A parallelized, automated platform enabling individual or sequential ChIP of histone marks and transcription factors.

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2 h), has a wide dynamic range (from 106 to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP's interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

Reduced NCOR2 expression accelerates androgen deprivation therapy failure in prostate cancer.

This study addresses the roles of nuclear receptor corepressor 2 (NCOR2) in prostate cancer (PC) progression in response to androgen deprivation therapy (ADT). Reduced NCOR2 expression significantly associates with shorter disease-free survival in patients with PC receiving adjuvant ADT. Utilizing the CWR22 xenograft model, we demonstrate that stably reduced NCOR2 expression accelerates disease recurrence following ADT, associates with gene expression patterns that include neuroendocrine features, and induces DNA hypermethylation. Stably reduced NCOR2 expression in isogenic LNCaP (androgen-sensitive) and LNCaP-C4-2 (androgen-independent) cells revealed that NCOR2 reduction phenocopies the impact of androgen treatment and induces global DNA hypermethylation patterns. NCOR2 genomic binding is greatest in LNCaP-C4-2 cells and most clearly associates with forkhead box (FOX) transcription factor FOXA1 binding. NCOR2 binding significantly associates with transcriptional regulation most when in active enhancer regions. These studies reveal robust roles for NCOR2 in regulating the PC transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PC disease progression.

The miR-96 and RARγ signaling axis governs androgen signaling and prostate cancer progression.

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ) are commonly reduced in prostate cancer (PCa). Therefore, we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome, which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ downregulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function. Capture of the miR-96 targetome by biotin-miR-96 identified that RARγ and a number of RARγ interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ target genes (e.g., SOX15) that significantly associated with worse disease-free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p = 0.015). In summary, miR-96 targets a RARγ network to govern AR signaling, PCa progression and disease outcome.