Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. This could result from genetic or functional constraints. We demonstrate that head sensory organ size trade-offs in Drosophila are genetically encoded and arise through differential subdivision of the head primordium into visual versus non-visual fields. We discover that changes in the temporal regulation of the highly conserved eyeless/Pax6 gene expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless in a binding site of its repressor Cut that is sufficient to alter its temporal regulation and eye size. Because eyeless/Pax6 is a conserved regulator of head sensory placode subdivision, we propose that its temporal regulation is key to define the relative size of head sensory organs.

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.

Alterations in the expression or function of cell adhesion molecules have been implicated in all steps of tumor progression. Among those, P-cadherin is highly enriched in basal-like breast carcinomas, playing a central role in cancer cell self-renewal, collective cell migration and invasion. To establish a clinically relevant platform for functional exploration of P-cadherin effectors in vivo, we generated a humanized P-cadherin Drosophila model. We report that actin nucleators, Mrtf and Srf, are main P-cadherin effectors in fly. We validated these findings in a human mammary epithelial cell line with conditional activation of the SRC oncogene. We show that, prior to promoting malignant phenotypes, SRC induces a transient increase in P-cadherin expression, which correlates with MRTF-A accumulation, its nuclear translocation and the upregulation of SRF target genes. Moreover, knocking down P-cadherin, or preventing F-actin polymerization, impairs SRF transcriptional activity. Furthermore, blocking MRTF-A nuclear translocation hampers proliferation, self-renewal and invasion. Thus, in addition to sustaining malignant phenotypes, P-cadherin can also play a major role in the early stages of breast carcinogenesis by promoting a transient boost of MRTF-A-SRF signaling through actin regulation.

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, we performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, we derived direct TF-DNA interactions using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, we found network motifs, cis-regulatory codes, and regulators of eye development. We validate the predicted target regions of Grainyhead by ChIP-seq and identify this factor as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions.

i-cisTarget is a web tool to predict regulators of a set of genomic regions, such as ChIP-seq peaks or co-regulated/similar enhancers. i-cisTarget can also be used to identify upstream regulators and their target enhancers starting from a set of co-expressed genes. Whereas the original version of i-cisTarget was focused on Drosophila data, the 2015 update also provides support for human and mouse data. i-cisTarget detects transcription factor motifs (position weight matrices) and experimental data tracks (e.g. from ENCODE, Roadmap Epigenomics) that are enriched in the input set of regions. As experimental data tracks we include transcription factor ChIP-seq data, histone modification ChIP-seq data and open chromatin data. The underlying processing method is based on a ranking-and-recovery procedure, allowing accurate determination of enrichment across heterogeneous datasets, while also discriminating direct from indirect target regions through a 'leading edge' analysis. We illustrate i-cisTarget on various Ewing sarcoma datasets to identify EWS-FLI1 targets starting from ChIP-seq, differential ATAC-seq, differential H3K27ac and differential gene expression data. Use of i-cisTarget is free and open to all, and there is no login requirement. Address: http://gbiomed.kuleuven.be/apps/lcb/i-cisTarget.

Studies of the role of actin in tumour progression have highlighted its key contribution in cell softening associated with cell invasion. Here, using a human breast cell line with conditional Src induction, we demonstrate that cells undergo a stiffening state prior to acquiring malignant features. This state is characterized by the transient accumulation of stress fibres and upregulation of Ena/VASP-like (EVL). EVL, in turn, organizes stress fibres leading to transient cell stiffening, ERK-dependent cell proliferation, as well as enhancement of Src activation and progression towards a fully transformed state. Accordingly, EVL accumulates predominantly in premalignant breast lesions and is required for Src-induced epithelial overgrowth in Drosophila. While cell softening allows for cancer cell invasion, our work reveals that stress fibre-mediated cell stiffening could drive tumour growth during premalignant stages. A careful consideration of the mechanical properties of tumour cells could therefore offer new avenues of exploration when designing cancer-targeting therapies.

Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

The field of regulatory genomics today is characterized by the generation of high-throughput data sets that capture genome-wide transcription factor (TF) binding, histone modifications, or DNAseI hypersensitive regions across many cell types and conditions. In this context, a critical question is how to make optimal use of these publicly available datasets when studying transcriptional regulation. Here, we address this question in Drosophila melanogaster for which a large number of high-throughput regulatory datasets are available. We developed i-cisTarget (where the 'i' stands for integrative), for the first time enabling the discovery of different types of enriched 'regulatory features' in a set of co-regulated sequences in one analysis, being either TF motifs or 'in vivo' chromatin features, or combinations thereof. We have validated our approach on 15 co-expressed gene sets, 21 ChIP data sets, 628 curated gene sets and multiple individual case studies, and show that meaningful regulatory features can be confidently discovered; that bona fide enhancers can be identified, both by in vivo events and by TF motifs; and that combinations of in vivo events and TF motifs further increase the performance of enhancer prediction.

Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. We further found that the Hippo pathway effector Yorkie was strongly activated in tumor cells and initiated cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.

Scoring the impact of noncoding variation on the function of cis-regulatory regions, on their chromatin state, and on the qualitative and quantitative expression levels of target genes is a fundamental problem in evolutionary genomics. A particular challenge is how to model the divergence of quantitative traits and to identify relationships between the changes across the different levels of the genome, the chromatin activity landscape, and the transcriptome. Here, we examine the use of the Ornstein-Uhlenbeck (OU) model to infer selection at the level of predicted cis-regulatory modules (CRMs), and link these with changes in transcription factor binding and chromatin activity. Using publicly available cross-species ChIP-Seq and STARR-Seq data we show how OU can be applied genome-wide to identify candidate transcription factors for which binding site and CRM turnover is correlated with changes in regulatory activity. Next, we profile open chromatin in the developing eye across three Drosophila species. We identify the recognition motifs of the chromatin remodelers, Trithorax-like and Grainyhead as mostly correlating with species-specific changes in open chromatin. In conclusion, we show in this study that CRM scores can be used as quantitative traits and that motif discovery approaches can be extended towards more complex models of divergence.

Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.

E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. Here we show that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down capping protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, our findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours.

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

The specification and growth of organs is controlled simultaneously by networks of transcription factors. While the connection between these transcription factors with fate determinants is increasingly clear, how they establish the link with the cell cycle is far less understood. Here we investigate this link in the developing Drosophila eye, where two transcription factors, the MEIS1 homologue hth and the Zn-finger tsh, synergize to stimulate the proliferation of naïve eye progenitors. Experiments combining transcriptomics, open-chromatin profiling, motif analysis and functional assays indicate that these progenitor transcription factors exert a global regulation of the proliferation program. Rather than directly regulating cell cycle genes, they control proliferation through an intermediary layer of nuclear receptors of the ecdysone/estrogen-signaling pathway. This regulatory subnetwork between hth, tsh and nuclear receptors might be conserved from Drosophila to mammals, as we find a significant co-overexpression of their human homologues in specific cancer types.

Identifying master regulators of biological processes and mapping their downstream gene networks are key challenges in systems biology. We developed a computational method, called iRegulon, to reverse-engineer the transcriptional regulatory network underlying a co-expressed gene set using cis-regulatory sequence analysis. iRegulon implements a genome-wide ranking-and-recovery approach to detect enriched transcription factor motifs and their optimal sets of direct targets. We increase the accuracy of network inference by using very large motif collections of up to ten thousand position weight matrices collected from various species, and linking these to candidate human TFs via a motif2TF procedure. We validate iRegulon on gene sets derived from ENCODE ChIP-seq data with increasing levels of noise, and we compare iRegulon with existing motif discovery methods. Next, we use iRegulon on more challenging types of gene lists, including microRNA target sets, protein-protein interaction networks, and genetic perturbation data. In particular, we over-activate p53 in breast cancer cells, followed by RNA-seq and ChIP-seq, and could identify an extensive up-regulated network controlled directly by p53. Similarly we map a repressive network with no indication of direct p53 regulation but rather an indirect effect via E2F and NFY. Finally, we generalize our computational framework to include regulatory tracks such as ChIP-seq data and show how motif and track discovery can be combined to map functional regulatory interactions among co-expressed genes. iRegulon is available as a Cytoscape plugin from http://iregulon.aertslab.org.

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.

The identification of transcription factor binding sites, enhancers, and transcriptional target genes often relies on the integration of gene expression profiling and computational cis-regulatory sequence analysis. Methods for the prediction of cis-regulatory elements can take advantage of comparative genomics to increase signal-to-noise levels. However, gene expression data are usually derived from only one species. Here we investigate tissue-specific cross-species gene expression profiling by high-throughput sequencing, combined with cross-species motif discovery. First, we compared different methods for expression level quantification and cross-species integration using Tag-seq data. Using the optimal pipeline, we derived a set of genes with conserved expression during retinal determination across Drosophila melanogaster, Drosophila yakuba, and Drosophila virilis. These genes are enriched for binding sites of eye-related transcription factors including the zinc-finger Glass, a master regulator of photoreceptor differentiation. Validation of predicted Glass targets using RNA-seq in homozygous glass mutants confirms that the majority of our predictions are expressed downstream from Glass. Finally, we tested nine candidate enhancers by in vivo reporter assays and found eight of them to drive GFP in the eye disc, of which seven colocalize with the Glass protein, namely, scrt, chp, dpr10, CG6329, retn, Lim3, and dmrt99B. In conclusion, we show for the first time the combined use of cross-species expression profiling with cross-species motif discovery as a method to define a core developmental program, and we augment the candidate Glass targetome from a single known target gene, lozenge, to at least 62 conserved transcriptional targets.

Signaling through the αβT cell receptor (TCR) is a crucial determinant of T-cell fate and can induce two opposite outcomes during thymocyte development: cell death or survival and differentiation. To date, the role played by T-cell receptor in the oncogenic transformation of developing T cells remains unclear. Here we show that human primary T-cell acute lymphoblastic leukemias expressing an αβT cell receptor are frequently deficient for phosphatase and tensin homolog protein (PTEN), and fail to respond strongly to T-cell receptor activation. Using Pten-deficient T-cell acute lymphoblastic leukemia mouse models, we confirm that T-cell receptor signaling is involved in leukemogenesis. We show that abrogation of T-cell receptor expression accelerated tumor onset, while enforced expression of a fit transgenic T-cell receptor led to the development of T-cell receptor-negative lymphoma and delayed tumorigenesis. We further demonstrate that pre-tumoral Pten-deficient thymocytes harboring fit T-cell receptors undergo early clonal deletion, thus preventing their malignant transformation, while cells with unfit T-cell receptors that should normally be deleted during positive selection, pass selection and develop T-cell acute lymphoblastic leukemias. Altogether, our data show that fit T-cell receptor signaling suppresses tumor development mediated by Pten loss-of-function and point towards a role of Pten in positive selection.

Aberrant expression of the Spectraplakin Dystonin (DST) has been observed in various cancers, including those of the breast. However, little is known about its role in carcinogenesis. In this report, we demonstrate that Dystonin is a candidate tumour suppressor in breast cancer and provide an underlying molecular mechanism. We show that in MCF10A cells, Dystonin is necessary to restrain cell growth, anchorage-independent growth, self-renewal properties and resistance to doxorubicin. Strikingly, while Dystonin maintains focal adhesion integrity, promotes cell spreading and cell-substratum adhesion, it prevents Zyxin accumulation, stabilizes LATS and restricts YAP activation. Moreover, treating DST-depleted MCF10A cells with the YAP inhibitor Verteporfin prevents their growth. In vivo, the Drosophila Dystonin Short stop also restricts tissue growth by limiting Yorkie activity. As the two Dystonin isoforms BPAG1eA and BPAG1e are necessary to inhibit the acquisition of transformed features and are both downregulated in breast tumour samples and in MCF10A cells with conditional induction of the Src proto-oncogene, they could function as the predominant Dystonin tumour suppressor variants in breast epithelial cells. Thus, their loss could deem as promising prognostic biomarkers for breast cancer.