Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

The little skate genome and the evolutionary emergence of wing-like fins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Dynamic antagonism between key repressive pathways maintains the placental epigenome.

DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape's molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.

Evolution of 3D chromatin organization at different scales.

Most animal genomes fold in 3D chromatin domains called topologically associated domains (TADs) that facilitate interactions between cis-regulatory elements (CREs) and promoters. Owing to their critical role in the control of developmental gene expression, we explore how TADs have shaped animal evolution. In the light of recent studies that profile TADs in disparate animal lineages, we discuss their phylogenetic distribution and the mechanisms that underlie their formation. We present evidence indicating that TADs are plastic entities composed of genomic strata of different ages: ancient cores are combined with newer regions and brought into extant TADs through genomic rearrangements. We highlight that newly incorporated TAD strata enable the establishment of new CRE-promoter interactions and in turn new expression patterns that can drive phenotypical innovation. We further highlight how subtle changes in chromatin folding may fine-tune the expression levels of developmental genes and hold a potential for evolutionary significance.

Cell adhesion and immune response, two main functions altered in the transcriptome of seasonally regressed testes of two mammalian species.

In species with seasonal breeding, male specimens undergo substantial testicular regression during the nonbreeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, nonapoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing, and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK, and TGF-ß signaling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the "immune privilege" that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.

C/EBPß regulates lipid metabolism and Pparg isoform 2 expression in alveolar macrophages.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPß-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPß protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPß as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation.

Vertebrate genomes organize into topologically associating domains, delimited by boundaries that insulate regulatory elements from nontarget genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CCCTC-binding factor (CTCF) boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding site (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors such as CBS number and orientation. Combined deletions demonstrated that CBSs cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by nonconvergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

The mole genome reveals regulatory rearrangements associated with adaptive intersexuality.

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Order and disorder: abnormal 3D chromatin organization in human disease.

A precise three-dimensional (3D) organization of chromatin is central to achieve the intricate transcriptional patterns that are required to form complex organisms. Growing evidence supports an important role of 3D chromatin architecture in development and delineates its alterations as prominent causes of disease. In this review, we discuss emerging concepts on the fundamental forces shaping genomes in space and on how their disruption can lead to pathogenic phenotypes. We describe the molecular mechanisms underlying a wide range of diseases, from the systemic effects of coding mutations on 3D architectural factors, to the more tissue-specific phenotypes resulting from genetic and epigenetic modifications at specific loci. Understanding the connection between the 3D organization of the genome and its underlying biological function will allow a better interpretation of human pathogenesis.

A (3D-Nuclear) Space Odyssey: Making Sense of Hi-C Maps.

Three-dimensional (3D)-chromatin organization is critical for proper enhancer-promoter communication and, therefore, for a precise execution of the transcriptional programs governing cellular processes. The emergence of Chromosome Conformation Capture (3C) methods, in particular Hi-C, has allowed the investigation of chromatin interactions on a genome-wide scale, revealing the existence of overlapping molecular mechanisms that we are just starting to decipher. Therefore, disentangling Hi-C signal into these individual components is essential to provide meaningful biological data interpretation. Here, we discuss emerging views on the molecular forces shaping the genome in 3D, with a focus on their respective contributions and interdependence. We discuss Hi-C data at both population and single-cell levels, thus providing criteria to interpret genomic function in the 3D-nuclear space.

GOPHER: Generator Of Probes for capture Hi-C Experiments at high Resolution.

BACKGROUND: Target enrichment combined with chromosome conformation capturing methodologies such as capture Hi-C (CHC) can be used to investigate spatial layouts of genomic regions with high resolution and at scalable costs. A common application of CHC is the investigation of regulatory elements that are in contact with promoters, but CHC can be used for a range of other applications. Therefore, probe design for CHC needs to be adapted to experimental needs, but no flexible tool is currently available for this purpose. RESULTS: We present a Java desktop application called GOPHER (Generator Of Probes for capture Hi-C Experiments at high Resolution) that implements three strategies for CHC probe design. GOPHER's simple approach is similar to the probe design of previous approaches that employ CHC to investigate all promoters, with one probe being placed at each margin of a single digest that overlaps the transcription start site (TSS) of each promoter. GOPHER's simple-patched approach extends this methodology with a heuristic that improves coverage of viewpoints in which the TSS is located near to one of the boundaries of the digest. GOPHER's extended approach is intended mainly for focused investigations of smaller gene sets. GOPHER can also be used to design probes for regions other than TSS such as GWAS hits or large blocks of genomic sequence. GOPHER additionally provides a number of features that allow users to visualize and edit viewpoints, and outputs a range of files useful for documentation, ordering probes, and downstream analysis. CONCLUSION: GOPHER is an easy-to-use and robust desktop application for CHC probe design. Source code and a precompiled executable can be downloaded from the GOPHER GitHub page at https://github.com/TheJacksonLaboratory/Gopher .

Unraveling the transcriptional regulation of TWIST1 in limb development.

The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.

Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis.

The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.

Structural variation in the 3D genome.

Structural and quantitative chromosomal rearrangements, collectively referred to as structural variation (SV), contribute to a large extent to the genetic diversity of the human genome and thus are of high relevance for cancer genetics, rare diseases and evolutionary genetics. Recent studies have shown that SVs can not only affect gene dosage but also modulate basic mechanisms of gene regulation. SVs can alter the copy number of regulatory elements or modify the 3D genome by disrupting higher-order chromatin organization such as topologically associating domains. As a result of these position effects, SVs can influence the expression of genes distant from the SV breakpoints, thereby causing disease. The impact of SVs on the 3D genome and on gene expression regulation has to be considered when interpreting the pathogenic potential of these variant types.

Polymer physics predicts the effects of structural variants on chromatin architecture.

Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog).

Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.

Breaking TADs: How Alterations of Chromatin Domains Result in Disease.

Spatial organization is an inherent property of the vertebrate genome to accommodate the roughly 2m of DNA in the nucleus of a cell. In this nonrandom organization, topologically associating domains (TADs) emerge as a fundamental structural unit that is thought to guide regulatory elements to their cognate promoters. In this review we summarize the most recent findings about TADs and the boundary regions separating them. We discuss how the disruption of these structures by genomic rearrangements can result in gene misexpression and disease.

Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice.

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.