Single-cell transcriptomics uncovers an instructive T-cell receptor role in adult γδ T-cell lineage commitment.

After entering the adult thymus, bipotent T-cell progenitors give rise to αß or γδ T cells. To determine whether the γδ T-cell receptor (TCR) has an instructive role in γδ T-cell lineage commitment or only "confirms" a pre-established γδ Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for γδ TCR signaling. Although these mice showed a T-cell development block at the CD4- CD8- double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of γδ TCR (further referred to as γδint ) at their surface. Single-cell transcriptomics of LAT-deficient DN3 γδint cells demonstrated no sign of commitment to the γδ T-cell lineage, apart from γδ TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 γδint cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the γδ T-cell lineage. Therefore, the γδ TCR-LAT signaling axis builds upon a γδ T-cell uncommitted lineage state to fully instruct adult γδ T-cell lineage specification.

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis.

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.

Fit αß T-cell receptor suppresses leukemogenesis of Pten-deficient thymocytes.

Signaling through the αßT 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 αßT 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.

Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open chromatin profiling.

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.

i-cisTarget 2015 update: generalized cis-regulatory enrichment analysis in human, mouse and fly.

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.

Identification of Lineage-Specific Cis-Regulatory Modules Associated with Variation in Transcription Factor Binding and Chromatin Activity Using Ornstein-Uhlenbeck Models.

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.

Comparative motif discovery combined with comparative transcriptomics yields accurate targetome and enhancer predictions.

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.

iRegulon: from a gene list to a gene regulatory network using large motif and track collections.

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.

Identification of cis-regulatory modules encoding temporal dynamics during development.

BACKGROUND: Developmental transcriptional regulatory networks are circuits of transcription factors (TFs) and cis-acting DNA elements (Cis Regulatory Modules, CRMs) that dynamically control expression of downstream genes. Comprehensive knowledge of these networks is an essential step towards our understanding of developmental processes. However, this knowledge is mostly based on genome-wide mapping of transcription factor binding sites, and therefore requires prior knowledge regarding the TFs involved in the network. RESULTS: Focusing on how temporal control of gene expression is integrated within a developmental network, we applied an in silico approach to discover regulatory motifs and CRMs of co-expressed genes, with no prior knowledge about the involved TFs. Our aim was to identify regulatory motifs and potential trans-acting factors which regulate the temporal expression of co-expressed gene sets during a particular process of organogenesis, namely adult heart formation in Drosophila. Starting from whole genome tissue specific expression dynamics, we used an in silico method, cisTargetX, to predict TF binding motifs and CRMs. Potential Nuclear Receptor (NR) binding motifs were predicted to control the temporal expression profile of a gene set with increased expression levels during mid metamorphosis. The predicted CRMs and NR motifs were validated in vivo by reporter gene essays. In addition, we provide evidence that three NRs modulate CRM activity and behave as temporal regulators of target enhancers. CONCLUSIONS: Our approach was successful in identifying CRMs and potential TFs acting on the temporal regulation of target genes. In addition, our results suggest a modular architecture of the regulatory machinery, in which the temporal and spatial regulation can be uncoupled and encoded by distinct CRMs.

i-cisTarget: an integrative genomics method for the prediction of regulatory features and cis-regulatory modules.

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.

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia.

BACKGROUND: Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. OBJECTIVES: To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. METHODS: Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. RESULTS: Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. CONCLUSION: These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

Calcium Signaling Is Impaired in PTEN-Deficient T Cell Acute Lymphoblastic Leukemia.

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 TCRαß+ T-ALL development. Herein, we explored the functional interaction between TCRαß 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.

Multiplexed single-cell RNA-sequencing of mouse thymic and splenic samples.

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here, we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample, and 10× Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein, we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy.

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.

MYC deficiency impairs the development of effector/memory T lymphocytes.

In the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation.

GATA1 pathogenic variants disrupt MYH10 silencing during megakaryopoiesis.

BACKGROUND: GATA1 is an essential transcription factor for both polyploidization and megakaryocyte (MK) differentiation. The polyploidization defect observed in GATA1 variant carriers is not well understood. OBJECTIVE: To extensively phenotype two pedigrees displaying different variants in the GATA1 gene and determine if GATA1 controls MYH10 expression levels, a key modulator of MK polyploidization. METHOD: A total of 146 unrelated propositi with constitutional thrombocytopenia were screened on a multigene panel. We described the genotype-phenotype correlation in GATA1 variant carriers and investigated the effect of these novel variants on MYH10 transcription using luciferase constructs. RESULTS: The clinical profile associated with the p.L268M variant localized in the C terminal zinc finger was unusual in that the patient displayed bleeding and severe platelet aggregation defects without early-onset thrombocytopenia. p.N206I localized in the N terminal zinc finger was associated, on the other hand, with severe thrombocytopenia (15G/L) in early life. High MYH10 levels were evidenced in platelets of GATA1 variant carriers. Analysis of MKs anti-GATA1 chromatin immunoprecipitation-sequencing data revealed two GATA1 binding sites, located in the 3' untranslated region and in intron 8 of the MYH10 gene. Luciferase reporter assays showed their respective role in the regulation of MYH10 gene expression. Both GATA1 variants significantly alter intron 8 driven MYH10 transcription. CONCLUSION: The discovery of an association between MYH10 and GATA1 is a novel one. Overall, this study suggests that impaired MYH10 silencing via an intronic regulatory element is the most likely cause of GATA1-related polyploidization defect.

Altering the Temporal Regulation of One Transcription Factor Drives Evolutionary Trade-Offs between Head Sensory Organs.

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.

Cortical Neurogenesis Requires Bcl6-Mediated Transcriptional Repression of Multiple Self-Renewal-Promoting Extrinsic Pathways.

During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition.

The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes.

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.

Nuclear receptors connect progenitor transcription factors to cell cycle control.

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.