TIP60 is required for tumorigenesis in non-small cell lung cancer.

Histone modifications play crucial roles in transcriptional activation, and aberrant epigenetic changes are associated with oncogenesis. Lysine (K) acetyltransferases 5 (TIP60, also known as KAT5) is reportedly implicated in cancer development and maintenance, although its function in lung cancer remains controversial. Here we demonstrate that TIP60 knockdown in non-small cell lung cancer cell lines decreased tumor cell growth, migration, and invasion. Furthermore, analysis of a mouse lung cancer model with lung-specific conditional Tip60 knockout revealed suppressed tumor formation relative to controls, but no apparent effects on normal lung homeostasis. RNA-seq and ChIP-seq analyses of inducible TIP60 knockdown H1975 cells relative to controls revealed transglutaminase enzyme (TGM5) as downstream of TIP60. Investigation of a connectivity map database identified several candidate compounds that decrease TIP60 mRNA, one that suppressed tumor growth in cell culture and in vivo. In addition, TH1834, a TIP60 acetyltransferase inhibitor, showed comparable antitumor effects in cell culture and in vivo. Taken together, suppression of TIP60 activity shows tumor-specific efficacy against lung cancer, with no overt effect on normal tissues. Our work suggests that targeting TIP60 could be a promising approach to treating lung cancer.

Transcriptional Circuitry of NKX2-1 and SOX1 Defines an Unrecognized Lineage Subtype of Small-Cell Lung Cancer.

Rationale: The current molecular classification of small-cell lung cancer (SCLC) on the basis of the expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. Objectives: To refine the current SCLC classification with epigenomic profiles and to identify features of the redefined SCLC subtypes. Methods: We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance of NKX2-1 (NK2 homeobox 1) was evaluated by cell growth, apoptosis, and xenograft using clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-associated protein 9)-mediated deletion. NKX2-1-specific cistromic profiles were determined using chromatin immunoprecipitation followed by sequencing, and its functional transcriptional partners were determined using coimmunoprecipitation followed by mass spectrometry. Rb1flox/flox; Trp53flox/flox and Rb1flox/flox; Trp53flox/flox; Nkx2-1flox/flox mouse models were engineered to explore the function of Nkx2-1 in SCLC tumorigenesis. Epigenomic landscapes of six human SCLC specimens and 20 tumors from two mouse models were characterized. Measurements and Main Results: We identified two epigenomic subclusters of the major SCLC-A subtype: SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found that NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. In addition, we found that maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor, SOX1 (SRY-box transcription factor 1). Conclusions: We comprehensively describe additional epigenomic heterogeneity of the major SCLC-A subtype and define the SCLC-Aα subtype by the core regulatory circuitry of NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

Nkx2-1 represses a latent gastric differentiation program in lung adenocarcinoma.

Tissue-specific differentiation programs become dysregulated during cancer evolution. The transcription factor Nkx2-1 is a master regulator of pulmonary differentiation that is downregulated in poorly differentiated lung adenocarcinoma. Here we use conditional murine genetics to determine how the identity of lung epithelial cells changes upon loss of their master cell-fate regulator. Nkx2-1 deletion in normal and neoplastic lungs causes not only loss of pulmonary identity but also conversion to a gastric lineage. Nkx2-1 is likely to maintain pulmonary identity by recruiting transcription factors Foxa1 and Foxa2 to lung-specific loci, thus preventing them from binding gastrointestinal targets. Nkx2-1-negative murine lung tumors mimic mucinous human lung adenocarcinomas, which express gastric markers. Loss of the gastrointestinal transcription factor Hnf4α leads to derepression of the embryonal proto-oncogene Hmga2 in Nkx2-1-negative tumors. These observations suggest that loss of both active and latent differentiation programs is required for tumors to reach a primitive, poorly differentiated state.

Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas.

Non-chemical fluorination of hexagonal boron nitride by high-energy ion irradiation.

Two-dimensional materials such as hexagonal boron nitride (h-BN) and graphene have attracted wide attention in nanoelectronics and spintronics. Since their electronic characteristics are strongly affected by the local atomic structure, the heteroatom doping could allow us to tailor the electronic and physical properties of two-dimensional materials. In this study, a non-chemical method of heteroatom doping into h-BN under high-energy ion irradiation was demonstrated for the LiF/h-BN/Cu heterostructure. Spectroscopic analysis of chemical states on the relevant atoms revealed that 6% ± 2% fluorinated h-BN is obtained by the irradiation of 2.4 MeV Cu2+ ions with the fluence up to 1014 ions cm-2. It was shown that the high-energy ion irradiation leads to a single-sided fluorination of h-BN by the formation of the fluorinated sp 3-hybridized BN.

Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.

Aberrations in epigenetic processes, such as histone methylation, can cause cancer. Retinoblastoma binding protein 2 (RBP2; also called JARID1A or KDM5A) can demethylate tri- and dimethylated lysine 4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by the retinoblastoma protein (pRB). Here, we show that genetic ablation of Rbp2 decreases tumor formation and prolongs survival in Rb1(+/-) mice and Men1-defective mice. These studies link RBP2 histone demethylase activity to tumorigenesis and nominate RBP2 as a potential target for cancer therapy.

Changes in intestinal microbiota and biochemical parameters in patients with inflammatory bowel disease and irritable bowel syndrome induced by the prolonged addition of soluble fibers to usual drug therapy.

OBJECTIVES: Partially hydrolyzed guar gum (PHGG) is a soluble dietary fiber;in addition to improving bowel movements, it maintains intestinal health by producing short-chain fatty acids. However, majority of clinical studies on PHGG have been concluded within a month and excluded usual drug therapy. Hence, this study aimed to determine the effects of long-term consumption of PHGG, in combination with drug therapy, on gut bacteria ratios, laboratory values for inflammatory response, and fecal characteristics. METHODS AND RESULTS: The study was performed in patients with irritable bowel syndrome (IBS), Crohn's disease (CD), and ulcerative colitis (UC), by the administration of PHGG for six months while they continued their usual treatment. PHGG treatment caused significant changes in patients with IBS, including an increase in the abundance of short-chain fatty acid-producing bacteria, a significant decrease in Bacteroides abundance, and normalization of the Bristol scale of stool. In patients with UC, non-significant normalization of soft stools and decrease in fecal calprotectin were observed. Adverse events were not observed in any of the groups. CONCLUSION: Thus, it would be beneficial to include PHGG in the usual drug therapies of patients with IBS. J. Med. Invest. 71 : 121-128, February, 2024.

Patho-transcriptomic analysis of invasive mucinous adenocarcinoma of the lung (IMA): comparison with lung adenocarcinoma with signet ring cell features (SRCC).

Background: Invasive mucinous adenocarcinoma (IMA) comprises ∼5% of lung adenocarcinoma. There is no effective therapy for IMA when surgical resection is not possible. IMA is sometimes confused with adenocarcinoma with signet ring cell features (SRCC) pathologically since both adenocarcinomas feature tumor cells with abundant intracellular mucin. The molecular mechanisms by which such mucin-producing lung adenocarcinomas develop remain unknown. Methods: Using a Visium spatial transcriptomics approach, we analyzed IMA and compared it with SRCC patho-transcriptomically. Combining spatial transcriptomics data with in vitro studies using RNA-seq and ChIP-seq, we assessed downstream targets of transcription factors HNF4A and SPDEF that are highly expressed in IMA and/or SRCC. Results: Spatial transcriptomics analysis indicated that there are 6 distinct cell clusters in IMA and SRCC. Notably, two clusters (C1 and C3) of mucinous tumor cells exist in both adenocarcinomas albeit at a different ratio. Importantly, a portion of genes (e.g., NKX2-1 , GKN1 , HNF4A and FOXA3 ) are distinctly expressed while some mucous-related genes (e.g., SPDEF and FOXA2 ) are expressed in both adenocarcinomas. We determined that HNF4A induces MUC3A/B and TM4SF4 and that BI 6015, an HNF4A antagonist, suppressed the growth of IMA cells. Using mutant SPDEF that is associated with COVID-19, we also determined that an intact DNA-binding domain of SPDEF is required for SPDEF-mediated induction of mucin genes ( MUC5AC , MUC5B and AGR2 ). Additionally, we found that XMU-MP-1, a SPDEF inhibitor, suppressed the growth of IMA cells. Conclusion: These results revealed that IMA and SRCC contain heterogenous tumor cell types, some of which are targetable.

Imaging Classification of Constipation.

Objectives: The diagnosis of patients with chronic constipation is very complicated. This study aimed to develop a simple imaging classification for the diagnosis of chronic constipation by abdominal computed tomography (CT). Methods: Sixty-two patients who underwent abdominal CT in our hospital between January and June 2022 were enrolled. The CT values of the stool in the rectum and cecum were measured in patients with chronic constipation (C group) and in those without (non-C group). Results: A strong correlation was observed between the Bristol Stool Form Scale (BSFS) and the CT value of rectal stool. Furthermore, the rectal stool CT value was significantly higher in patients with chronic constipation than in those without. The CT value of cecal stool did not differ between the two groups. The cecal stool CT value was significantly higher in patients with severe constipation (BSFS 1) than in those with BSFS 2-6. A cutoff CT value of 100 was selected as the optimal value for indicating chronic constipation. Conclusions: Abdominal CT was useful in the diagnosis of chronic constipation. If the patient had constipation, the optimal cutoff CT value was 100.

Effectiveness and safety of lubiprostone after switching from stimulant laxatives in elderly patients with chronic constipation.

Background and Aim: Stimulant laxatives may cause electrolyte abnormalities, dehydration, and abdominal pain; their long-term use can lead to tolerance and subsequent refractory constipation. We investigated the effectiveness, safety, and quality of life after switching from stimulant laxatives to lubiprostone in elderly patients with chronic constipation (CC). Methods: This multicenter, interventional, open-label, single-arm, before-and-after comparison study enrolled 99 Japanese patients aged 65-90 years with CC who took stimulant laxatives for ≥2 weeks prior to switching to lubiprostone monotherapy. Results: The mean ± SD spontaneous defecations at Week 1 of 7.8 ± 6.2 times/week was not significantly different from that at baseline (8.3 ± 4.7). Spontaneous defecations were significantly reduced at Weeks 2 (-1.5 ± 4.0, P < 0.001) and 4 (-1.5 ± 3.7, P < 0.001). The Bristol Stool Form Scale score did not change from baseline (4.7 ± 0.9) at Weeks 1 (4.5 ± 1.3) or 4 (4.3 ± 1.3), but it did at Week 2 (4.3 ± 1.5, P < 0.05). The Patient Assessment of Constipation Quality of Life questionnaire score increased (0.36 ± 0.07, P < 0.001) after 28 days. Nausea was the only symptom that worsened from baseline and was the most frequently reported adverse drug reaction (15.2%). Conclusion: Switching to lubiprostone monotherapy for CC was not associated with significant concerns in short-term spontaneous defecation frequency and safety, but it might affect the efficacy and patient quality of life over 2 weeks. Careful treatment strategies facilitating gradual switching to lubiprostone monotherapy may be needed in patients using stimulant laxatives.

Early-Stage Lung Adenocarcinoma MDM2 Genomic Amplification Predicts Clinical Outcome and Response to Targeted Therapy.

Lung cancer is the most common cause of cancer-related deaths in both men and women, accounting for one-quarter of total cancer-related mortality globally. Lung adenocarcinoma is the major subtype of non-small cell lung cancer (NSCLC) and accounts for around 40% of lung cancer cases. Lung adenocarcinoma is a highly heterogeneous disease and patients often display variable histopathological morphology, genetic alterations, and genomic aberrations. Recent advances in transcriptomic and genetic profiling of lung adenocarcinoma by investigators, including our group, has provided better stratification of this heterogeneous disease, which can facilitate devising better treatment strategies suitable for targeted patient cohorts. In a recent study we have shown gene expression profiling identified novel clustering of early stage LUAD patients and correlated with tumor invasiveness and patient survival. In this study, we focused on copy number alterations in LUAD patients. SNP array data identified amplification at chromosome 12q15 on MDM2 locus and protein overexpression in a subclass of LUAD patients with an invasive subtype of the disease. High copy number amplification and protein expression in this subclass correlated with poor overall survival. We hypothesized that MDM2 copy number and overexpression predict response to MDM2-targeted therapy. In vitro functional data on a panel of LUAD cells showed that MDM2-targeted therapy effectively suppresses cell proliferation, migration, and invasion in cells with MDM2 amplification/overexpression but not in cells without MDM2 amplification, independent of p53 status. To determine the key signaling mechanisms, we used RNA sequencing (RNA seq) to examine the response to therapy in MDM2-amplified/overexpressing p53 mutant and wild-type LUAD cells. RNA seq data shows that in MDM2-amplified/overexpression with p53 wild-type condition, the E2F → PEG10 → MMPs pathway is operative, while in p53 mutant genetic background, MDM2-targeted therapy abrogates tumor progression in LUAD cells by suppressing epithelial to mesenchymal transition (EMT) signaling. Our study provides a potentially clinically relevant strategy of selecting LUAD patients for MDM2-targeted therapy that may provide for increased response rates and, thus, better survival.

Integrative network analysis of early-stage lung adenocarcinoma identifies aurora kinase inhibition as interceptor of invasion and progression.

Here we focus on the molecular characterization of clinically significant histological subtypes of early-stage lung adenocarcinoma (esLUAD), which is the most common histological subtype of lung cancer. Within lung adenocarcinoma, histology is heterogeneous and associated with tumor invasion and diverse clinical outcomes. We present a gene signature distinguishing invasive and non-invasive tumors among esLUAD. Using the gene signatures, we estimate an Invasiveness Score that is strongly associated with survival of esLUAD patients in multiple independent cohorts and with the invasiveness phenotype in lung cancer cell lines. Regulatory network analysis identifies aurora kinase as one of master regulators of the gene signature and the perturbation of aurora kinases in vitro and in a murine model of invasive lung adenocarcinoma reduces tumor invasion. Our study reveals aurora kinases as a therapeutic target for treatment of early-stage invasive lung adenocarcinoma.

Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer.

Comprehensive genomic analyses of small cell lung cancer (SCLC) have revealed frequent mutually exclusive genomic amplification of MYC family members. Hence, it has been long suggested that they are functionally equivalent; however, more recently, their expression has been associated with specific neuroendocrine markers and distinct histopathology. Here, we explored a previously undescribed role of L-Myc and c-Myc as lineage-determining factors contributing to SCLC molecular subtypes and histology. Integrated transcriptomic and epigenomic analyses showed that L-Myc and c-Myc impart neuronal and non-neuroendocrine-associated transcriptional programs, respectively, both associated with distinct SCLC lineage. Genetic replacement of c-Myc with L-Myc in c-Myc-SCLC induced a neuronal state but was insufficient to induce ASCL1-SCLC. In contrast, c-Myc induced transition from ASCL1-SCLC to NEUROD1-SCLC characterized by distinct large-cell neuroendocrine carcinoma-like histopathology. Collectively, we characterize a role of historically defined general oncogenes, c-Myc and L-Myc, for regulating lineage plasticity across molecular and histological subtypes.

Upregulation of FGF9 in Lung Adenocarcinoma Transdifferentiation to Small Cell Lung Cancer.

Transdifferentiation of lung adenocarcinoma to small cell lung cancer (SCLC) has been reported in a subset of lung cancer cases that bear EGFR mutations. Several studies have reported the prerequisite role of TP53 and RB1 alterations in transdifferentiation. However, the mechanism underlying transdifferentiation remains understudied, and definitive additional events, the third hit, for transdifferentiation have not yet been identified. In addition, no prospective experiments provide direct evidence for transdifferentiation. In this study, we show that FGF9 upregulation plays an essential role in transdifferentiation. An integrative omics analysis of paired tumor samples from a patient with transdifferentiated SCLC exhibited robust upregulation of FGF9. Furthermore, FGF9 upregulation was confirmed at the protein level in four of six (66.7%) paired samples. FGF9 induction transformed mouse lung adenocarcinoma-derived cells to SCLC-like tumors in vivo through cell autonomous activation of the FGFR pathway. In vivo treatment of transdifferentiated SCLC-like tumors with the pan-FGFR inhibitor AZD4547 inhibited growth. In addition, FGF9 induced neuroendocrine differentiation, a pathologic characteristic of SCLC, in established human lung adenocarcinoma cells. Thus, the findings provide direct evidence for FGF9-mediated SCLC transdifferentiation and propose the FGF9-FGFR axis as a therapeutic target for transdifferentiated SCLC. SIGNIFICANCE: This study demonstrates that FGF9 plays a role in the transdifferentiation of lung adenocarcinoma to small cell lung cancer.

Intrinsic Immunogenicity of Small Cell Lung Carcinoma Revealed by Its Cellular Plasticity.

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861.

Epigenomic Profiling Discovers Trans-lineage SOX2 Partnerships Driving Tumor Heterogeneity in Lung Squamous Cell Carcinoma.

Molecular characterization of lung squamous cell carcinoma (LUSC), one of the major subtypes of lung cancer, has not sufficiently improved its nonstratified treatment strategies over decades. Accumulating evidence suggests that lineage-specific transcriptional regulators control differentiation states during cancer evolution and underlie their distinct biological behaviors. In this study, by investigating the super-enhancer landscape of LUSC, we identified a previously undescribed "neural" subtype defined by Sox2 and a neural lineage factor Brn2, as well as the classical LUSC subtype defined by Sox2 and its classical squamous partner p63. Robust protein-protein interaction and genomic cooccupancy of Sox2 and Brn2, in place for p63 in the classical LUSC, indicated their transcriptional cooperation imparting this unique lineage state in the "neural" LUSC. Forced expression of p63 downregulated Brn2 in the "neural" LUSC cells and invoked the classical LUSC lineage with more squamous/epithelial features, which were accompanied by increased activities of ErbB/Akt and MAPK-ERK pathways, suggesting differential dependency. Collectively, our data demonstrate heterogeneous cell lineage states of LUSC featured by Sox2 cooperation with Brn2 or p63, for which distinct therapeutic approaches may be warranted. SIGNIFICANCE: Epigenomic profiling reveals a novel subtype of lung squamous cell carcinoma with neural differentiation.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/24/6084/F1.large.jpg.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells.

BACKGROUND: Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs) have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. RESULTS: We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1) that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. CONCLUSION: Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

Regulatory Architecture of the LßT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone.

The LßT2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying the developmental processes and the response to gonadotropin-releasing hormone (GnRH). The global epigenetic landscape, which contributes to cell-specific gene regulatory mechanisms, and the single-cell transcriptome response variation of LßT2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of LßT2 cells during GnRH stimulation. In addition, we examine cell-to-cell variability in the transcriptional response to GnRH using Gel bead-in-Emulsion Drop-seq technology. Analysis of a bulk RNA-seq data set obtained 45 min after exposure to either GnRH or vehicle identified 112 transcripts that were regulated >4-fold by GnRH (FDR < 0.05). The top regulated transcripts constitute, as determined by Bayesian massive public data integration analysis, a human pituitary-relevant coordinated gene program. Chromatin accessibility [assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)] data sets generated from GnRH-treated LßT2 cells identified more than 58,000 open chromatin regions, some containing notches consistent with bound transcription factor footprints. The study of the most prominent open regions showed that 75% were in transcriptionally active promoters or introns, supporting their involvement in active transcription. Lhb, Cga, and Egr1 showed significantly open chromatin over their promoters. While Fshb was closed over its promoter, several discrete significantly open regions were found at -40 to -90 kb, which may represent novel upstream enhancers. Chromatin accessibility determined by ATAC-seq was associated with high levels of gene expression determined by RNA-seq. We obtained high-quality single-cell Gel bead-in-Emulsion Drop-seq transcriptome data, with an average of >4,000 expressed genes/cell, from 1,992 vehicle- and 1,889 GnRH-treated cells. While the individual cell expression patterns showed high cell-to-cell variation, representing both biological and measurement variation, the average expression patterns correlated well with bulk RNA-seq data. Computational assignment of each cell to its precise cell cycle phase showed that the response to GnRH was unaffected by cell cycle. To our knowledge, this study represents the first genome-wide epigenetic and single-cell transcriptomic characterization of this important gonadotrope model. The data have been deposited publicly and should provide a resource for hypothesis generation and further study.