Progesterone receptor modulates ERα action in breast cancer.

Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.We would like to acknowledge the support of the University of Cambridge, Cancer Research UK and Hutchison Whampoa Limited. Research reported in this manuscript was supported by the National Cancer Institute of the National Institutes of Health under award number 5P30CA142543 (to UT Southwestern) and Department of Defense grants W81XWH-12-1-0288-03 (GVR). W.D.T. is supported by grants from the National Health and Medical Research Council of Australia (ID 1008349; ID 1084416) and Cancer Australia (ID 627229) T.E.H held a Fellowship Award from the US Department of Defense Breast Cancer Research Program (BCRP; #W81XWH-11-1-0592) and currently is supported by a Florey Fellowship from the Royal Adelaide Hospital Research Foundation. J.S.C is supported by an ERC starting grant and an EMBO Young investigator award.This is the accepted manuscript. The final version is available at www.nature.com/nature/journal/v523/n7560/full/nature14583.htm

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer

Funder: National Health and Medical Research Council; doi: http://dx.doi.org/10.13039/501100000925Abstract: Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC

Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA

International audienceThis protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as 'selective chemical labeling' (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis

Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.

ChIP-exonuclease (ChIP-exo) is a modified ChIP-seq approach for high resolution mapping of transcription factor DNA sites. We describe an Illumina-based ChIP-exo method which provides a global improvement of the data quality of estrogen receptor (ER) ChIP and insights into the motif structure for key ER-associated factors. ChIP-exo of the ER pioneer factor FoxA1 identifies protected DNA with a predictable 8 bp overhang from the Forkhead motif, which we term mesas. We show that mesas occur in multiple cellular contexts and exist as single or overlapping motifs. Our Illumina-based ChIP-exo provides high resolution mapping of transcription factor binding sites

Endogenous purification reveals GREB1 as a key estrogen receptor regulatory factor.

Estrogen receptor-α (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts