To date, most reports on hormone receptor genomics, including ERα and AR, made use of cell line models, and only slowly, reports on genome-wide chromatin binding in the context of human tumor tissue are being released. But even when activated, ERα ( Swinstead et al, 2016) as well as AR ( Kang et al, 2002) binds the chromatin in a dynamic and transient fashion, which is in contrast to the stable histone modifications that make up a structural and stable factor of the chromatin. When activated, AR translocates into the nucleus to facilitate chromatin binding and testosterone-driven transcription. Analogous to this, AR in prostate cancer cells is confined in the cytosol prior to testosterone binding ( Brinkmann et al, 1999). ERα chromatin interactions are stabilized upon ligand binding, a step crucial for estradiol-mediated gene transcription and breast cancer cell proliferation ( Tan et al, 2011). Many transcription factors, such as steroid hormone receptors and FOXA1 ( Swinstead et al, 2016), are intrinsically dynamic when it comes to chromatin interactions. Both ERα and AR require direct functional involvement of pioneer factors, such as Forkhead box protein A1 (FOXA1), to facilitate chromatin accessibility at designated binding sites for ERα and AR ( Robinson & Carroll, 2012). AR is considered the oncogenic driver in prostate cancer development and progression ( Lonergan & Tindall, 2011). This mode of activation is shared by practically all steroid hormone receptors, including AR. ERα is a hormone-dependent transcription factor, which upon activation by its natural ligand estradiol, binds regulatory regions throughout the genome to orchestrate responsive gene activity by chromatin looping ( Fullwood et al, 2009 Flach & Zwart, 2016). Around 75% of all human breast tumors are positive for, and considered dependent on, ERα. The steroid hormone receptor family is composed of multiple members, including estrogen receptor α (ERα), estrogen receptor β, androgen receptor (AR), glucocorticoid receptor, progesterone receptor, and mineralocorticoid receptor. Steroid hormone receptors are not only critical regulators in human physiology, but also central players in multiple diseases, including cancer. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ∼100% success rate for all transcription factors analyzed. Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio.
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