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Background  DNA methylation variability regions (MVRs) across the oestrogen receptor alpha ( ESR1 ) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in  ESR1  in breast cancer cell lines, tumour biopsies and exfoliated epithelial cells from expressed breast milk (EBM), to determine their significance for  ESR1  transcription.    Methods  DNA methylation levels in eight MVRs across  ESR1  were assessed by pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with DAC (1 μM), or DMSO (controls).  ESR1  methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumours (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450 K (n = 36) and pyrosequencing in a further 53 donor samples.  ESR1  mRNA levels were measured by qRT-PCR.    Results  We show that ER-positive cell lines had unmethylated  ESR1  promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The  ESR1  promoter was unmethylated in breast tumour biopsies with high levels of intragenic methylation, independent of ER status. However,  ESR1  methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumour cell lines.    Conclusion  DAC treatment inhibited  ESR1  transcription in cells with an unmethylated  ESR1  promoter and reduced intragenic DNA methylation. Intragenic methylation levels correlated with ESR1 expression in homogenous cell populations (cell lines and exfoliated primary breast epithelial cells), but not in heterogeneous tumour biopsies, highlighting the significant differences between the  in vivo  tumour microenvironment and individual homogenous cell types. These findings emphasise the need for care when choosing material for epigenetic research and highlights the presence of aberrant intragenic methylation levels in tumour tissue.    Electronic supplementary material  The online version of this article (doi:10.1186/s12885-015-1335-5) contains supplementary material, which is available to authorized users.

Transcriptional implications of intragenic DNA methylation in the oestrogen receptor alpha gene in breast cancer cells and tissues