RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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.

Latent regulatory potential of human-specific repetitive elements.

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

Independence of HIF1a and androgen signaling pathways in prostate cancer.

BACKGROUND: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. METHODS: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. RESULTS: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. CONCLUSIONS: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation.

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer.

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Differential oestrogen receptor binding is associated with clinical outcome in breast cancer.

Oestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems. Here we map genome-wide ER-binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcomes and in distant ER-positive metastases. We find that drug-resistant cancers still recruit ER to the chromatin, but that ER binding is a dynamic process, with the acquisition of unique ER-binding regions in tumours from patients that are likely to relapse. The acquired ER regulatory regions associated with poor clinical outcome observed in primary tumours reveal gene signatures that predict clinical outcome in ER-positive disease exclusively. We find that the differential ER-binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FOXA1-mediated reprogramming of ER binding on a rapid timescale. The parallel redistribution of ER and FOXA1 binding events in drug-resistant cellular contexts is supported by histological co-expression of ER and FOXA1 in metastatic samples. By establishing transcription-factor mapping in primary tumour material, we show that there is plasticity in ER-binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.

Cooperative interaction between retinoic acid receptor-alpha and estrogen receptor in breast cancer.

Retinoic acid receptor-alpha (RAR alpha) is a known estrogen target gene in breast cancer cells. The consequence of RAR alpha induction by estrogen was previously unknown. We now show that RAR alpha is required for efficient estrogen receptor-alpha (ER)-mediated transcription and cell proliferation. RAR alpha can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RAR alpha that is independent of its classic role. We show, on a genome-wide scale, that RAR alpha and ER can co-occupy regulatory regions together within the chromatin. This transcriptionally active co-occupancy and dependency occurs when exposed to the predominant breast cancer hormone, estrogen--an interaction that is promoted by the estrogen-ER induction of RAR alpha. These findings implicate RAR alpha as an essential component of the ER complex, potentially by maintaining ER-cofactor interactions, and suggest that different nuclear receptors can cooperate for effective transcriptional activity in breast cancer cells.

GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.

Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline.

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.

The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer.

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABPα, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABPα enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABPα has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABPα bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABPα in CRPC and reveals potential targets for therapeutic intervention.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis.

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Smoking-associated gene expression alterations in nasal epithelium reveal immune impairment linked to lung cancer risk.

BACKGROUND: Lung cancer is the leading cause of cancer-related death in the world. In contrast to many other cancers, a direct connection to modifiable lifestyle risk in the form of tobacco smoke has long been established. More than 50% of all smoking-related lung cancers occur in former smokers, 40% of which occur more than 15 years after smoking cessation. Despite extensive research, the molecular processes for persistent lung cancer risk remain unclear. We thus set out to examine whether risk stratification in the clinic and in the general population can be improved upon by the addition of genetic data and to explore the mechanisms of the persisting risk in former smokers. METHODS: We analysed transcriptomic data from accessible airway tissues of 487 subjects, including healthy volunteers and clinic patients of different smoking statuses. We developed a computational model to assess smoking-associated gene expression changes and their reversibility after smoking is stopped, comparing healthy subjects to clinic patients with and without lung cancer. RESULTS: We find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Integrating previous GWAS data using a transcriptional network approach, we demonstrate that the same immune- and interferon-related pathways are strongly enriched for genes linked to known genetic risk factors, demonstrating a causal relationship between immune alteration and lung cancer risk. Finally, we used accessible airway transcriptomic data to derive a non-invasive lung cancer risk classifier. CONCLUSIONS: Our results provide initial evidence for germline-mediated personalized smoke injury response and risk in the general population, with potential implications for managing long-term lung cancer incidence and mortality.

BayesPeak--an R package for analysing ChIP-seq data.

MOTIVATION: Identification of genomic regions of interest in ChIP-seq data, commonly referred to as peak-calling, aims to find the locations of transcription factor binding sites, modified histones or nucleosomes. The BayesPeak algorithm was developed to model the data structure using Bayesian statistical techniques and was shown to be a reliable method, but did not have a full-genome implementation. RESULTS: In this note we present BayesPeak, an R package for genome-wide peak-calling that provides a flexible implementation of the BayesPeak algorithm and is compatible with downstream BioConductor packages. The BayesPeak package introduces a new method for summarizing posterior probability output, along with methods for handling overfitting and support for parallel processing. We briefly compare the package with other common peak-callers. AVAILABILITY: Available as part of BioConductor version 2.6. URL: http://bioconductor.org/packages/release/bioc/html/BayesPeak.html.

ZFTA Translocations Constitute Ependymoma Chromatin Remodeling and Transcription Factors.

ZFTA (C11orf95)-a gene of unknown function-partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation-including ZFTA zinc fingers and partner gene transactivation domains-thereby unmasking vulnerabilities for therapeutic targeting. SIGNIFICANCE: Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting.This article is highlighted in the In This Issue feature, p. 2113.

BayesPeak: Bayesian analysis of ChIP-seq data.

BACKGROUND: High-throughput sequencing technology has become popular and widely used to study protein and DNA interactions. Chromatin immunoprecipitation, followed by sequencing of the resulting samples, produces large amounts of data that can be used to map genomic features such as transcription factor binding sites and histone modifications. METHODS: Our proposed statistical algorithm, BayesPeak, uses a fully Bayesian hidden Markov model to detect enriched locations in the genome. The structure accommodates the natural features of the Solexa/Illumina sequencing data and allows for overdispersion in the abundance of reads in different regions. Moreover, a control sample can be incorporated in the analysis to account for experimental and sequence biases. Markov chain Monte Carlo algorithms are applied to estimate the posterior distributions of the model parameters, and posterior probabilities are used to detect the sites of interest. CONCLUSION: We have presented a flexible approach for identifying peaks from ChIP-seq reads, suitable for use on both transcription factor binding and histone modification data. Our method estimates probabilities of enrichment that can be used in downstream analysis. The method is assessed using experimentally verified data and is shown to provide high-confidence calls with low false positive rates.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target.

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer.

BACKGROUND: The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise. RESULTS: Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. CONCLUSIONS: Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo data.

With the advent of ChIP-seq multiplexing technologies and the subsequent increase in ChIP-seq throughput, the development of working standards for the quality assessment of ChIP-seq studies has received significant attention. The ENCODE consortium's large scale analysis of transcription factor binding and epigenetic marks as well as concordant work on ChIP-seq by other laboratories has established a new generation of ChIP-seq quality control measures. The use of these metrics alongside common processing steps has however not been evaluated. In this study, we investigate the effects of blacklisting and removal of duplicated reads on established metrics of ChIP-seq quality and show that the interpretation of these metrics is highly dependent on the ChIP-seq preprocessing steps applied. Further to this we perform the first investigation of the use of these metrics for ChIP-exo data and make recommendations for the adaptation of the NSC statistic to allow for the assessment of ChIP-exo efficiency.