Publications by authors named "Iros Barozzi"

55 Publications

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning.

Development 2021 Dec 3;148(23). Epub 2021 Dec 3.

Developmental Genetics, Department of Biomedicine, University of Basel, 4058 Basel, Switzerland.

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.200182DOI Listing
December 2021

A first exon termination checkpoint preferentially suppresses extragenic transcription.

Nat Struct Mol Biol 2021 04 25;28(4):337-346. Epub 2021 Mar 25.

European Institute of Oncology (IEO) IRCCS, Milan, Italy.

Interactions between the splicing machinery and RNA polymerase II increase protein-coding gene transcription. Similarly, exons and splicing signals of enhancer-generated long noncoding RNAs (elncRNAs) augment enhancer activity. However, elncRNAs are inefficiently spliced, suggesting that, compared with protein-coding genes, they contain qualitatively different exons with a limited ability to drive splicing. We show here that the inefficiently spliced first exons of elncRNAs as well as promoter-antisense long noncoding RNAs (pa-lncRNAs) in human and mouse cells trigger a transcription termination checkpoint that requires WDR82, an RNA polymerase II-binding protein, and its RNA-binding partner of previously unknown function, ZC3H4. We propose that the first exons of elncRNAs and pa-lncRNAs are an intrinsic component of a regulatory mechanism that, on the one hand, maximizes the activity of these cis-regulatory elements by recruiting the splicing machinery and, on the other, contains elements that suppress pervasive extragenic transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-021-00572-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610630PMC
April 2021

Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation.

EMBO Rep 2021 03 15;22(3):e50852. Epub 2021 Feb 15.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Transition from proliferative-to-invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down-regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR-depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re-expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor-initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient-rich conditions by repressing translation of selected ISR RNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.202050852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926219PMC
March 2021

Phenotypic Mapping of Pathologic Cross-Talk between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing.

Cancer Discov 2021 03 23;11(3):754-777. Epub 2020 Dec 23.

Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany.

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, and . Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. SIGNIFICANCE: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611210PMC
March 2021

Evolution of Advanced Chronic Lymphoid Leukemia Unveiled by Single-Cell Transcriptomics: A Case Report.

Front Oncol 2020 30;10:584607. Epub 2020 Oct 30.

Department of Surgery and Cancer, Imperial College London, London, United Kingdom.

Genetic and transcriptional heterogeneity of Chronic lymphocytic leukaemia (CLL) limits prevention of disease progression. Longitudinal single-cell transcriptomics represents the state-of-the-art method to profile the disease heterogeneity at diagnosis and to inform about disease evolution. Here, we apply single-cell RNA-seq to a CLL case, sampled at diagnosis and relapse, that was treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) and underwent a dramatic decrease in CD19 expression during disease progression. Computational analyses revealed a major switch in clones' dominance during treatment. The clone that expanded at relapse showed 17p and 3p chromosomal deletions, and up-regulation of pathways related to motility, cytokine signaling and antigen presentation. Single-cell RNA-seq uniquely revealed that this clone was already present at low frequency at diagnosis, and it displays feature of plasma cell differentiation, consistent with a more aggressive phenotype. This study shows the benefit of single-cell profiling of CLL heterogeneity at diagnosis, to identify clones that might otherwise not be recognized and to determine the best treatment options.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2020.584607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664833PMC
October 2020

Supervised enhancer prediction with epigenetic pattern recognition and targeted validation.

Nat Methods 2020 08 29;17(8):807-814. Epub 2020 Jul 29.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.

Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated that our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-020-0907-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073243PMC
August 2020

An atlas of dynamic chromatin landscapes in mouse fetal development.

Nature 2020 07 29;583(7818):744-751. Epub 2020 Jul 29.

Center for Epigenomics, University of California, San Diego School of Medicine, La Jolla, CA, USA.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2093-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398618PMC
July 2020

Exploiting evolutionary steering to induce collateral drug sensitivity in cancer.

Nat Commun 2020 04 21;11(1):1923. Epub 2020 Apr 21.

Evolutionary Genomics and Modelling Lab, Centre for Evolution and Cancer, The Institute of Cancer Research, London, UK.

Drug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased fecundity or increased sensitivity to another drug. These evolutionary trade-offs can be exploited using 'evolutionary steering' to control the tumour population and delay resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here, we present an approach for evolutionary steering based on a combination of single-cell barcoding, large populations of 10-10 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary steering in a lung cancer model, showing that it shifts the clonal composition of the tumour in our favour, leading to collateral sensitivity and proliferative costs. Genomic profiling revealed some of the mechanisms that drive evolved sensitivity. This approach allows modelling evolutionary steering strategies that can potentially control treatment resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15596-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174377PMC
April 2020

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

Sci Adv 2020 04 8;6(15):eaax2746. Epub 2020 Apr 8.

Department of Experimental Oncology, European Institute of Oncology (IEO), IRCCS, Via Adamello 16, Milan 20139, Italy.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aax2746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141832PMC
April 2020

High-resolution label-free 3D mapping of extracellular pH of single living cells.

Nat Commun 2019 12 6;10(1):5610. Epub 2019 Dec 6.

Department of Medicine, Imperial College London, London, W12 0NN, UK.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13535-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898398PMC
December 2019

Single-cell transcriptomics reveals multi-step adaptations to endocrine therapy.

Nat Commun 2019 09 2;10(1):3840. Epub 2019 Sep 2.

Department of Surgery and Cancer, Imperial College London, London, UK.

Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11721-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718416PMC
September 2019

Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells.

Genes Dev 2019 09 1;33(17-18):1159-1174. Epub 2019 Aug 1.

Humanitas University (Hunimed), Pieve Emanuele, Milano 20090, Italy.

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of -regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated -regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.326348.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719622PMC
September 2019

Noncoding deletions reveal a gene that is critical for intestinal function.

Nature 2019 07 19;571(7763):107-111. Epub 2019 Jun 19.

Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1312-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061489PMC
July 2019

Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Nat Immunol 2018 09 20;19(9):932-941. Epub 2018 Aug 20.

Lymphocyte Development Group, Epigenetics Section, MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-018-0184-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195188PMC
September 2018

Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer.

Nat Med 2018 09 23;24(9):1469-1480. Epub 2018 Jul 23.

Department of Surgery and Cancer, The Imperial Centre for Translational and Experimental Medicine, Imperial College London, London, UK.

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-018-0091-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130800PMC
September 2018

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures.

Genome Res 2018 02 2. Epub 2018 Feb 2.

University of Cambridge.

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis- regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.227272.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848609PMC
February 2018

Enhancer redundancy provides phenotypic robustness in mammalian development.

Nature 2018 02 31;554(7691):239-243. Epub 2018 Jan 31.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature25461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808607PMC
February 2018

Ultraconserved Enhancers Are Required for Normal Development.

Cell 2018 01 18;172(3):491-499.e15. Epub 2018 Jan 18.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; School of Natural Sciences, University of California Merced, Merced, CA 95343, USA. Electronic address:

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786478PMC
January 2018

Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb.

PLoS Comput Biol 2017 Aug 21;13(8):e1005720. Epub 2017 Aug 21.

Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.

Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers in vivo. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the Limb-Enhancer Genie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of >50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously in vivo validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through in vivo validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1005720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578682PMC
August 2017

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

Cell Rep 2017 05;19(8):1602-1613

Developmental Genetics, Department of Biomedicine, University of Basel, 4058 Basel, Switzerland. Electronic address:

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2017.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5523860PMC
May 2017

High constitutive activity of a broad panel of housekeeping and tissue-specific -regulatory elements depends on a subset of ETS proteins.

Genes Dev 2017 02 8;31(4):399-412. Epub 2017 Mar 8.

Department of Experimental Oncology, European Institute of Oncology (IEO), 20139 Milan, Italy.

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of -regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the -regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping -regulatory elements with the ability to efficiently promote transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.293134.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358759PMC
February 2017

Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

Nat Genet 2017 03 23;49(3):444-450. Epub 2017 Jan 23.

Department of Experimental Oncology, European Institute of Oncology Milan, Italy.

Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1). Relapsed patients also developed numerous mutations targeting key breast cancer-associated genes, including ESR1 and CYP19A1. Notably, CYP19A1 cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1 cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1 and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.3773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326683PMC
March 2017

Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes.

Genome Res 2017 03 5;27(3):479-490. Epub 2017 Jan 5.

Friedrich Miescher Institute for Biomedical Research, Basel, CH-4058, Switzerland.

Understanding how regulatory sequences interact in the context of chromosomal architecture is a central challenge in biology. Chromosome conformation capture revealed that mammalian chromosomes possess a rich hierarchy of structural layers, from multi-megabase compartments to sub-megabase topologically associating domains (TADs) and sub-TAD contact domains. TADs appear to act as regulatory microenvironments by constraining and segregating regulatory interactions across discrete chromosomal regions. However, it is unclear whether other (or all) folding layers share similar properties, or rather TADs constitute a privileged folding scale with maximal impact on the organization of regulatory interactions. Here, we present a novel algorithm named CaTCH that identifies hierarchical trees of chromosomal domains in Hi-C maps, stratified through their reciprocal physical insulation, which is a single and biologically relevant parameter. By applying CaTCH to published Hi-C data sets, we show that previously reported folding layers appear at different insulation levels. We demonstrate that although no structurally privileged folding level exists, TADs emerge as a functionally privileged scale defined by maximal boundary enrichment in CTCF and maximal cell-type conservation. By measuring transcriptional output in embryonic stem cells and neural precursor cells, we show that the likelihood that genes in a domain are coregulated during differentiation is also maximized at the scale of TADs. Finally, we observe that regulatory sequences occur at genomic locations corresponding to optimized mutual interactions at the same scale. Our analysis suggests that the architectural functionality of TADs arises from the interplay between their ability to partition interactions and the specific genomic position of regulatory sequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.212803.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340975PMC
March 2017

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13.

Cell Rep 2016 12;17(11):2913-2926

Laboratory of Genetics and Development, Institut de Recherches Cliniques de Montréal (IRCM), 110 avenue des Pins Ouest, Montréal, QC H2W1R7, Canada; Department of Medicine, Université de Montréal, Montréal, H3T1J4 QC, Canada. Electronic address:

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13; Hoxd13 limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697718PMC
December 2016

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

Cell 2016 Oct;167(3):633-642.e11

MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; School of Natural Sciences, University of California, Merced, CA 95343, USA. Electronic address:

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2016.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484524PMC
October 2016
-->