blocks MTA1-mediated repression of BRCA1 (SM, mutated ERE-1; DM, mutated ERE-1 + ERE-2; TM, mutated ERE-1 + ERE-2 + ERE-3). E, ChIP assay with MTA1-specific antibody of MCF7 cells treated with control siRNA and ERα specific siRNA. Results demonstrated a loss of MTA1 recruitment to the BRCA1 promoter under ERα knockdown conditions in comparison to the cells transfected with control RNAi. F, Schematic diagram of regulation of BRCA1 expression by the MTA1/NuRD complex.

Figure 6. MTA1 over expression and resulting increases in centrosome number

A, Western blot analysis showing endogenous MTA1, T7-pcDNA, T7-MTA1 plasmid expression in transfected MCF7 and HC11 cells. Significant decreases in the protein BRCA1 level in the presence of MTA1 over expression in HC11 cells. Tubulin was used as an internal control. B, Representative immunofluorescent images for α-tubulin (green), pericentrin (red) and DNA (blue) as seen under a confocal laser microscope are shown here. Cells were synchronized with double thymidine block. A total of 100 cells were counted. C, Bar chart showing quantified results. MTA1 over expression led to an increase in number of cells with multiple spindle poles.

Figure 7. Role of BRCA1 in the multiple spindle phenotype observed in MTA1-over expressing cells

A, MCF7/MTA1 cells transfected with GFP vector or GFP-BRCA1 was assessed by immunofluorescence staining for GFP (green), α-tubulin (Magenta), pericentrin (red) and DAPI stained DNA (blue). MTA1 over expression led to an increase in the number of centrosomes; this phenotype was compromised, however, by the reintroduction of BRCA1, thus implicating BRCA1 repression in the centrosome amplification observed in MTA1-overexpressing cells. B, Bar chart showing quantified results.