Genome-Wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in SCLC.

INTRODUCTION: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. METHODS: Transcriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitive site sequencing (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors. RESULTS: Considerable commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DHS-seq to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment. CONCLUSIONS: NFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.

Long-range gene regulation in hormone-dependent cancer.

The human genome is organized into multiple structural layers, ranging from chromosome territories to progressively smaller substructures, such as topologically associating domains (TADs) and chromatin loops. These substructures, collectively referred to as long-range chromatin interactions (LRIs), have a significant role in regulating gene expression. TADs are regions of the genome that harbour groups of genes and regulatory elements that frequently interact with each other and are insulated from other regions, thereby preventing widespread uncontrolled DNA contacts. Chromatin loops formed within TADs through enhancer and promoter interactions are elastic, allowing transcriptional heterogeneity and stochasticity. Over the past decade, it has become evident that the 3D genome structure, also referred to as the chromatin architecture, is central to many transcriptional cellular decisions. In this Review, we delve into the intricate relationship between steroid receptors and LRIs, discussing how steroid receptors interact with and modulate these chromatin interactions. Genetic alterations in the many processes involved in organizing the nuclear architecture are often associated with the development of hormone-dependent cancers. A better understanding of the interplay between architectural proteins and hormone regulatory networks can ultimately be exploited to develop improved approaches for cancer treatment.

SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck.

Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

Extrachromosomal DNA Amplification Contributes to Small Cell Lung Cancer Heterogeneity and Is Associated with Worse Outcomes.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications. SIGNIFICANCE: MYC drives SCLC progression, but the genetic basis of MYC-driven SCLC evolution is unknown. Using SCLC as a paradigm, we report how ecDNA amplifications function as MYC-amplifying units, fostering tumor plasticity and a high degree of tumor heterogeneity. This article is highlighted in the In This Issue feature, p. 799.

Identification of a novel GR-ARID1a-P53BP1 protein complex involved in DNA damage repair and cell cycle regulation.

ARID1a (BAF250), a component of human SWI/SNF chromatin remodeling complexes, is frequently mutated across numerous cancers, and its loss of function has been putatively linked to glucocorticoid resistance. Here, we interrogate the impact of siRNA knockdown of ARID1a compared to a functional interference approach in the HeLa human cervical cancer cell line. We report that ARID1a knockdown resulted in a significant global decrease in chromatin accessibility in ATAC-Seq analysis, as well as affecting a subset of genome-wide GR binding sites determined by analyzing GR ChIP-Seq data. Interestingly, the specific effects on gene expression were limited to a relatively small subset of glucocorticoid-regulated genes, notably those involved in cell cycle regulation and DNA repair. The vast majority of glucocorticoid-regulated genes were largely unaffected by ARID1a knockdown or functional interference, consistent with a more specific role for ARID1a in glucocorticoid function than previously speculated. Using liquid chromatography-mass spectrometry, we have identified a chromatin-associated protein complex comprising GR, ARID1a, and several DNA damage repair proteins including P53 binding protein 1 (P53BP1), Poly(ADP-Ribose) Polymerase 1 (PARP1), DNA damage-binding protein 1 (DDB1), DNA mismatch repair protein MSH6 and splicing factor proline and glutamine-rich protein (SFPQ), as well as the histone acetyltransferase KAT7, an epigenetic regulator of steroid-dependent transcription, DNA damage repair and cell cycle regulation. Not only was this protein complex ablated with both ARID1a knockdown and functional interference, but spontaneously arising DNA damage was also found to accumulate in a manner consistent with impaired DNA damage repair mechanisms. Recovery from dexamethasone-dependent cell cycle arrest was also significantly impaired. Taken together, our data demonstrate that although glucocorticoids can still promote cell cycle arrest in the absence of ARID1a, the purpose of this arrest to allow time for DNA damage repair is hindered.

Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates.

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation.

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

Mechanical Regulation of Transcription: Recent Advances.

Mechanotransduction is the ability of a cell to sense mechanical cues from its microenvironment and convert them into biochemical signals to elicit adaptive transcriptional and other cellular responses. Here, we describe recent advances in the field of mechanical regulation of transcription, highlight mechanical regulation of the epigenome as a key novel aspect of mechanotransduction, and describe recent technological advances that could further elucidate the link between mechanical stimuli and gene expression. In this review, we emphasize the importance of mechanotransduction as one of the governing principles of cancer progression, underscoring the need to conduct further studies of the molecular mechanisms involved in sensing mechanical cues and coordinating transcriptional responses.

Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model.

Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs-one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.

Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.

High Quality ATAC-Seq Data Recovered from Cryopreserved Breast Cell Lines and Tissue.

DNA accessibility to transcription regulators varies between cells and modulates gene expression patterns. Several "open" chromatin profiling methods that provide valuable insight into the activity of these regulatory regions have been developed. However, their application to clinical samples has been limited despite the discovery that the Analysis of Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) method can be performed using fewer cells than other techniques. Obtaining fresh rather than stored samples and a lack of adequate optimization and quality controls are major barriers to ATAC's clinical implementation. Here, we describe an optimized ATAC protocol in which we varied nuclear preparation conditions and transposase concentrations and applied rigorous quality control measures before testing fresh, flash frozen, and cryopreserved breast cells and tissue. We obtained high quality data from small cell number. Furthermore, the genomic distribution of sequencing reads, their enrichment at transcription start sites, and transcription factor footprint analyses were similar between cryopreserved and fresh samples. This updated method is applicable to clinical samples, including cells from fine needle aspiration and tissues obtained via core needle biopsy or surgery. Chromatin accessibility analysis using patient samples will greatly expand the range of translational research and personalized medicine by identification of clinically-relevant epigenetic features.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression.

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1-/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

Chromatin reprogramming in breast cancer.

Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. In this review, we describe what is currently known about the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. Emphasis has been placed on characterizing the different modes of action of TFs in regulating enhancer activity, specifically, how different SRs target enhancer regions to reprogram chromatin in breast cancer cells. In addition, we discuss current techniques employed to study enhancer function at a genome-wide level. Further, we have noted recent advances in live cell imaging technology. These single-cell approaches enable the coupling of population-based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in breast cancer.

Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo.

Glucocorticoid hormone plays a major role in metabolism and disease. The hormone-bound glucocorticoid receptor (GR) binds to a specific set of enhancers in different cell types, resulting in unique patterns of gene expression. We have addressed the role of chromatin structure in GR binding by mapping nucleosome positions in mouse adenocarcinoma cells. Before hormone treatment, GR-enhancers exist in one of three chromatin states: (i) Nucleosome-depleted enhancers that are DNase I-hypersensitive, associated with the Brg1 chromatin remodeler and flanked by nucleosomes incorporating histone H2A.Z. (ii) Nucleosomal enhancers that are DNase I-hypersensitive, marked by H2A.Z and associated with Brg1. (iii) Nucleosomal enhancers that are inaccessible to DNase I, incorporate little or no H2A.Z and lack Brg1. Hormone-induced GR binding results in nucleosome shifts at all types of GR-enhancer, coinciding with increased recruitment of Brg1. We propose that nucleosome-depleted GR-enhancers are formed and maintained by other transcription factors which recruit Brg1 whereas, at nucleosomal enhancers, GR behaves like a pioneer factor, interacting with nucleosomal sites and recruiting Brg1 to remodel the chromatin.

The Three Ds of Transcription Activation by Glucagon: Direct, Delayed, and Dynamic.

Upon lowered blood glucose occurring during fasting, glucagon is secreted from pancreatic islets, exerting various metabolic effects to normalize glucose levels. A considerable portion of these effects is mediated by glucagon-activated transcription factors (TFs) in liver. Glucagon directly activates several TFs via immediate cyclic adenosine monophosphate (cAMP)- and calcium-dependent signaling events. Among these TFs, cAMP response element-binding protein (CREB) is a major factor. CREB recruits histone-modifying enzymes and cooperates with other TFs on the chromatin template to increase the rate of gene transcription. In addition to direct signal transduction, the transcriptional effects of glucagon are also influenced by dynamic TF cross talk. Specifically, assisted loading of one TF by a companion TF leads to increased binding and activity. Lastly, transcriptional regulation by glucagon is also exerted by TF cascades by which a primary TF induces the gene expression of secondary TFs that bring about their activity a few hours after the initial glucagon signal. This mechanism of a delayed response may be instrumental in establishing the temporal organization of the fasting response by which distinct metabolic events separate early from prolonged fasting. In this mini-review, we summarize recent advances and critical discoveries in glucagon-dependent gene regulation with a focus on direct TF activation, dynamic TF cross talk, and TF cascades.

Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading.

The cytokines interleukin 1ß and 6 (IL-1ß, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins. Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression. By mapping changes in enhancer landscape and transcription factor occupancy (using ChIP-seq), we show that synergistic gene induction is achieved by assisted loading of STAT3 on chromatin by NF-κB. With IL-6 treatment alone, STAT3 does not efficiently bind 20% of its coordinated binding sites. In the presence of IL-1ß, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac. This facilitates STAT3 binding and synergistic gene expression. Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others. This model reconciles seemingly contradictory reports of NF-κB-STAT3 crosstalk.

Anti-Inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action.

Despite the widespread use of glucocorticoids (GCs), their anti-inflammatory effects are not understood mechanistically. Numerous investigations have examined the effects of glucocorticoid receptor (GR) activation prior to inflammatory challenges. However, clinical situations are emulated by a GC intervention initiated in the midst of rampant inflammatory responses. To characterize the effects of a late GC treatment, we profiled macrophage transcriptional and chromatinscapes with Dexamethasone (Dex) treatment before or after stimulation by lipopolysaccharide (LPS). The late activation of GR had a similar gene-expression profile as from GR pre-activation, while ameliorating the disruption of metabolic genes. Chromatin occupancy of GR was not predictive of Dex-regulated gene expression, contradicting the "trans-repression by tethering" model. Rather, GR activation resulted in genome-wide blockade of NF-κB interaction with chromatin and directly induced inhibitors of NF-κB and AP-1. Our investigation using GC treatments with clinically relevant timing highlights mechanisms underlying GR actions for modulating the "inflamed epigenome."

Identifying environmental chemicals as agonists of the androgen receptor by using a quantitative high-throughput screening platform.

The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy's disease and Klinefelter's syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR ß-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation.