Maria Tsompana

Chromatin accessibility: a window into the genome

Transcriptional activation throughout the eukaryotic lineage has been tightly linked with disruption of nucleosome organization at promoters, enhancers, silencers, insulators and locus control regions due to transcription factor binding. Regulatory DNA thus coincides with open or accessible genomic sites of remodeled chromatin. Current chromatin accessibility assays are used to separate the genome by enzymatic or chemical means and isolate either the accessible or protected locations. The isolated DNA is then quantified using a next-generation sequencing platform. Wide application of these assays has recently focused on the identification of the instrumental epigenetic changes responsible for differential gene expression, cell proliferation, functional diversification and disease development. Here we discuss the limitations and advantages of current genome-wide chromatin accessibility assays with especial attention on experimental precautions and sequence data analysis. We conclude with our perspective on future improvements necessary for moving the field of chromatin profiling forward.

Sunitinib Dose Escalation Overcomes Transient Resistance in Clear Cell Renal Cell Carcinoma and Is Associated with Epigenetic Modifications

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinibs clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.

Suppressed hepatic bile acid signalling despite elevated production of primary and secondary bile acids in NAFLD

Objective Bile acids are regulators of lipid and glucose metabolism, and modulate inflammation in the liver and other tissues. Primary bile acids such as cholic acid and chenodeoxycholic acid (CDCA) are produced in the liver, and converted into secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid by gut microbiota. Here we investigated the possible roles of bile acids in non-alcoholic fatty liver disease (NAFLD) pathogenesis and the impact of the gut microbiome on bile acid signalling in NAFLD. Design Serum bile acid levels and fibroblast growth factor 19 (FGF19), liver gene expression profiles and gut microbiome compositions were determined in patients with NAFLD, high-fat diet-fed rats and their controls. Results Serum concentrations of primary and secondary bile acids were increased in patients with NAFLD. In per cent, the farnesoid X receptor (FXR) antagonistic DCA was increased, while the agonistic CDCA was decreased in NAFLD. Increased mRNA expression for cytochrome P450 7A1, Na+-taurocholate cotransporting polypeptide and paraoxonase 1, no change in mRNA expression for small heterodimer partner and bile salt export pump, and reduced serum FGF19 were evidence of impaired FXR and fibroblast growth factor receptor 4 (FGFR4)-mediated signalling in NAFLD. Taurine and glycine metabolising bacteria were increased in the gut of patients with NAFLD, reflecting increased secondary bile acid production. Similar changes in liver gene expression and the gut microbiome were observed in high-fat diet-fed rats. Conclusions The serum bile acid profile, the hepatic gene expression pattern and the gut microbiome composition consistently support an elevated bile acid production in NAFLD. The increased proportion of FXR antagonistic bile acid explains, at least in part, the suppression of hepatic FXR-mediated and FGFR4-mediated signalling. Our study suggests that future NAFLD intervention may target the components of FXR signalling, including the bile acid converting gut microbiome.

Multi-targeting therapeutic mechanisms of the Chinese herbal medicine QHD in the treatment of non-alcoholic fatty liver disease

Beneficial effects of the Chinese herbal medicine Qushi Huayu Decoction (QHD) were observed with non-alcoholic fatty liver disease (NAFLD) patients and animal models. The impact of QHD or its active components (geniposide and chlorogenic acid, GC) on NAFLD liver transcriptome and gut microbiota was examined with NAFLD rats. Increased expression for genes required for glutathione production and decreased expression for genes required for lipid synthesis was observed in NAFLD livers treated with QHD and GC. GC treatment decreased serum LPS, which could be explained by reduced mucosal damage in the colon of GC-treated rats. Further, our data suggest an increased abundance of Treg-inducing bacteria that stimulated the Treg activity in GC treated colon, which in turn down-regulated inflammatory signals, improved gut barrier function and consequently reduced hepatic exposure to microbial products. Our study suggests that QHD simultaneously enhanced the hepatic anti-oxidative mechanism, decreased hepatic lipid synthesis, and promoted the regulatory T cell inducing microbiota in the gut.

An automated method for efficient, accurate and reproducible construction of RNA-seq libraries

BackgroundIntegration of RNA-seq expression data with knowledge on chromatin accessibility, histone modifications, DNA methylation, and transcription factor binding has been instrumental for the unveiling of cell-specific local and long-range regulatory patterns, facilitating further investigation on the underlying rules of transcription regulation at an individual and allele-specific level. However, full genome transcriptome characterization has been partially limited by the complexity and increased time-requirements of available RNA-seq library construction protocols.FindingsUse of the SX-8G IP-Star® Compact System significantly reduces the hands-on time for RNA-seq library synthesis, adenylation, and adaptor ligation providing with high quality RNA-seq libraries tailored for Illumina high-throughput next-generation sequencing. Generated data exhibits high technical reproducibility compared to data from RNA-seq libraries synthesized manually for the same samples. Obtained results are consistent regardless the researcher, day of the experiment, and experimental run.ConclusionsOverall, the SX-8G IP-Star® Compact System proves an efficient, fast and reliable tool for the construction of next-generation RNA-seq libraries especially for trancriptome-based annotation of larger genomes.

Correction: Salivary inflammatory markers and microbiome in normoglycemic lean and obese children compared to obese children with type 2 diabetes

[This corrects the article DOI: 10.1371/journal.pone.0172647.].

Gut microbiome may contribute to insulin resistance and systemic inflammation in obese rodents: a meta-analysis

A number of studies have associated obesity with altered gut microbiota, although results are discordant regarding compositional changes in the gut microbiota of obese animals. Herein we used a meta-analysis to obtain an unbiased evaluation of structural and functional changes of the gut microbiota in diet-induced obese rodents. The raw sequencing data of nine studies generated from high-fat diet (HFD)-induced obese rodent models were processed with QIIME to obtain gut microbiota compositions. Biological functions were predicted and annotated with KEGG pathways with PICRUSt. No significant difference was observed for alpha diversity and Bacteroidetes-to-Firmicutes ratio between obese and lean rodents. Bacteroidia, Clostridia, Bacilli, and Erysipelotrichi were dominant classes, but gut microbiota compositions varied among studies. Meta-analysis of the nine microbiome data sets identified 15 differential taxa and 57 differential pathways between obese and lean rodents. In obese rodents, increased abundance was observed for Dorea, Oscillospira, and Ruminococcus, known for fermenting polysaccharide into short chain fatty acids (SCFAs). Decreased Turicibacter and increased Lactococcus are consistent with elevated inflammation in the obese status. Differential functional pathways of the gut microbiome in obese rodents included enriched pyruvate metabolism, butanoate metabolism, propanoate metabolism, pentose phosphate pathway, fatty acid biosynthesis, and glycerolipid metabolism pathways. These pathways converge in the function of carbohydrate metabolism, SCFA metabolism, and biosynthesis of lipid. HFD-induced obesity results in structural and functional dysbiosis of gut microbiota. The altered gut microbiome may contribute to obesity development by promoting insulin resistance and systemic inflammation.

Abstract 07: An oncogenic transcriptional network anchored by ETS1, p63 and subtype specific drivers of HNSCC revealed by epigenomic and genomic interrogation

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease of significant mortality and morbidity, with a 5-year survival rate that has shown only modest improvement. This persistent patient mortality and morbidity can be attributed, in part, to an overall lack of novel targeted treatment options. Indeed, recent genome sequencing studies have highlighted the striking heterogeneity of HNSCC tumors and have failed to identify pervasive and broadly actionable driver mutations and/or other genetic alterations. Despite this genetic heterogeneity, HNSCC tumors can be classified into gene-expression defined subtypes, raising the possibility that such distinct gene expression programs and regulatory networks are closely interwoven with an intrinsic heritable and signature epigenetic state. We hypothesized that there exist key oncogenic transcriptional regulators of subtype dependency and that defining the epigenetic landscape of HNSCC tumors will reveal the identity of these crucial factors. This will be impactful since targeting of tumor addiction to key oncogenic Transcription Factors (TFs), although successful in a number of other tumor types, remains largely unexplored in HNSCC due to limited knowledge of the identity of these regulators in this cancer. To decipher the genomic and epigenomic drivers of HNSCC tumor subtypes, we examined a large cohort of cell lines that represent the Atypical, Basal and Mesenchymal subtypes. By using global H3K27 acetylation levels as a surrogate marker, we first generated a consensus genome-wide regulatory enhancer and Super-Enhancer map. Interestingly, we observed a differential enrichment pattern of H3K27Ac signals that recapitulated the segregation of cell-lines according to subtype based on differential gene expression profiles. We leveraged the differential enhancer map to identify enriched DNA-Binding Motifs to generate a Transcription Factor Combinatorial Regulatory Network, based on the co-occurrence frequency of TFs relative to genomic background. Our analysis highlighted a Cis-Regulatory Module (CRM) Network, specific to each molecular subtype of HNSCC. Furthermore, we identified key TF motifs that are embedded within these CRMs and enriched for each subtype-specific network, which we posit represent the pertinent TF drivers of the distinct gene-expression network found in various HNSCC tumors. Here, as proof of principle, we have examined the potential interaction between TP63 and ETS1, two oncogenic TFs revealed by our epigenomic and genomic analysis to be crucial mediators of HPV-negative HNSCC tumors. To better understand the TP63-ETS1 driven molecular processes in HPV-negative tumors, we performed transcriptomic analysis of the effects of knockdown of TP63 and ETS1 by RNA-Seq and identified their global targets by ChIP-Seq. Interestingly, our comprehensive analysis showed that TP63 and ETS1 share a significant number of genomic binding sites, exhibit a marked preference for binding to Super-Enhancers and together act in a concerted fashion to drive expression of unique set of pathways and processes. By integrating our findings with meta-analysis of HNSCC patient datasets, we have uncovered a core TP63-ETS1 driven signature network that serves as a regulatory anchor of common facets of cancer biology, such as proliferation and angiogenesis, as well as drug-targetable tumor-specific processes involving cancer stem cells. Our study has harnessed the power of an integrative genomic/epigenomic approach to characterize HNSCC subtypes and heterogeneity and to provide a robust framework for the discovery of novel tumor drivers that can be leveraged for targeted therapeutics. Citation Format: Christian Gluck, Isha Sethi, Maria Tsompana, Satrajit Sinha. An oncogenic transcriptional network anchored by ETS1, p63 and subtype specific drivers of HNSCC revealed by epigenomic and genomic interrogation [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 07.

Distinct and Shared Determinants of Cardiomyocyte Contractility in Multi-Lineage Competent Ethnically Diverse Human iPSCs.

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics, epigenomics, and bioinformatics that inform on genetic pathways directing tissue development and function. When possible, population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American, Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype, verified teratoma formation, pluripotency biomarkers, and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural, pancreatic, and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential.

Abstract B13: Genomic mistargeting of p63 drives the cancer phenotype in head and neck squamous cell carcinoma

TP63 is a member of the p53 family and is a master regulator of epithelial development and differentiation. Overexpression and/or genomic amplification of TP63 is commonly found in a large number of epithelial tumors, including the majority of head and neck squamous cell carcinomas (HNSCC). In many tumors, p63 appears to function as an oncogene, contributing to both increased tumor cell proliferation and survival. To address the mechanisms of p63 function, we studied the interplay of p63 with the chromatin microenvironment at its genomic targets, under distinct physiological and pathological states. We generated transcriptomic (RNA-Seq) and epigenomic (ChIP-Seq, ATAC-Seq, and DNase-Seq) datasets in representative normal oral and epidermal keratinocytes (HGEP, NHEK) and HNSCC (SCC25, SCC13) cell-lines, to examine whether there exists a HNSCC-specific molecular pattern of p63 targeting. We utilized machine learning methods including Random Forests for classification and regression, to determine the contribution of epigenetic states, transcriptional co-factors and the p63 DNA binding motif in differential p63 binding between normal and cancer states. We found that a core epigenomic signature (H3K4me1 and H3K27me3 binding status and chromatin accessibility) is highly predictive of p63 binding. Furthermore, our analysis revealed that SCC25-specific p63 targets (corresponding to 1239 genomic regions) are inaccessible in normal cells, but in SCC25 they are marked by H3K4me1 and H3K27ac marks, indicative of active regulatory regions. Our data suggests that the complex interplay between local chromatin microenvironment, presence and/or absence of distinct co-factors and potential cell-type specific p63 activity might be key drivers of the differential binding events between normal and cancer epithelial cells. Our investigation of SCC-specific p63 binding events also led to the identification of several oncogenic genes (for e.g. SOX2, TRIM32, HAS2, PRKCE) that are direct targets of p63. By performing Gene Set Enrichment Analysis we found that these p63 misregulated genes encompass both known (G-protein coupled receptor signaling pathway) and novel cancer pathways thus providing a framework for the oncogenic function of p63. To further evaluate the importance of these findings in the tumor context, we derived a p63 target genes signature and used it to cluster RNA-Seq data of HNSCC patients (obtained from The Cancer Genome Atlas). The gene signature could both classify normal samples away from tumors and importantly distinguish between different molecular subtypes of HNSCCs. Collectively our studies suggest that aberrant targeting of p63 leads to dynamic remodeling of the chromatin at its target sites, trigger the expression of a cascade of pro-tumorigenic genes, ultimately condemning the cell to an oncogenic fate. These novel insights potentially provide new avenues to be harnessed for tumor subtype classification, development of biomarkers and for targeted therapies. Citation Format: Isha Sethi, Maria Tsompana, Christian Gluck, Michael J. Buck, Satrajit Sinha. Genomic mistargeting of p63 drives the cancer phenotype in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B13.