Genome-wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in Small Cell Lung Cancer.

BACKGROUND: 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 to normal human small airway epithelial cells (SAEC) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitivity (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 examine growth and tumorigenencity as well as changes in transcriptomes and glucose metabolism of SCLC cells following Nuclear Factor 1C (NFIC) knockdown, and to examine NFIC expression in SCLC cells following exposure to BET inhibitors. RESULTS: Significant 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 DNase-seq to RNA-seq 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 Bromodomain-containing protein 4 (BRD4) in the NFIC promoter region. Knock-down of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETi) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes downregulated by BETi treatment were repressed by NFIC knockdown in SCLC, while 34% of genes repressed following NFIC knockdown were also downregulated in SCLC cells following 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.

Unraveling the mechanism of centromere inactivation in human aging.

Aging involves a range of genetic, epigenetic, and physiological alterations. A key characteristic of aged cells is the loss of global heterochromatin, accompanied by a reduction in canonical histone levels. In this study, we track the fate of centromeres during aging in human cells. Our findings reveal that the centromeric histone H3 variant CENP-A is downregulated in aged cells, in a p53-dependent manner. We observe repression of centromeric noncoding transcription through an epigenetic mechanism via recruitment of a lysine-specific demethylase 1 (LSD1/KDM1A) to centromeres. This suppression results in defective de novo CENP-A loading at aging centromeres. By dual inhibition of p53 and LSD1/KDM1A in aged cells, we mitigate the reduction in centromeric proteins and centromeric transcripts, leading to mitotic rejuvenation of these cells. These results offer insights into a novel mechanism for centromeric inactivation during aging and provide potential strategies to reactivate centromeres.

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.

Single-cell transcriptome and accessible chromatin dynamics during endocrine pancreas development.

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate ß-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.

Oncogenic lncRNAs alter epigenetic memory at a fragile chromosomal site in human cancer cells.

Chromosome instability is a critical event in cancer progression. Histone H3 variant CENP-A plays a fundamental role in defining centromere identity, structure, and function but is innately overexpressed in several types of solid cancers. In the cancer background, excess CENP-A is deposited ectopically on chromosome arms, including 8q24/cMYC locus, by invading transcription-coupled H3.3 chaperone pathways. Up-regulation of lncRNAs in many cancers correlates with poor prognosis and recurrence in patients. We report that transcription of 8q24-derived oncogenic lncRNAs plays an unanticipated role in altering the 8q24 chromatin landscape by H3.3 chaperone-mediated deposition of CENP-A-associated complexes. Furthermore, a transgene cassette carrying specific 8q24-derived lncRNA integrated into a naïve chromosome locus recruits CENP-A to the new location in a cis-acting manner. These data provide a plausible mechanistic link between locus-specific oncogenic lncRNAs, aberrant local chromatin structure, and the generation of new epigenetic memory at a fragile site in human cancer cells.

Corticosterone pattern-dependent glucocorticoid receptor binding and transcriptional regulation within the liver.

Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction.

WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a malignancy with poor outcomes, thus novel therapies are urgently needed. We recently showed that WHSC1 is necessary for the viability of SCCHN cells through H3K36 di-methylation. Here, we report the identification of its novel substrate, histone H1, and that WHSC1-mediated H1.4K85 mono-methylation may enhance stemness features in SCCHN cells. To identify proteins interacting with WHSC1 in SCCHN cells, WHSC1 immunoprecipitation and mass spectrometry identified H1 as a WHSC1-interacting candidate. In vitro methyltransferase assays showed that WHSC1 mono-methylates H1 at K85. We generated an H1K85 mono-methylation-specific antibody and confirmed that this methylation occurs in vivo. Sphere formation assays using SCC-35 cells stably expressing either wild-type (FLAG-H1.4-WT) or mutated (FLAG-H1.4K85A) vector with lysine 85 to alanine substitution which is not methylated, indicated a higher number of spheres in SCC-35 cells expressing the wild type than those with the mutant vector. SCC-35 cells expressing the wild type H1.4 proliferated faster than those expressing the mutated vector. RNA sequencing, RT-PCR and Western blotting of the FLAG-H1.4-WT or FLAG-H1.4K85A SCC-35 cells revealed that OCT4 levels were higher in wild type compared to mutant cells. These results were reproduced in SCC-35 cells genetically modified with CRISPR to express H1.4K85R. Chromatin immunoprecipitation showed that FLAG-H1.4K85A had decreased occupancy in the OCT4 gene compared to FLAG-H1.4-WT. This study supports that WHSC1 mono-methylates H1.4 at K85, it induces transcriptional activation of OCT4 and stemness features in SCCHN cells, providing rationale to target H1.4K85 mono-methylation through WHSC1 in SCCHN.

XL-DNase-seq: improved footprinting of dynamic transcription factors.

BACKGROUND: As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. RESULTS: Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. CONCLUSIONS: XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.

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.

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."

Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity.

In response to activating signals, transcription factors (TFs) bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint). Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot), efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq), all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined.

Genome-Wide Identification of Basic Helix-Loop-Helix and NF-1 Motifs Underlying GR Binding Sites in Male Rat Hippocampus.

Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites are dependent on the activity of additional signal-activated transcription factors that prime chromatin toward context-specific organization. We hypothesized a stress context dependency for GR binding in hippocampus as a consequence of rapidly induced stress mediators priming chromatin accessibility. Using chromatin immunoprecipitation sequencing to interrogate GR binding, we found no effect of restraint stress context on GR binding, although analysis of sequences underlying GR binding sites revealed mechanistic detail for hippocampal GR function. We note enrichment of GR binding sites proximal to genes linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix-loop-helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding sites and is proposed as a candidate lineage-determining transcription factor directing hippocampal chromatin access for GRs. Of our GR binding sites, 54% additionally contained half-sites for nuclear factor (NF)-1 that we propose as a collaborative or general transcription factor involved in hippocampal GR function. Our findings imply a dose-dependent and context-independent action of GRs in the hippocampus. Alterations in the expression or activity of NF-1/basic helix-loop-helix factors may play an as yet undetermined role in glucocorticoid-related disease susceptibility and outcome by altering GR access to hippocampal binding sites.

High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss.

Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response.

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting.

Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

Cajal bodies are linked to genome conformation.

The mechanisms underlying nuclear body (NB) formation and their contribution to genome function are unknown. Here we examined the non-random positioning of Cajal bodies (CBs), major NBs involved in spliceosomal snRNP assembly and their role in genome organization. CBs are predominantly located at the periphery of chromosome territories at a multi-chromosome interface. Genome-wide chromosome conformation capture analysis (4C-seq) using CB-interacting loci revealed that CB-associated regions are enriched with highly expressed histone genes and U small nuclear or nucleolar RNA (sn/snoRNA) loci that form intra- and inter-chromosomal clusters. In particular, we observed a number of CB-dependent gene-positioning events on chromosome 1. RNAi-mediated disassembly of CBs disrupts the CB-targeting gene clusters and suppresses the expression of U sn/snoRNA and histone genes. This loss of spliceosomal snRNP production results in increased splicing noise, even in CB-distal regions. Therefore, we conclude that CBs contribute to genome organization with global effects on gene expression and RNA splicing fidelity.