Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Extensive DNA methylome rearrangement during early lamprey embryogenesis.

DNA methylation (5mC) is a repressive gene regulatory mark widespread in vertebrate genomes, yet the developmental dynamics in which 5mC patterns are established vary across species. While mammals undergo two rounds of global 5mC erasure, teleosts, for example, exhibit localized maternal-to-paternal 5mC remodeling. Here, we studied 5mC dynamics during the embryonic development of sea lamprey, a jawless vertebrate which occupies a critical phylogenetic position as the sister group of the jawed vertebrates. We employed 5mC quantification in lamprey embryos and tissues, and discovered large-scale maternal-to-paternal epigenome remodeling that affects ~30% of the embryonic genome and is predominantly associated with partially methylated domains. We further demonstrate that sequences eliminated during programmed genome rearrangement (PGR), are hypermethylated in sperm prior to the onset of PGR. Our study thus unveils important insights into the evolutionary origins of vertebrate 5mC reprogramming, and how this process might participate in diverse developmental strategies.

Evolutionary conservation of embryonic DNA methylome remodelling in distantly related teleost species.

Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilization, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the global erasure and re-establishment of DNA methylation patterns. However, in non-mammalian vertebrates like zebrafish, no global erasure has been observed. To investigate the evolutionary conservation and divergence of DNA methylation remodelling in teleosts, we generated base resolution DNA methylome datasets of developing medaka and medaka-zebrafish hybrid embryos. In contrast to previous reports, we show that medaka display comparable DNA methylome dynamics to zebrafish with high gametic mCG levels (sperm: ∼90%; egg: ∼75%), and adoption of a paternal-like methylome during early embryogenesis, with no signs of prior DNA methylation erasure. We also demonstrate that non-canonical DNA methylation (mCH) reprogramming at TGCT tandem repeats is a conserved feature of teleost embryogenesis. Lastly, we find remarkable evolutionary conservation of DNA methylation remodelling patterns in medaka-zebrafish hybrids, indicative of compatible DNA methylation maintenance machinery in far-related teleost species. Overall, these results suggest strong evolutionary conservation of DNA methylation remodelling pathways in teleosts, which is distinct from the global DNA methylome erasure and reestablishment observed in mammals.

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.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

The Australasian dingo archetype: de novo chromosome-length genome assembly, DNA methylome, and cranial morphology.

BACKGROUND: One difficulty in testing the hypothesis that the Australasian dingo is a functional intermediate between wild wolves and domesticated breed dogs is that there is no reference specimen. Here we link a high-quality de novo long-read chromosomal assembly with epigenetic footprints and morphology to describe the Alpine dingo female named Cooinda. It was critical to establish an Alpine dingo reference because this ecotype occurs throughout coastal eastern Australia where the first drawings and descriptions were completed. FINDINGS: We generated a high-quality chromosome-level reference genome assembly (Canfam_ADS) using a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. Compared to the previously published Desert dingo assembly, there are large structural rearrangements on chromosomes 11, 16, 25, and 26. Phylogenetic analyses of chromosomal data from Cooinda the Alpine dingo and 9 previously published de novo canine assemblies show dingoes are monophyletic and basal to domestic dogs. Network analyses show that the mitochondrial DNA genome clusters within the southeastern lineage, as expected for an Alpine dingo. Comparison of regulatory regions identified 2 differentially methylated regions within glucagon receptor GCGR and histone deacetylase HDAC4 genes that are unmethylated in the Alpine dingo genome but hypermethylated in the Desert dingo. Morphologic data, comprising geometric morphometric assessment of cranial morphology, place dingo Cooinda within population-level variation for Alpine dingoes. Magnetic resonance imaging of brain tissue shows she had a larger cranial capacity than a similar-sized domestic dog. CONCLUSIONS: These combined data support the hypothesis that the dingo Cooinda fits the spectrum of genetic and morphologic characteristics typical of the Alpine ecotype. We propose that she be considered the archetype specimen for future research investigating the evolutionary history, morphology, physiology, and ecology of dingoes. The female has been taxidermically prepared and is now at the Australian Museum, Sydney.

The Australasian dingo archetype: De novo chromosome-length genome assembly, DNA methylome, and cranial morphology.

Background: One difficulty in testing the hypothesis that the Australasian dingo is a functional intermediate between wild wolves and domesticated breed dogs is that there is no reference specimen. Here we link a high-quality de novo long read chromosomal assembly with epigenetic footprints and morphology to describe the Alpine dingo female named Cooinda. It was critical to establish an Alpine dingo reference because this ecotype occurs throughout coastal eastern Australia where the first drawings and descriptions were completed. Findings: We generated a high-quality chromosome-level reference genome assembly (Canfam_ADS) using a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. Compared to the previously published Desert dingo assembly, there are large structural rearrangements on Chromosomes 11, 16, 25 and 26. Phylogenetic analyses of chromosomal data from Cooinda the Alpine dingo and nine previously published de novo canine assemblies show dingoes are monophyletic and basal to domestic dogs. Network analyses show that the mtDNA genome clusters within the southeastern lineage, as expected for an Alpine dingo. Comparison of regulatory regions identified two differentially methylated regions within glucagon receptor GCGR and histone deacetylase HDAC4 genes that are unmethylated in the Alpine dingo genome but hypermethylated in the Desert dingo. Morphological data, comprising geometric morphometric assessment of cranial morphology place dingo Cooinda within population-level variation for Alpine dingoes. Magnetic resonance imaging of brain tissue show she had a larger cranial capacity than a similar-sized domestic dog. Conclusions: These combined data support the hypothesis that the dingo Cooinda fits the spectrum of genetic and morphological characteristics typical of the Alpine ecotype. We propose that she be considered the archetype specimen for future research investigating the evolutionary history, morphology, physiology, and ecology of dingoes. The female has been taxidermically prepared and is now at the Australian Museum, Sydney.

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins.

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module.

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability.

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

The Australian dingo is an early offshoot of modern breed dogs.

Dogs are uniquely associated with human dispersal and bring transformational insight into the domestication process. Dingoes represent an intriguing case within canine evolution being geographically isolated for thousands of years. Here, we present a high-quality de novo assembly of a pure dingo (CanFam_DDS). We identified large chromosomal differences relative to the current dog reference (CanFam3.1) and confirmed no expanded pancreatic amylase gene as found in breed dogs. Phylogenetic analyses using variant pairwise matrices show that the dingo is distinct from five breed dogs with 100% bootstrap support when using Greenland wolf as the outgroup. Functionally, we observe differences in methylation patterns between the dingo and German shepherd dog genomes and differences in serum biochemistry and microbiome makeup. Our results suggest that distinct demographic and environmental conditions have shaped the dingo genome. In contrast, artificial human selection has likely shaped the genomes of domestic breed dogs after divergence from the dingo.

A developmentally programmed splicing failure contributes to DNA damage response attenuation during mammalian zygotic genome activation.

Transition from maternal to embryonic transcriptional control is crucial for embryogenesis. However, alternative splicing regulation during this process remains understudied. Using transcriptomic data from human, mouse, and cow preimplantation development, we show that the stage of zygotic genome activation (ZGA) exhibits the highest levels of exon skipping diversity reported for any cell or tissue type. Much of this exon skipping is temporary, leads to disruptive noncanonical isoforms, and occurs in genes enriched for DNA damage response in the three species. Two core spliceosomal components, Snrpb and Snrpd2, regulate these patterns. These genes have low maternal expression at ZGA and increase sharply thereafter. Microinjection of Snrpb/d2 messenger RNA into mouse zygotes reduces the levels of exon skipping at ZGA and leads to increased p53-mediated DNA damage response. We propose that mammalian embryos undergo an evolutionarily conserved, developmentally programmed splicing failure at ZGA that contributes to the attenuation of cellular responses to DNA damage.

Nanopore Sequencing and Data Analysis for Base-Resolution Genome-Wide 5-Methylcytosine Profiling.

Whole-genome bisulfite sequencing (WGBS) is currently the gold standard for DNA methylation (5-methylcytosine, 5mC) profiling; however, the destructive nature of sodium bisulfite results in DNA fragmentation and subsequent biases in sequencing data. Such issues have led to the development of bisulfite-free methods for 5mC detection. Nanopore sequencing is a long read nondestructive approach that directly analyzes DNA and RNA fragments in real time. Recently, computational tools have been developed that enable base-resolution detection of 5mC from Oxford Nanopore sequencing data. In this chapter, we provide a detailed protocol for preparation, sequencing, read assembly, and analysis of genome-wide 5mC using Nanopore sequencing technologies.

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish.

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.

Sequence determinants, function, and evolution of CpG islands.

In vertebrates, cytosine-guanine (CpG) dinucleotides are predominantly methylated, with ∼80% of all CpG sites containing 5-methylcytosine (5mC), a repressive mark associated with long-term gene silencing. The exceptions to such a globally hypermethylated state are CpG-rich DNA sequences called CpG islands (CGIs), which are mostly hypomethylated relative to the bulk genome. CGIs overlap promoters from the earliest vertebrates to humans, indicating a concerted evolutionary drive compatible with CGI retention. CGIs are characterised by DNA sequence features that include DNA hypomethylation, elevated CpG and GC content and the presence of transcription factor binding sites. These sequence characteristics are congruous with the recruitment of transcription factors and chromatin modifying enzymes, and transcriptional activation in general. CGIs colocalize with sites of transcriptional initiation in hypermethylated vertebrate genomes, however, a growing body of evidence indicates that CGIs might exert their gene regulatory function in other genomic contexts. In this review, we discuss the diverse regulatory features of CGIs, their functional readout, and the evolutionary implications associated with CGI retention in vertebrates and possibly in invertebrates.

TAB-seq and ACE-seq Data Processing for Genome-Wide DNA hydroxymethylation Profiling.

5-Methylcytosine (5mC) is one of the most abundant and well-studied chemical DNA modifications of vertebrate genomes. 5mC plays an essential role in genome regulation including: silencing of retroelements, X chromosome inactivation, and heterochromatin stability. Furthermore, 5mC shapes the activity of cis-regulatory elements crucial for cell fate determination. TET enzymes can oxidize 5mC to form 5-hydroxymethylcytosine (5hmC), thereby adding an additional layer of complexity to the DNA methylation landscape dynamics. The advent of techniques enabling genome-wide 5hmC profiling provided critical insights into its genomic distribution, scope, and function. These methods include immunoprecipitation, chemical labeling and capture-based approaches, as well as single-nucleotide 5hmC profiling techniques such as TET-assisted bisulfite sequencing (TAB-seq) and APOBEC-coupled epigenetic sequencing (ACE-seq). Here we provide a detailed protocol for computational analysis required for the genomic alignment of TAB-seq and ACE-seq data, 5hmC calling, and statistical analysis.

Generation and Molecular Characterization of Transient tet1/2/3 Zebrafish Knockouts.

5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).

Krüppel-like factor 1 is a core cardiomyogenic trigger in zebrafish.

Cardiac regeneration requires dedifferentiation and proliferation of mature cardiomyocytes, but the mechanisms underlying this plasticity remain unclear. Here, we identify a potent cardiomyogenic role for Krüppel-like factor 1 (Klf1/Eklf), which is induced in adult zebrafish myocardium upon injury. Myocardial inhibition of Klf1 function does not affect heart development, but it severely impairs regeneration. Transient Klf1 activation is sufficient to expand mature myocardium in uninjured hearts. Klf1 directs epigenetic reprogramming of the cardiac transcription factor network, permitting coordinated cardiomyocyte dedifferentiation and proliferation. Myocardial expansion is supported by Klf1-induced rewiring of mitochondrial metabolism from oxidative respiration to anabolic pathways. Our findings establish Klf1 as a core transcriptional regulator of cardiomyocyte renewal in adult zebrafish hearts.