DNase I is an enzyme preferentially cleaving DNA in highly accessible regions. Recently, Next-Generation Sequencing has been applied to DNase I assays (DNase-seq) to obtain genome-wide maps of these accessible chromatin regions. With high-depth sequencing, DNase I cleavage sites can be identified with base-pair resolution, revealing the presence of protected regions ("footprints"), corresponding to bound molecules on the DNA. Integrating footprint positions close to transcription start sites with motif analysis can reveal the presence of regulatory interactions between specific transcription factors (TFs) and genes. However, this inference heavily relies on the accuracy of the footprint call and on the sequencing depth of the DNase-seq experiment. Using ENCODE data, we comprehensively evaluate the performances of two recent footprint callers (Wellington and DNaseR) and one metric (the Footprint Occupancy Score, or FOS), and assess the consequences of different footprint calls on the reconstruction of TF-TF regulatory networks. We rate Wellington as the method of choice among those tested: not only its predictions are the best in terms of accuracy, but also the properties of the inferred networks are robust against sequencing depth.

Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC-myomiR-BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a "latent" myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles.

The Yin Yang 1 (YY1) transcription factor is a master regulator of development, essential for early embryogenesis and adult tissues formation. YY1 is the mammalian orthologue of Pleiohomeotic, one of the transcription factors that binds Polycomb DNA response elements in Drosophila melanogaster and mediates Polycomb group proteins (PcG) recruitment to DNA. Despite several publications pointing at YY1 having a similar role in mammalians, others showed features of YY1 that are not compatible with PcG functions. Here, we show that, in mouse Embryonic Stem (ES) cells, YY1 has genome-wide PcG-independent activities while it is still stably associated with the INO80 chromatin-remodeling complex, as well as with novel RNA helicase activities. YY1 binds chromatin in close proximity of the transcription start site of highly expressed genes. Loss of YY1 functions preferentially led to a down-regulation of target genes expression, as well as to an up-regulation of several small non-coding RNAs, suggesting a role for YY1 in regulating small RNA biogenesis. Finally, we found that YY1 is a novel player of Myc-related transcription factors and that its coordinated binding at promoters potentiates gene expression, proposing YY1 as an active component of the Myc transcription network that links ES to cancer cells.

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 infants1,2. 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.

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 cis-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 cis-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.

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 (CYP19A1amp). Relapsed patients also developed numerous mutations targeting key breast cancer-associated genes, including ESR1 and CYP19A1. Notably, CYP19A1amp 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 CYP19A1amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.

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.

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.

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.

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

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.

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

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.

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.

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II binding sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation, and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes.

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

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.