Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 promoter

Abstract

BRCA1 expression is repressed by aberrant cytosine methylation in sporadic breast cancer. We hypothesized that aberrant cytosine methylation of the BRCA1 promoter was associated with the transcriptionally repressive effects of histone hypoacetylation and chromatin condensation. To address this question, we developed an in vitro model of study using normal cells and sporadic breast cancer cells with known levels of BRCA1 transcript to produce a 1.4 kb 5-methylcytosine map of the BRCA1 5' CpG island. While all cell types were densely methylated upstream of -728 relative to BRCA1 transcription start, all normal and BRCA1 expressing cells were non-methylated downstream of -728 suggesting that this region contains the functional BRCA1 5' regulatory region. In contrast, the non-BRCA1 expressing UACC3199 cells were completely methylated at all 75 CpGs. Chromatin immunoprecipitations showed that the UACC3199 cells were hypoacetylated at both histones H3 and H4 in the BRCA1 promoter compared to non-methylated BRCA1 expressing cells. The chromatin of the methylated UACC3199 BRCA1 promoter was inaccessible to DNA-protein interactions. These data indicate that the epigenetic effects of aberrant cytosine methylation, histone hypoacetylation and chromatin condensation act together in a discrete region of the BRCA1 5' CpG island to repress BRCA1 transcription in sporadic breast cancer.

Figure 1

High resolution 5-methylcytosine map of the BRCA1 5′ region. Bisulfite modified DNA from PBL, HFF, HMEC and the sporadic breast cancer cell lines MCF7 and UACC3199 were PCR amplified, cloned and sequenced. Clones from each cell type were analyzed to obtain a percent methylation of the 75 CpG dinucleotides in the BRCA1 5′ region located on the y-axis. The x-axis represents the nucleotide position relative to the BRCA1 transcriptional start site (GenBank accession no. U37574).

Figure 2

Acetylation status of histones H3 and H4 in the BRCA1 and GAPDH promoters in UACC3199, MCF7 and HMEC. Chromatin immunoprecipitations using acetyl-histone H3 and H4 antibodies were performed on UACC3199, MCF7 and HMEC. Following isolation of the acetyl-histone H3 and H4 enriched fraction of genomic DNA, the BRCA1 or GAPDH promoters were PCR amplified. Presence of a PCR product indicates acetylation of the immunoprecipitated histone. Acetylation status of histone H3 (A) or histone H4 (B) for the BRCA1 (top) and GAPDH (bottom) promoters in UACC3199, MCF7 and HMEC is shown. The different cell types analyzed are shown across the top and are grouped according to their incubation with acetyl-histone H3 or H4 antibody (+H3 Ab or +H4 Ab), no acetyl-histone antibody (–H3 Ab or –H4 Ab), or control sample. These experiments were performed three times each with similar results.

Figure 3

EcoRI chromatin accessibility assay of the BRCA1 5′ region. (A) A schematic showing the in vivo EcoRI and in vitro BamHI restriction sites relative to the BRCA1 transcription start site (bent arrow), the BRCA1 probe, exons 1a and 1b, and the four possible cleavage products and their predicted sizes. (B) A Southern blot that shows each cell type and the resultant in vivo EcoRI digest products. The lanes, from left to right, are 0, 25, 75 and 225 U of EcoRI; the in vitro control EcoRI digest is shown in the fifth lane for each cell type. This experiment was performed three times with similar results.

Figure 4

SstI chromatin accessibility assay of the BRCA1 5′ region. (A) A schematic showing the in vivo SstI and in vitro BamHI restriction sites relative to the BRCA1 transcription start site (bent arrow), the BRCA1 probe, exons 1a and 1b, and the four possible in vivo SstI cleavage products and their predicted sizes. (B) A Southern blot that shows each cell type and the resultant in vivo SstI digest products. The lanes, from left to right, are 0, 25, 75 and 225 U of SstI; the in vitro control SstI digest is shown in the fifth lane of each cell type. This experiment was performed three times with similar results.

Figure 5

Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 5′ regulatory region in sporadic breast cancer. Top, a schematic of the BRCA1 5′ flanking region. The BRCA1 upstream region (hashed box) contains a repetitive element (Alu) and extends to the beginning of the BRCA1 5′ regulatory region at –728 (black vertical line). The numbers represent the nucleotide position relative to the BRCA1 transcription start site (bent arrow). BRCA1-positive normal breast cells (middle) are non-methylated, contain acetylated histones H3 and H4 (Ac) and have a protein accessible chromatin conformation (open ovals) in the BRCA1 5′ regulatory region. In contrast, the BRCA1-negative tumor cells (bottom) have an aberrantly methylated BRCA15′ regulatory region (meC), hypoacetylated histones H3 and H4 (deAc), and condensed chromatin (bricked ovals) coincident with BRCA1 repression. Both normal and tumor cells have a methylated (meC) and chromatin condensed (bricked ovals) BRCA1 upstream region.