Optimized ChIP-seq method facilitates transcription factor profiling in human tumors.

Abhishek A Singh,Simon Linder,Karianne Schuurman,Henk Van Der Poel,Joyce Sanders,Marjolein Droog,Ekaterina Nevedomskaya,Andries M Bergman,Lodewyk Fa Wessels,Suzan Stelloo,Yongsoo Kim,Wilbert Zwart

doi:10.26508/lsa.201800115

Abstract

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. 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. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. 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 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.

Highlights

Steroid hormone receptors are critical regulators in human physiology, and central players in multiple diseases, including cancer
Proliferating cells were used in these analyses and fixed either using standard 1% FA or through a two-step fixation procedure with 45-min 2-mM disuccinimidyl glutarate (DSG) fixation, of which the last 10 min the cells are co-incubated with 1% FA
These peaks were shared with Forkhead box protein A1 (FOXA1) and histone modification H3K27ac, while this histone modification was found at promoter regions along with H3K4me3 (Fig 2A)

Summary

Introduction

Steroid hormone receptors are critical regulators in human physiology, and central players in multiple diseases, including cancer. 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). This mode of activation is shared by practically all steroid hormone receptors, including AR. AR is considered the oncogenic driver in prostate cancer development and progression (Lonergan & Tindall, 2011) 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)

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Conclusion