Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue

ABSTRACT

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.

KEYWORDS:

• Epigenetics
• DNA methylation
• 5-hydroxymethylcytosine

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability

The datasets supporting the conclusions of this article are available both at the repositories listed below, as well as within the article as additional files. Raw (IDAT files) and normalized DNA methylation data (450 K platform) used to estimate 5hmC and 5mC proportions from the 18 normal breast tissue samples are available under accession GSE100653 in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/). Raw DNA methylation data for DCIS and adjacent-normal tissue samples from the NHMN is also available in GEO (GSE66313). Level 1 TCGA breast (BRCA) intensity files derived from the 450 K platform were downloaded from the TCGA data portal and are currently available through the National Cancer Institute (NCI) Genomic Data Commons (GDC) data portal (https://portal.gdc.cancer.gov/). Coordinates of DNase hypersensitivity sites, histone modifications (derived from ChIP-seq experiments), and ChromHMM states in breast myoepithelial cells (E028), HMECs (E119), and vHMECs (E027) were downloaded from the NIH Roadmap Epigenomics Project [50] and are also available in GEO. Specific GEO accession numbers from each Roadmap Epigenomics experiment can be found at https://www.ncbi.nlm.nih.gov/geo/roadmap/epigenomics/. All other data are available within the article or Supporting Information. R code used for all analyses is available in the ‘Normal-Breast-5hmC’ repository on GitHub (https://github.com/Christensen-Lab-Dartmouth).

Ethics approval and consent to participate

Informed consent for use of tissue samples in biomedical research from donors or next-of-kin has been obtained for all breast tissue samples sourced from the NDRI. For study donors from the NHMN and TCGA projects, written informed consent was provided at the time of surgery for use of their tissue specimens in research. All work was performed with Institutional Review Board (IRB) approval and is in accordance with the ethical principles outlined in the Declaration of Helsinki.

Authors’ Contributions

OMW conceived and designed the approach, performed statistical analyses, interpreted the results, wrote and revised the manuscript. KCJ conceived and designed the approach, performed statistical analyses, interpreted the results, wrote and revised the manuscript. EAH conceived and designed the approach, generated statistical models, performed statistical analyses, interpreted the results, and revised the manuscript. JEK carried out laboratory experiments and revised the manuscript. CJM conceived and designed the approach and revised the manuscript. BCC conceived and designed the approach, interpreted the results, wrote and revised the manuscript. All authors have read and approved the final manuscript.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Institutes of Health [grants R01DE022772 and R01CA216265] [to B.C.C.], R01MH094609 [to E.A.H], and the Center for Molecular Epidemiology COBRE program funded by [P20GM104416].