ChIP-seq Assays Research Articles

Cistrome Data Browser: expanded datasets and new tools for gene regulatory analysis.

The Cistrome Data Browser (DB) is a resource of human and mouse cis-regulatory information derived from ChIP-seq, DNase-seq and ATAC-seq chromatin profiling assays, which map the genome-wide locations of transcription factor binding sites, histone post-translational modifications and regions of chromatin accessible to endonuclease activity. Currently, the Cistrome DB contains approximately 47,000 human and mouse samples with about 24,000 newly collected datasets compared to the previous release two years ago. Furthermore, the Cistrome DB has a new Toolkit module with several features that allow users to better utilize the large-scale ChIP-seq, DNase-seq, and ATAC-seq data. First, users can query the factors which are likely to regulate a specific gene of interest. Second, the Cistrome DB Toolkit facilitates searches for factor binding, histone modifications, and chromatin accessibility in any given genomic interval shorter than 2Mb. Third, the Toolkit can determine the most similar ChIP-seq, DNase-seq, and ATAC-seq samples in terms of genomic interval overlaps with user-provided genomic interval sets. The Cistrome DB is a user-friendly, up-to-date, and well maintained resource, and the new tools will greatly benefit the biomedical research community. The database is freely available at http://cistrome.org/db, and the Toolkit is at http://dbtoolkit.cistrome.org.

Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects

Neural tube defects (NTDs) are serious congenital malformations. Excessive maternal homocysteine (Hcy) increases the risk of NTDs, while its mechanism remains elusive. Here we report the role of histone homocysteinylation in neural tube closure (NTC). A total of 39 histone homocysteinylation sites are identified in samples from human embryonic brain tissue using mass spectrometry. Elevated levels of histone KHcy and H3K79Hcy are detected at increased cellular Hcy levels in human fetal brains. Using ChIP-seq and RNA-seq assays, we demonstrate that an increase in H3K79Hcy level down-regulates the expression of selected NTC-related genes including Cecr2, Smarca4, and Dnmt3b. In human NTDs brain tissues, decrease in expression of CECR2, SMARCA4, and DNMT3B is also detected along with high levels of Hcy and H3K79Hcy. Our results suggest that higher levels of Hcy contribute to the onset of NTDs through up-regulation of histone H3K79Hcy, leading to abnormal expressions of selected NTC-related genes.

PO-086 BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous cell carcinoma

IntroductionLung cancer accounts for the highest rate of cancer related diagnosis and mortality worldwide. Small cell lung cancer and non-small cell lung cancer (NSCLC) make up the major subtypes of lung cancer. NSCLC patients only have a 15% five-year survival rate. The major subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUSC accounts for more than 4 00 000 deaths each year and unlike LUAD there are limited targeted therapies. Therefore, a great deal of work still needs to be done to understand the drivers for this cancer.Material and methods shRNA was utilised to modulate the levels of BCL11A and SOX2 in LUSC cell lines. In vitro 3D colony assays and xenograft mouse models were employed to understand the role of BCL11A in driving LUSC tumorigenesis. To explore the role of BCL11A in vivo, a Cre-inducible BCL11A overexpression mouse model was used. To further investigate BCL11A in LUSC, a mouse tracheal basal cells organoid assay was employed. CHIP-seq, immunoprecipitation and immunoblotting assays were designed to dissect the mechanism by which BCL11A elicits its function in LUSC. Drug dose response assays were used to test the efficacy of SETD8 inhibitors and cisplatin on an array of lung cancer cell lines.Results and discussionsAnalysis of TCGA has revealed BCL11A to be upregulated in LUSC but not LUAD. Subsequently reducing BCL11A levels in LUSC cell lines results in diminished xenograft tumour growth. Inversely, its overexpression in vivo led to lung airway hyperplasia and the development of reserve cell hyperplastic lesions which is a precursor to squamous metaplasia. Moreover, deleting Bcl11a in mouse tracheal basal cells abolished the development of tracheosphere organoids while its overexpression led to solid tracheospheres expressing markers of squamous cells.At the molecular level we found BCL11A to be a target of SOX2 and we show that it is required for the oncogenic role of SOX2 in LUSC. Furthermore, we showed that BCL11A and SOX2 interact at the protein level and that together they co-regulated the expression of several transcription factors. We demonstrate that pharmacological inhibition of SETD8, a gene co-regulated by BCL11A and SOX2, alone or in combination with cisplatin treatment, shows significant selectivity to LUSC in comparison to LUAD cells.ConclusionCollectively, these results indicate that the disruption of the BCL11A-SOX2 transcriptional program provides a future framework for the development of targeted therapeutic intervention for LUSC patients.

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

PO-090 Expression of the cocaine- and amphetamine-regulated transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the oestrogen receptor

IntroductionLung cancer accounts for the highest rate of cancer related diagnosis and mortality worldwide. Small cell lung cancer and non-small cell lung cancer (NSCLC) make up the major subtypes of lung cancer. NSCLC patients only have a 15% five-year survival rate. The major subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUSC accounts for more than 4 00 000 deaths each year and unlike LUAD there are limited targeted therapies. Therefore, a great deal of work still needs to be done to understand the drivers for this cancer.Material and methods shRNA was utilised to modulate the levels of BCL11A and SOX2 in LUSC cell lines. In vitro 3D colony assays and xenograft mouse models were employed to understand the role of BCL11A in driving LUSC tumorigenesis. To explore the role of BCL11A in vivo, a Cre-inducible BCL11A overexpression mouse model was used. To further investigate BCL11A in LUSC, a mouse tracheal basal cells organoid assay was employed. CHIP-seq, immunoprecipitation and immunoblotting assays were designed to dissect the mechanism by which BCL11A elicits its function in LUSC. Drug dose response assays were used to test the efficacy of SETD8 inhibitors and cisplatin on an array of lung cancer cell lines.Results and discussionsAnalysis of TCGA has revealed BCL11A to be upregulated in LUSC but not LUAD. Subsequently reducing BCL11A levels in LUSC cell lines results in diminished xenograft tumour growth. Inversely, its overexpression in vivo led to lung airway hyperplasia and the development of reserve cell hyperplastic lesions which is a precursor to squamous metaplasia. Moreover, deleting Bcl11a in mouse tracheal basal cells abolished the development of tracheosphere organoids while its overexpression led to solid tracheospheres expressing markers of squamous cells.At the molecular level we found BCL11A to be a target of SOX2 and we show that it is required for the oncogenic role of SOX2 in LUSC. Furthermore, we showed that BCL11A and SOX2 interact at the protein level and that together they co-regulated the expression of several transcription factors. We demonstrate that pharmacological inhibition of SETD8, a gene co-regulated by BCL11A and SOX2, alone or in combination with cisplatin treatment, shows significant selectivity to LUSC in comparison to LUAD cells.ConclusionCollectively, these results indicate that the disruption of the BCL11A-SOX2 transcriptional program provides a future framework for the development of targeted therapeutic intervention for LUSC patients.

TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor κB p65: Single Nucleotide Polymorphism and Estrogen Dependence.

T-cell leukemia 1A (TCL1A) single-nucleotide polymorphisms (SNPs) have been associated with aromatase inhibitor-induced musculoskeletal adverse events. We previously demonstrated that TCL1A is inducible by estradiol (E2) and plays a critical role in the regulation of cytokines, chemokines, and Toll-like receptors in a TCL1A SNP genotype and estrogen-dependent fashion. Furthermore, TCLIA SNP-dependent expression phenotypes can be "reversed" by exposure to selective estrogen receptor modulators such as 4-hydroxytamoxifen (4OH-TAM). The present study was designed to comprehensively characterize the role of TCL1A in transcriptional regulation across the genome by performing RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays with lymphoblastoid cell lines. RNA-seq identified 357 genes that were regulated in a TCL1A SNP- and E2-dependent fashion with expression patterns that were 4OH-TAM reversible. ChIP-seq for the same cells identified 57 TCL1A binding sites that could be regulated by E2 in a SNP-dependent fashion. Even more striking, nuclear factor-κB (NF-κB) p65 bound to those same DNA regions. In summary, TCL1A is a novel transcription factor with expression that is regulated in a SNP- and E2-dependent fashion-a pattern of expression that can be reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq results suggest that TCL1A also acts as a transcriptional coregulator with NF-κB p65, an important immune system transcription factor.

Nuclear galectin-1-FOXP3 interaction dampens the tumor-suppressive properties of FOXP3 in breast cancer

FOXP3 is an important X-linked suppressor of breast cancer. It is reported that FOXP3 is usually mutant, absent, or cytoplasmic distribution in breast cancer cells, which increases the risk of breast cancer. However, in our study the full-length FOXP3 transcript can be detected in breast cancer cells and nuclear FOXP3 is expressed in some breast cancer samples. Therefore, an important question is how the tumor-suppressive function of wild-type FOXP3 is negated in these cancers. We found that Gal-1 is a novel interacting protein of FOXP3 in breast cancer. Furthermore, our results show that the FKH domain in FOXP3 is essential for its interaction with Gal-1. Through ChIP-seq assay, we found that the expression of Gal-1 could inhibit a variety of target genes which were directly regulated by FOXP3. More importantly, these FOXP3-bound genes are involved in the development and metastasis of cancer. Furthermore, functional studies revealed that blocking the FOXP3/Gal-1 interaction restores the tumor-suppressive properties of FOXP3 in breast cancer cells. Finally, we observed that the nuclear abundance of Gal-1 was significantly higher in breast cancer tissues than that in adjacent normal tissues. In addition, we identified that the acidic extracellular microenvironment in breast cancer tissues causes Gal-1 to accumulate in the nucleus. Altogether, nuclear Gal-1 interferes with the binding of FOXP3 to DNA by interacting with the FKH domain of FOXP3, and it indicates a possible mechanism for the loss of the tumor-suppressive properties of FOXP3 in wild-type FOXP3-positive breast cancer.

Integrating ChIP-seq with other functional genomics data.

Transcription is regulated by transcription factor (TF) binding at promoters and distal regulatory elements and histone modifications that control the accessibility of these elements. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard assay for identifying genome-wide protein–DNA interactions in vitro and in vivo. As large-scale ChIP-seq data sets have been collected for different TFs and histone modifications, their potential to predict gene expression can be used to test hypotheses about the mechanisms of gene regulation. In addition, complementary functional genomics assays provide a global view of chromatin accessibility and long-range cis-regulatory interactions that are being combined with TF binding and histone remodeling to study the regulation of gene expression. Thus, ChIP-seq analysis is now widely integrated with other functional genomics assays to better understand gene regulatory mechanisms. In this review, we discuss advances and challenges in integrating ChIP-seq data to identify context-specific chromatin states associated with gene activity. We describe the overall computational design of integrating ChIP-seq data with other functional genomics assays. We also discuss the challenges of extending these methods to low-input ChIP-seq assays and related single-cell assays.

Inactivation of PBX3 and HOXA9 by down-regulating H3K79 methylation represses NPM1-mutated leukemic cell survival.

Acute myeloid leukemia (AML) with an NPM1 mutation (NPMc+) has a distinct gene expression signature and displays molecular abnormalities similar to mixed lineage leukemia (MLL), including aberrant expression of the PBX3 and HOXA gene cluster. However, it is unclear if the aberrant expression of PBX3 and HOXA is essential for the survival of NPM1-mutated leukemic cells.Methods: Using the gene expression profiling of TCGA and E-MTAB-3444 datasets, we screened for high co-expression of PBX3 and HOXA9 in NPMc+ leukemia patients. We performed NPMc+ depletion and overexpression experiments to examine aberrant H3K79 methylation through epigenetic regulation. Through RNA interference technology and small-molecule inhibitor treatment, we evaluated the effect of methyl-modified H3K79 on cell survival and explored the possible underlying mechanism.Results: We showed that NPMc+ increased the expression of PBX3 and HOXA9, which are both poor prognosis indicators in AML. High PBX3 and HOXA9 expression was accompanied by increased dimethylated and trimethylated H3K79 in transgenic murine Lin-Sca-1+c-Kit+ cells and human NPMc+ leukemia cells. Using chromatin immunoprecipitation sequencing (ChIP-seq) assays of NPMc+ cells, we determined that hypermethylated H3K79 was present at the expressed HOXA9 gene but not the PBX3 gene. PBX3 expression was positively regulated by HOXA9, and a reduction in either PBX3 or HOXA9 resulted in NPMc+ cell apoptosis. Importantly, an inhibitor of DOT1L, EPZ5676, effectively and selectively promoted NPMc+ human leukemic cell apoptosis by reducing HOXA9 and PBX3 expression.Conclusion: Our data indicate that NPMc+ leukemic cell survival requires upregulation of PBX3 and HOXA9, and this action can be largely attenuated by a DOT1L inhibitor.

Abstract LB-B27: Novel antitumor agent GZ17-6.02 exerts discrete effects on transcriptional regulation in pancreatic cancer cells and cancer associated fibroblasts

Abstract GZ17-6.02 is a synthetic formulation of Arum palaestinum extracts that has shown significant antitumor activity against a variety of cancer types including pancreatic ductal adenocarcinoma (PDAC). Previous studies have showed that GZ17-6.02 significantly inhibited PDAC tumor growth in an orthotopic xenograft mouse model for PDAC. Mechanistically, GZ17-6.02 suppressed the expression of multiple tumor stem cell markers in PDAC cells. However, the exact mechanism of action or cellular targets of GZ17-6.02 is not well understood. Considering the substantial effect of GZ17-6.02 on transcription, we hypothesized that GZ17-6.02 might affect the super-enhancers (SEs) networks in pancreatic cancer cells and/or stromal fibroblasts. In this study, we sought to evaluate the effect of GZ17-6.02 on global gene expression, particularly those associated with super-enhancers in PDAC cells and cancer associated fibroblasts (CAFs) using whole genome RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) assays. Our results showed that GZ17-6.02 induced expression changes in a large number of genes in both pancreatic cancer cells and CAFs. Overall, GZ17-6.02 induced expression changes in more genes in the cancer cells (MIA PaCa-2) than in the CAFs (CAF08). Clustering analysis showed that the top differentially expressed genes are very different between the cancer and CAF cells. In MIA PaCa-2 cells, GZ17-6.02 most significantly affected pathways related to gap junctions whereas in CAF08 cells, pathways related to extracellular matrix proteins such as hyaluronan were among the most significantly dysregulated by the drug. The ChIP-seq results indicated that GZ17-6.02 treatment at 50 μg/mL for 6 hours disrupted many SEs, which may have caused changes in the expression of genes associated with those SEs in the pancreatic cancer cells. The effect of GZ17-6.02 at 50 μg/mL for 6 hours on SEs in the CAF08 was not significant. This is consistent with the fact that relatively small effect on global gene expression in CAF08 cells was observed in the transcriptome analysis. Overall, our study demonstrates that GZ17-6.02 exerts discrete effects on pancreatic cancer cells and CAFs, indicating that the drug may target both the tumors and their associated fibroblasts (stroma). Furthermore, GZ17-6.02 may disrupt super-enhancer networks in the tumor cells to affect genes that play important roles in pancreatic cancer. Citation Format: Emily Rodela, Pawan Noel, Shaimaa Hussein, Yin C. Lin, Daniel D. Von Hoff, Haiyong Han. Novel antitumor agent GZ17-6.02 exerts discrete effects on transcriptional regulation in pancreatic cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B27.

The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis

BackgroundThe stem cell factor spalt-like transcription factor 4 (SALL4) plays important roles in normal hematopoiesis and also in leukemogenesis. We previously reported that SALL4 exerts its effect by recruiting important epigenetic factors such as DNA methyltransferases DNMT1 and lysine-specific demethylase 1 (LSD1/KDM1A). Both of these proteins are critically involved in mixed lineage leukemia (MLL)-rearranged (MLL-r) leukemia, which has a very poor clinical prognosis. Recently, SALL4 has been further linked to the functions of MLL and its target gene homeobox A9 (HOXA9). However, it remains unclear whether SALL4 is indeed a key player in MLL-r leukemia pathogenesis.MethodsUsing a mouse bone marrow retroviral transduction/ transplantation approach combined with tamoxifen-inducible, CreERT2-mediated Sall4 gene deletion, we studied SALL4 functions in leukemic transformation that was induced by MLL-AF9—one of the most common MLL-r oncoproteins found in patients. In addition, the underlying transcriptional and epigenetic mechanisms were explored using chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq), mRNA microarray, qRT-PCR, histone modification, co-immunoprecipitation (co-IP), cell cycle, and apoptosis assays. The effects of SALL4 loss on normal hematopoiesis in mice were also investigated.ResultsIn vitro and in vivo studies revealed that SALL4 expression is critically required for MLL-AF9-induced leukemic transformation and disease progression in mice. Loss of SALL4 in MLL-AF9-transformed cells induced apoptosis and cell cycle arrest at G1. ChIP-Seq assay identified that Sall4 binds to key MLL-AF9 target genes and important MLL-r or non-MLL-r leukemia-related genes. ChIP-PCR assays indicated that SALL4 affects the levels of the histone modification markers H3K79me2/3 and H3K4me3 at MLL-AF9 target gene promoters by physically interacting with DOT1-like histone H3K79 methyltransferase (DOT1l) and LSD1/KDM1A, and thereby regulates transcript expression. Surprisingly, normal Sall4f/f/CreERT2 mice treated with tamoxifen or vav-Cre-mediated (hematopoietic-specific) Sall4−/− mice were healthy and displayed no significant hematopoietic defects.ConclusionsOur findings indicate that SALL4 critically contributes to MLL-AF9-induced leukemia, unraveling the underlying transcriptional and epigenetic mechanisms in this disease and suggesting that selectively targeting the SALL4 pathway may be a promising approach for managing human MLL-r leukemia.

Identification of the centromeres of Leishmania major: revealing the hidden pieces.

Leishmania affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in Trypanosoma brucei (KKTs) leads to the identification of a Leishmania KKT gene candidate (LmKKT1). The GFP-tagged LmKKT1 displays "kinetochore-like" dynamics of intranuclear localization throughout the cell cycle. By ChIP-Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of Leishmania chromosomes opens avenues to explore their role in mosaic aneuploidy.

The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis

BackgroundThe tumor acidic microenvironment, a common biochemical event in solid tumors, offers evolutional advantage for tumors cells and even enhances their aggressive phenotype. However, little is known about the molecular mechanism underlying the acidic microenvironment-induced invasion and metastasis.MethodsWe examined the expression of the acid-sending ion channel (ASIC) family members after acidic exposure using RT-PCR and immunofluoresence. Gene manipulation was applied to reveal the potential of ASIC2 on invasion, proliferation, colony formation of colorectal cancer (CRC). We assessed the in vivo tumor growth by subcutaneous transplantation and metastasis by spleen xenografts. Chromatin immunoprecipitation-sequencing was used to uncover the binding sites of NFAT1. Finally, we examined the expression of ASIC2 in CRC tissues using immunohistochemistry.ResultsAcidic exposure led to up-regulation of the acid-sensing ion channel, ASIC2, in colorectal cancer (CRC) cells. ASIC2 overexpression in CRC cell lines, SW480 and HCT116, significantly enhanced cell proliferation in vitro and in vivo, while ASIC2 knockdown had the reverse effect. Importantly, ASIC2 promoted CRC cell invasion under acidosis in vitro and liver metastasis in vivo. Mechanistically, ASIC2 activated the calcineurin/NFAT1 signaling pathway under acidosis. Inhibition of the calcineurin/NFAT pathway by cyclosporine A (CsA) profoundly attenuated ASIC2-induced invasion under acidosis. ChIP-seq assay revealed that the nuclear factor, NFAT1, binds to genes clustered in pathways involved in Rho GTPase signaling and calcium signaling. Furthermore, immunohistochemistry showed that ASIC2 expression is increased in CRC samples compared to that in adjacent tissues, and ASIC2 expression correlates with T-stage, distant metastasis, recurrence, and poor prognosis.ConclusionASIC2 promotes metastasis of CRC cells by activating the calcineurin/NFAT1 pathway under acidosis and high expression of ASIC2 predicts poor outcomes of patients with CRC.

1679P - Mutant KIT translocates into the nucleus and induces NFKBIB expression that leads to KIT expression in imatinib-resistant gastrointestinal stromal tumors

Background: Gastrointestinal stromal tumor (GIST) is a dominantly mutant KIT-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Increasing evidences show that membrane-bound receptors, as EGFR, can translocate into the nucleus, mediate genes expression, and lead to tumor survival and drug resistance. However, it’s barely known the nuclear role of KIT in GIST. Methods: In this study, two imatinib (IM)-resistant GIST cell lines, GIST48 and GIST430, were used as a model. Results: In this study, we first showed that KIT is distributed both in the cytoplasm and the nucleus in IM-resistant GIST cells. Using ChIP-seq and ChIP assay, we identified that nuclear KIT bound to the NFKBIB promoter region and regulated its expression. The expression levels of NFKBIB and phosopho-KIT were significantly correlated with NCCN-risk category in surgically resected GISTs stained by immunohistochemistry. The cell viabilities were inhibited as accompanying with KIT reduction in GIST cells while NFKBIB was silenced or RELA was overexpressed. Moreover, RELA was activated, translocated into the nucleus, and bound to KIT promoter region in NFKBIB-silenced or RELA-overexpressed GIST cells. Valproic acid, acted as a NFKB inducer, could induce RELA nucleus translocation and binding to KIT promoter region that led to the reduction of protein and RNA expression level of KIT and the cell viabilities of GIST cells. Furthermore, the combination of IM with low-dose valproic acid showed synergistically inhibitory effect on cell viabilities of GIST cells and comparable effects on reducing phospho-KIT level and inhibiting tumor growth as high-dose valproic acid did in GIST430 xenograft model. Conclusions: Taken together, we first demonstrated that phosphorylated KIT could translocate into the nucleus and drive itself expression in IM-resistant GIST cells through mediating NFKBIB expression. In addition, our findings identified a novel and druggable KIT-NFKBIB-NFKB regulatory axis that provides a new insight on tumorigenesis and therapeutic option for IM-resistant, mutant KIT-expressing GISTs. Legal entity responsible for the study: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan Funding: National Health Research Institutes, Taiwan and the Ministry of Science and Technology, Taiwan. Disclosure: Y-S. Hsueh: Research funds were provided by the Ministry of Science and Technology, Taiwan. L-T. Chen: Research funds were provided by the National Health Research Institutes, Taiwan. All other authors have declared no conflicts of interest.

Abstract 1524: BCL6 modulates the TP53 and STAT pathways in glioma

Abstract Glioblastoma multiforme (GBM) remains the most aggressive brain malignancy with little improvement in prognosis or therapy for decades. Recently, we identified BCL6, also known as ZBTB27, to be a novel oncogene in GBM. In this study, we performed IHC analysis of 153 primary human glioma specimens and 8 normal brain samples. BCL6 expression is robustly elevated in tumor samples and positively correlated with glioma pathological grade. High BCL6 expression strongly predicts a worse prognosis of GBM patients. Depletion of BCL6 in human GBM cells reduced the incorporation of BrdU, promoted the cellular senescence and inhibited the growth of human GBM cells in vivo. Next, genome-wide occupancy of BCL6 in GBM cells was characterized by ChIP-seq assay. Genomic regions centered on BCL6 peaks are co-enriched with RNA-Pol II and flanked with strong H3K27ac and H3K4me3 modifications. MYC and two long non-coding RNAs MALAT1 and NEAT1 were identified as novel BCL6 targets in GBM. Moreover, pathway enrichment analysis of BCL6 peak-associated genes reveals a significant enrichment of JAK-STAT, TP53, ERBB and MAPK pathways. We demostrated further that BCL6 represses the TP53 pathway and promotes the JAK-STAT pathway activation in GBM cells. Together, our findings uncover potential downstream targets and provide a better understanding of BCL6 function in GBM. Citation Format: Ye Chen, Liang Xu, Marina Dutra-Clarke, Anand Mayakonda, De-Chen Lin, Lynnette Koh, Yuk Kien Chong, Edwin Sandanaraj, Vikas Madan, Henry Yang, Ngan Doan, Jonathan W. Said, William H. Yong, Markus Müschen, Beng Ti Ang, Carol Tang, Joshua J. Breunig, Phillip Koeffler. BCL6 modulates the TP53 and STAT pathways in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1524. doi:10.1158/1538-7445.AM2017-1524

Abstract 3578: Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation

Abstract DNA methylation and histone modifications are interconnected and interdependent epigenetic mechanisms that regulate chromatin accessibility in cells. They establish cell’s developmental identity and modulate individual response to endogenous developmental stimuli and environmental changes. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a popular assay to study genomewide histone modification profiles. However, ChIP-seq assays require relatively large amounts of starting materials (ranging from ~105 cells using a low cell number protocol to 107 cells in a standard protocol), which remains as a major obstacle for precious biological samples including primary tumor specimens. On the other hand, tagmentation-based whole genome bisulfite sequencing (TWGBS) protocol enables unbiased assessment of the genomewide methylation pattern using ~3,000 cells. Therefore, a quantitative relationship between DNA methylation and epigenetic/transcriptomic activity is in great demand. In the current study, we developed Methyl2Acetyl, a machine learning framework to infer promoter epigenetic activities (e.g. H3K27Ac enrichment) using TWGBS data as input. The proposed model utilizes random forest learners, which allow automatic extraction of complex interactions among DNA methylation features surrounding transcription start sites. We applied Methyl2Acetyl to a set of pediatric solid tumor samples with high quality H3K27Ac ChIPseq data to evaluate its performance. The model accurately predicted promoter H3K27Ac enrichment in independent test samples (Pearson correlation against measured enrichment: 0.81 ± 0.02). To further test the robustness of our model, we trained a Methyl2Acetyl model on solid tumors and applied it to a set of 10 pediatric leukemia samples. Despite dramatic global methylome differences between solid tumors and leukemia, Methyl2Acetyl predicted promoter H3K27Ac enrichment showed strong concordance with target gene expression level in the same sample (Pearson correlation: 0.75 ± 0.02, classification AUC: 0.89 ± 0.01). Collectively, our data suggested that DNA methylation is predictive of promoter active histone modification enrichment and gene expression activity. Epigenetically active promoters can be imputed from TWGBS data in samples with limiting starting materials. Citation Format: Xiang Chen. Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2017-3578

CXCL13 is androgen-responsive and involved in androgen induced prostate cancer cell migration and invasion.

Androgen receptor (AR) is a key transcription factor playing a critical role in prostate cancer (PCa) initiation and progression. However, the molecular mechanisms of AR action in prostate cancer are not very clear. CXCL13, known as B cell attracting chemokine1 (BCA-1), is a member of CXC chemokine family and relevant to cancer metastasis. This study shows that CXCL13 is an androgen-responsive gene and involved in AR-induced PCa cell migration and invasion. In clinical specimens, expression of CXCL13 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of CXCL13 is up-regulated by androgen-AR axis at both mRNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen responsive elements (ARE) at CXCL13 enhancer and dual-luciferase reporter assays reveals that the ARE is highly responsive to androgen while mutations of the ARE abolish the reporter activity. Additional chromatin immunoprecipitation (ChIP) assays also identify that the ARE presents androgen responsiveness. In addition, CXCL13 promotes G2/M phase transition by increasing Cyclin B1 levels in PCa cells. Functional studies demonstrate that reducing endogenous CXCL13 expression in LNCaP cells largely weakens androgen-AR axis induced cell migration and invasion. Taken together, our study implicates for the first time that CXCL13 is an AR target gene and involved in AR-mediated cell migration and invasion in primary PCa.

Abstract AP31: TARGETING PD–L1/PD1 PATHWAY THROUGH BET PROTEIN INHIBITION IN EPITHELIAL OVARIAN CANCER

Abstract BACKGROUND: Expression of programmed death ligand 1 (PD-L1) is up-regulated in many cancers and has been found to suppress antitumor immunity through binding to its receptor programmed death 1 (PD-1). Blockade of PD-1/PD-L1 signaling axis enhances antitumor immunity in many cancers including epithelial ovarian cancer (EOC). Despite the importance of PD-L1 in tumor immunity, the epigenetic regulation of PD-L1 expression remains poorly understood. AIMS: To determine the roles of epigenetic components in regulating PD-L1 gene expression and modulating antitumor immunity. METHODS: Toward this goal, I screened a small molecule library of epigenetic inhibitors and examined their inhibition on PD-L1 expression in EOC cells to identify the positive hits. To further confirm PD-L1 inhibition in vivo, C57BL/6 mice are injected with luciferase expressing mouse ovarian cancer ID8 cells and treated with or without epigenetic inhibitors, PD-L1 expression in both tumor cells and immune cells are examined, tumor growth and survival are also monitored. RESULTS: BET inhibitors suppresses both constitutive and IFNγ induced PD-L1 expression in EOC cells through targeting BRD4. Further ChIP-seq and ChIP assay demonstrate that BRD4 directly binds to PD-L1 gene promoter and regulated its transcription. BET inhibitor JQ1 suppresses PD-L1 expression in both tumor cells and immune cells including dendritic cells and macrophages in mice bearing ID8 mouse ovarian cancer cells. Moreover, the BET inhibitor suppresses the tumor growth and improves the survival of EOC bearing mice through activating antitumor T cells. CONCLUSION: Taken together, we demonstrated that BRD4 can directly regulate PD-L1 expression in EOC cells by binding to the promoter of PD-L1 encoding gene, suppressing PD-L1 expression with BET inhibitors blocks PD1/PD-L1 signaling and enhances antitumor immunity in vivo. These findings indicate that pharmacological inhibition of BET proteins represent a new therapeutic strategy for EOC by targeting PD-L1 expression. Citation Format: Hengrui Zhu, Fee Bengsch, Nikolaos Svoronos, Melanie R. Rutkowski, Benjamin G. Bitler, Michael J. Allegrezza, Yuhki Yokoyama, James E. Bradner, Jose R. Conejo-Garcia, Rugang Zhang. TARGETING PD–L1/PD1 PATHWAY THROUGH BET PROTEIN INHIBITION IN EPITHELIAL OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP31.

A computational approach for the functional classification of the epigenome

BackgroundIn the last decade, advanced functional genomics approaches and deep sequencing have allowed large-scale mapping of histone modifications and other epigenetic marks, highlighting functional relationships between chromatin organization and genome function. Here, we propose a novel approach to explore functional interactions between different epigenetic modifications and extract combinatorial profiles that can be used to annotate the chromatin in a finite number of functional classes. Our method is based on non-negative matrix factorization (NMF), an unsupervised learning technique originally employed to decompose high-dimensional data in a reduced number of meaningful patterns. We applied the NMF algorithm to a set of different epigenetic marks, consisting of ChIP-seq assays for multiple histone modifications, Pol II binding and chromatin accessibility assays from human H1 cells.ResultsWe identified a number of chromatin profiles that contain functional information and are biologically interpretable. We also observe that epigenetic profiles are characterized by specific genomic contexts and show significant association with distinct genomic features. Moreover, analysis of RNA-seq data reveals that distinct chromatin signatures correlate with the level of gene expression.ConclusionsOverall, our study highlights the utility of NMF in studying functional relationships between different epigenetic modifications and may provide new biological insights for the interpretation of the chromatin dynamics.

Integrative analysis unveils new functions for the Drosophila Cutoff protein in noncoding RNA biogenesis and gene regulation.

Piwi-interacting RNAs (piRNAs) are central components of the piRNA pathway, which directs transposon silencing and guarantees genome integrity in the germ cells of several metazoans. In Drosophila, piRNAs are produced from discrete regions of the genome termed piRNA clusters, whose expression relies on the RDC complex comprised of the core proteins Rhino, Deadlock, and Cutoff. To date, the RDC complex has been exclusively implicated in the regulation of the piRNA loci. Here we further elucidate the function of Cutoff and the RDC complex by performing genome-wide ChIP-seq and RNA-seq assays in the Drosophila ovaries and analyzing these data together with other publicly available data sets. In agreement with previous studies, we confirm that Cutoff is involved in the transcriptional regulation of piRNA clusters and in the repression of transposable elements in germ cells. Surprisingly, however, we find that Cutoff is enriched at and affects the expression of other noncoding RNAs, including spliceosomal RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). At least in some instances, Cutoff appears to act at a transcriptional level in concert with Rhino and perhaps Deadlock. Finally, we show that mutations in Cutoff result in the deregulation of hundreds of protein-coding genes in germ cells. Our study uncovers a broader function for the RDC complex in the Drosophila germline development.