ChroKit: a Shiny-based framework for interactive analysis, visualization and integration of genomic data.

We developed ChroKit (the Chromatin toolKit), an interactive web-based framework written in R that enables intuitive exploration, multidimensional analyses, and visualization of genomic data from ChIP-Seq, DNAse-Seq or any other NGS experiment that reports the enrichment of aligned reads over genomic regions. This program takes preprocessed NGS data and performs operations on genomic regions of interest, including resetting their boundaries, their annotation based on proximity to genomic features, the association to gene ontologies, and signal enrichment calculations. Genomic regions can be further refined or subsetted by user-defined logical operations and unsupervised classification algorithms. ChroKit generates a full range of plots that are easily manipulated by point and click operations, thus allowing 'on the fly' re-analysis and fast exploration of the data. Working sessions can be exported for reproducibility, accountability, and easy sharing within the bioinformatics community. ChroKit is multiplatform and can be deployed on a server to enhance computational speed and provide simultaneous access by multiple users. ChroKit is a fast and intuitive genomic analysis tool suited for a wide range of users due to its architecture and its user-friendly graphical interface. ChroKit source code is available at https://github.com/ocroci/ChroKit and the Docker image at https://hub.docker.com/r/ocroci/chrokit.

Transcriptional integration of mitogenic and mechanical signals by Myc and YAP.

Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation.

Decoding YAP dependent transcription in the liver.

The transcriptional coactivator YAP is emerging as a master regulator of cell growth. In the liver, YAP activity is linked to hepatomegaly, regeneration, dedifferentiation, and aggressive tumor growth. Here we present genomic studies to address how YAP may elicit such profound biological changes in murine models. YAP bound the genome in a TEAD-dependent manner, either at loci constitutively occupied by TEAD or by pioneering enhancers, which comprised a fraction of HNF4a/FOXA-bound embryonic enhancers active during embryonic development but silent in the adult. YAP triggered transcription on promoters by recruiting BRD4, enhancing H3K122 acetylation, and promoting RNApol2 loading and pause-release. YAP also repressed HNF4a target genes by binding to their promoters and enhancers, thus preventing RNApol2 pause-release. YAP activation led to the induction of hepatocyte proliferation, accompanied by tissue remodeling, characterized by polarized macrophages, exhausted T-lymphocytes and dedifferentiation of endothelial cells into proliferative progenitors. Overall, these analyses suggest that YAP is a master regulator of liver function that reshapes the enhancer landscape to control transcription of genes involved in metabolism, proliferation, and inflammation, subverts lineage specification programs by antagonizing HNF4a and modulating the immune infiltrate and the vascular architecture of the liver.

A dual role of YAP in driving TGFß-mediated endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFß/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFß signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFß signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFß signaling. We demonstrate that YAP is required to trigger TGFß-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3ß-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFß-induced EndMT at early stages.

Association rule mining to identify transcription factor interactions in genomic regions.

MOTIVATION: Genome regulatory networks have different layers and ways to modulate cellular processes, such as cell differentiation, proliferation, and adaptation to external stimuli. Transcription factors and other chromatin-associated proteins act as combinatorial protein complexes that control gene transcription. Thus, identifying functional interaction networks among these proteins is a fundamental task to understand the genome regulation framework. RESULTS: We developed a novel approach to infer interactions among transcription factors in user-selected genomic regions, by combining the computation of association rules and of a novel Importance Index on ChIP-seq datasets. The hallmark of our method is the definition of the Importance Index, which provides a relevance measure of the interaction among transcription factors found associated in the computed rules. Examples on synthetic data explain the index use and potential. A straightforward pre-processing pipeline enables the easy extraction of input data for our approach from any set of ChIP-seq experiments. Applications on ENCODE ChIP-seq data prove that our approach can reliably detect interactions between transcription factors, including known interactions that validate our approach. AVAILABILITY AND IMPLEMENTATION: A R/Bioconductor package implementing our association rules and Importance Index-based method is available at http://bioconductor.org/packages/release/bioc/html/TFARM.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cooperation Between MYC and ß-Catenin in Liver Tumorigenesis Requires Yap/Taz.

BACKGROUND AND AIMS: Activation of MYC and catenin beta-1 (CTNNB1, encoding ß-catenin) can co-occur in liver cancer, but how these oncogenes cooperate in tumorigenesis remains unclear. APPROACH AND RESULTS: We generated a mouse model allowing conditional activation of MYC and WNT/ß-catenin signaling (through either ß-catenin activation or loss of APC - adenomatous polyposis coli) upon expression of CRE recombinase in the liver and monitored their effects on hepatocyte proliferation, apoptosis, gene expression profiles, and tumorigenesis. Activation of WNT/ß-catenin signaling strongly accelerated MYC-driven carcinogenesis in the liver. Both pathways also cooperated in promoting cellular transformation in vitro, demonstrating their cell-autonomous action. Short-term induction of MYC and ß-catenin in hepatocytes, followed by RNA-sequencing profiling, allowed the identification of a "Myc/ß-catenin signature," composed of a discrete set of Myc-activated genes whose expression increased in the presence of active ß-catenin. Notably, this signature enriched for targets of Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz), two transcriptional coactivators known to be activated by WNT/ß-catenin signaling and to cooperate with MYC in mitogenic activation and liver transformation. Consistent with these regulatory connections, Yap/Taz accumulated upon Myc/ß-catenin activation and were required not only for the ensuing proliferative response, but also for tumor cell growth and survival. Finally, the Myc/ß-catenin signature was enriched in a subset of human hepatocellular carcinomas characterized by comparatively poor prognosis. CONCLUSIONS: Myc and ß-catenin show a strong cooperative action in liver carcinogenesis, with Yap and Taz serving as mediators of this effect. These findings warrant efforts toward therapeutic targeting of Yap/Taz in aggressive liver tumors marked by elevated Myc/ß-catenin activity.

MYC-Induced Replicative Stress: A Double-Edged Sword for Cancer Development and Treatment.

MYC is a transcription factor that controls the expression of a large fraction of cellular genes linked to cell cycle progression, metabolism and differentiation. MYC deregulation in tumors leads to its pervasive genome-wide binding of both promoters and distal regulatory regions, associated with selective transcriptional control of a large fraction of cellular genes. This pairs with alterations of cell cycle control which drive anticipated S-phase entry and reshape the DNA-replication landscape. Under these circumstances, the fine tuning of DNA replication and transcription becomes critical and may pose an intrinsic liability in MYC-overexpressing cancer cells. Here, we will review the current understanding of how MYC controls DNA and RNA synthesis, discuss evidence of replicative and transcriptional stress induced by MYC and summarize preclinical data supporting the therapeutic potential of triggering replicative stress in MYC-driven tumors.

Integrative analysis of RNA polymerase II and transcriptional dynamics upon MYC activation.

Overexpression of the MYC transcription factor causes its widespread interaction with regulatory elements in the genome but leads to the up- and down-regulation of discrete sets of genes. The molecular determinants of these selective transcriptional responses remain elusive. Here, we present an integrated time-course analysis of transcription and mRNA dynamics following MYC activation in proliferating mouse fibroblasts, based on chromatin immunoprecipitation, metabolic labeling of newly synthesized RNA, extensive sequencing, and mathematical modeling. Transcriptional activation correlated with the highest increases in MYC binding at promoters. Repression followed a reciprocal scenario, with the lowest gains in MYC binding. Altogether, the relative abundance (henceforth, "share") of MYC at promoters was the strongest predictor of transcriptional responses in diverse cell types, predominating over MYC's association with the corepressor ZBTB17 (also known as MIZ1). MYC activation elicited immediate loading of RNA polymerase II (RNAPII) at activated promoters, followed by increases in pause-release, while repressed promoters showed opposite effects. Gains and losses in RNAPII loading were proportional to the changes in the MYC share, suggesting that repression by MYC may be partly indirect, owing to competition for limiting amounts of RNAPII. Secondary to the changes in RNAPII loading, the dynamics of elongation and pre-mRNA processing were also rapidly altered at MYC regulated genes, leading to the transient accumulation of partially or aberrantly processed mRNAs. Altogether, our results shed light on how overexpressed MYC alters the various phases of the RNAPII cycle and the resulting transcriptional response.

Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis.

The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this "clock" function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

The methyltransferase Set7/9 (Setd7) is dispensable for the p53-mediated DNA damage response in vivo.

p53 is the central regulator of cell fate following genotoxic stress and oncogene activation. Its activity is controlled by several posttranslational modifications. Originally defined as a critical layer of p53 regulation in human cell lines, p53 lysine methylation by Set7/9 (also called Setd7) was proposed to fulfill a similar function in vivo in the mouse, promoting p53 acetylation, stabilization, and activation upon DNA damage (Kurash et al., 2008). We tested the physiological relevance of this circuit in an independent Set7/9 knockout mouse strain. Deletion of Set7/9 had no effect on p53-dependent cell-cycle arrest or apoptosis following sublethal or lethal DNA damage induced by radiation or genotoxic agents. Set7/9 was also dispensable for p53 acetylation following irradiation. c-myc oncogene-induced apoptosis was also independent of Set7/9, and analysis of p53 target genes showed that Set7/9 is not required for the p53-dependent gene expression program. Our data indicate that Set7/9 is dispensable for p53 function in the mouse.

Mutual epithelium-macrophage dependency in liver carcinogenesis mediated by ST18.

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18-depleted tumors before involution revealed down-regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B-dependent transcription. CONCLUSION: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708-1719).

Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy.

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.

Myc induced replicative stress response: How to cope with it and exploit it.

Myc is a cellular oncogene frequently deregulated in cancer that has the ability to stimulate cellular growth by promoting a number of proliferative and pro-survival pathways. Here we will focus on how Myc controls a number of diverse cellular processes that converge to ensure processivity and robustness of DNA synthesis, thus preventing the inherent replicative stress responses usually evoked by oncogenic lesions. While these processes provide cancer cells with a long-term proliferative advantage, they also represent cancer liabilities that can be exploited to devise innovative therapeutic approaches to target Myc overexpressing tumors. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

Identification of BRCC3 and BRCA1 as Regulators of TAZ Stability and Activity.

TAZ (WWTR1) is a transcriptional co-activator regulated by Hippo signaling, mechano-transduction, and G-protein couple receptors. Once activated, TAZ and its paralogue, YAP1, regulate gene expression programs promoting cell proliferation, survival, and differentiation, thus controlling embryonic development, tissue regeneration, and aging. YAP and TAZ are also frequently activated in tumors, particularly in poorly differentiated and highly aggressive malignancies. Yet, mutations of YAP/TAZ or of their upstream regulators do not fully account for their activation in cancer, raising the possibility that other upstream regulatory pathways, still to be defined, are altered in tumors. In this work, we set out to identify novel regulators of TAZ by means of a siRNA-based screen. We identified 200 genes able to modulate the transcriptional activity of TAZ, with prominence for genes implicated in cell-cell contact, cytoskeletal tension, cell migration, WNT signaling, chromatin remodeling, and interleukins and NF-kappaB signaling. Among these genes we identified was BRCC3, a component of the BRCA1 complex that guards genome integrity and exerts tumor suppressive activity during cancer development. The loss of BRCC3 or BRCA1 leads to an increased level and activity of TAZ. Follow-up studies indicated that the cytoplasmic BRCA1 complex controls the ubiquitination and stability of TAZ. This may suggest that, in tumors, inactivating mutations of BRCA1 may unleash cell transformation by activating the TAZ oncogene.

c-MYC-dependent transcriptional inhibition of autophagy is implicated in cisplatin sensitivity in HPV-positive head and neck cancer.

Autophagy is important for the removal, degradation and recycling of damaged organelles, proteins, and lipids through the degradative action of lysosomes. In addition to its catabolic function, autophagy is important in cancer and viral-mediated tumorigenesis, including Human Papillomavirus (HPV) positive cancers. HPV infection is a major risk factor in a subset of head and neck cancer (HNC), for which no targeted therapies are currently available. Herein, we assessed autophagy function in HPV-positive HNC. We showed that HPV-positive HNC cells presented a transcriptional and functional impairment of the autophagic process compared to HPV-negative cells, which were reactivated by knocking down HPV E6/E7 oncoproteins, the drivers of cellular transformation. We found that the oncoprotein c-MYC was stabilized and triggered in HPV-positive cell lines. This resulted in the reduced binding of the MiT/TFE transcription factors to their autophagy targets due to c-MYC competition. Thus, the knock-down of c-MYC induced the upregulation of autophagic and lysosomal genes in HPV-positive HNC cells, as well as the increase of autophagic markers at the protein level. Moreover, HPV oncoprotein E7 upregulated the expression of the phosphatase inhibitor CIP2A, accounting for c-MYC upregulation and stability in HPV+ HNC cells. CIP2A mRNA expression negatively correlated with autophagy gene expression in tumor tissues from HNC patients, showing, for the first time, its implication in a transcriptional autophagic context. Both CIP2A and c-MYC knock-down, as well as pharmacological downregulation of c-MYC, resulted in increased resistance to cisplatin treatment. Our results not only show a novel way by which HPV oncoproteins manipulate the host machinery but also provide more insights into the role of autophagy in chemoresistance, with possible implications for targeted HPV-positive HNC therapy.

Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity.

Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)-the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate "young-like" HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs.

GZMKhigh CD8+ T effector memory cells are associated with CD15high neutrophil abundance in non-metastatic colorectal tumors and predict poor clinical outcome.

CD8+ T cells are a major prognostic determinant in solid tumors, including colorectal cancer (CRC). However, understanding how the interplay between different immune cells impacts on clinical outcome is still in its infancy. Here, we describe that the interaction of tumor infiltrating neutrophils expressing high levels of CD15 with CD8+ T effector memory cells (TEM) correlates with tumor progression. Mechanistically, stromal cell-derived factor-1 (CXCL12/SDF-1) promotes the retention of neutrophils within tumors, increasing the crosstalk with CD8+ T cells. As a consequence of the contact-mediated interaction with neutrophils, CD8+ T cells are skewed to produce high levels of GZMK, which in turn decreases E-cadherin on the intestinal epithelium and favors tumor progression. Overall, our results highlight the emergence of GZMKhigh CD8+ TEM in non-metastatic CRC tumors as a hallmark driven by the interaction with neutrophils, which could implement current patient stratification and be targeted by novel therapeutics.

Emerging Principles in the Transcriptional Control by YAP and TAZ.

Yes-associated protein (YAP) and TAZ are transcriptional cofactors that sit at the crossroad of several signaling pathways involved in cell growth and differentiation. As such, they play essential functions during embryonic development, regeneration, and, once deregulated, in cancer progression. In this review, we will revise the current literature and provide an overview of how YAP/TAZ control transcription. We will focus on data concerning the modulation of the basal transcriptional machinery, their ability to epigenetically remodel the enhancer-promoter landscape, and the mechanisms used to integrate transcriptional cues from multiple pathways. This reveals how YAP/TAZ activation in cancer cells leads to extensive transcriptional control that spans several hallmarks of cancer. The definition of the molecular mechanism of transcriptional control and the identification of the pathways regulated by YAP/TAZ may provide therapeutic opportunities for the effective treatment of YAP/TAZ-driven tumors.

Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab.

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.