Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation.

To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.

Chromatin gene-gene loops support the cross-regulation of genes with related function.

Chromatin loops between gene pairs have been observed in diverse contexts in both flies and vertebrates. Combining high-resolution Capture-C, DNA fluorescence in situ hybridization, and genetic perturbations, we dissect the functional role of three loops between genes with related function during Drosophila embryogenesis. By mutating the loop anchor (but not the gene) or the gene (but not loop anchor), we disentangle loop formation and gene expression and show that the 3D proximity of paralogous gene loci supports their co-regulation. Breaking the loop leads to either an attenuation or enhancement of expression and perturbs their relative levels of expression and cross-regulation. Although many loops appear constitutive across embryogenesis, their function can change in different developmental contexts. Taken together, our results indicate that chromatin gene-gene loops act as architectural scaffolds that can be used in different ways in different contexts to fine-tune the coordinated expression of genes with related functions and sustain their cross-regulation.

CTCF, BEAF-32, and CP190 are not required for the establishment of TADs in early Drosophila embryos but have locus-specific roles.

The boundaries of topologically associating domains (TADs) are delimited by insulators and/or active promoters; however, how they are initially established during embryogenesis remains unclear. Here, we examined this during the first hours of Drosophila embryogenesis. DNA-FISH confirms that intra-TAD pairwise proximity is established during zygotic genome activation (ZGA) but with extensive cell-to-cell heterogeneity. Most newly formed boundaries are occupied by combinations of CTCF, BEAF-32, and/or CP190. Depleting each insulator individually from chromatin revealed that TADs can still establish, although with lower insulation, with a subset of boundaries (~10%) being more dependent on specific insulators. Some weakened boundaries have aberrant gene expression due to unconstrained enhancer activity. However, the majority of misexpressed genes have no obvious direct relationship to changes in domain-boundary insulation. Deletion of an active promoter (thereby blocking transcription) at one boundary had a greater impact than deleting the insulator-bound region itself. This suggests that cross-talk between insulators and active promoters and/or transcription might reinforce domain boundary insulation during embryogenesis.

Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.

Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.

Factors impacting producer marketing through community supported agriculture.

Nowadays, consumers have become increasingly aware of their local food system as a result of concerning about health and nutrition, food safety and sustainability, and local economic development. This transitional shift from global to direct-to-consumer farm operations has increased the demand for locally produced foods. As an alternative, community supported agriculture (CSA), a direct and sustainable food channel, has gained tremendous popularity in the US. Despite the interest garnered by local agriculture and CSA, relatively few studies have empirically tested the determinants of why this marketing phenomenon has grown so rapidly. The purpose of this study is to explore the factors that drive producers to market their products through CSA by using a county-level data set from the US. Results using a Tobit model indicate that specific operator characteristics, such as young and female operators and those engaged in farming as primary occupation, play a strongly positive role in the likelihood of marketing through CSA; farms with small size, rented land, and engagement in growing vegetables, melons, fruits and tree nut crops are more interested in marketing via CSA; households with higher income and females significantly increase the share of farms marketing through CSA; presence of children and seniors and being married are negatively related to the demand for CSA foods. Moreover, counties with higher density of population, establishments-supermarket and other grocery stores, and legislation or active programs that encourage local food consumption tend to encourage more farms marketing through CSA.

Modulation of Apoptosis Controls Inhibitory Interneuron Number in the Cortex.

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors, and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic and cell-type specific and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast.

The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5'Hoxd-Gli3 antagonism.

The number of phalanges and joints are key features of digit 'identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5'Hoxd-Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5'Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation.

Recurrent chromosomal gains and heterogeneous driver mutations characterise papillary renal cancer evolution.

Papillary renal cell carcinoma (pRCC) is an important subtype of kidney cancer with a problematic pathological classification and highly variable clinical behaviour. Here we sequence the genomes or exomes of 31 pRCCs, and in four tumours, multi-region sequencing is undertaken. We identify BAP1, SETD2, ARID2 and Nrf2 pathway genes (KEAP1, NHE2L2 and CUL3) as probable drivers, together with at least eight other possible drivers. However, only ~10% of tumours harbour detectable pathogenic changes in any one driver gene, and where present, the mutations are often predicted to be present within cancer sub-clones. We specifically detect parallel evolution of multiple SETD2 mutations within different sub-regions of the same tumour. By contrast, large copy number gains of chromosomes 7, 12, 16 and 17 are usually early, monoclonal changes in pRCC evolution. The predominance of large copy number variants as the major drivers for pRCC highlights an unusual mode of tumorigenesis that may challenge precision medicine approaches.

Spatial and temporal diversity in genomic instability processes defines lung cancer evolution.

Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC.

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution.

BACKGROUND: Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution. RESULTS: We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma. CONCLUSIONS: In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

Manipulating gene expression and signaling activity in cultured mouse limb bud cells.

BACKGROUND: The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. RESULTS: In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. CONCLUSIONS: Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements.

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing.

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.

Development of a high-throughput Gaussia luciferase reporter assay for the activation of the GADD45a gene by mutagens, promutagens, clastogens, and aneugens.

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

Integration of the transcriptional networks regulating limb morphogenesis.

The developing limb is one of the best described vertebrate systems for understanding how coordinated gene expression during embryogenesis leads to the structures present in the mature organism. This knowledge, derived from decades of research, is largely based upon gain- and loss-of-function experiments. These studies have provided limited information about how the key signaling pathways interact with each other and the downstream effectors of these pathways. We summarize our current understanding of known genetic interactions in the context of three temporally defined gene regulatory networks. These networks crystallize our current knowledge, depicting a dynamic process involving multiple feedback loops between the ectoderm and mesoderm. At the same time, they highlight the fact that many essential processes are still largely undescribed. Much of the dynamic transcriptional activity occurring during development is regulated by distal cis-regulatory elements. Modern genomic tools have provided new approaches for studying the function of cis-regulatory elements and we discuss the results of these studies in regard to understanding limb development. Ultimately, these genomic techniques will allow scientists to understand how multiple signaling pathways are integrated in space and time to drive gene expression and regulate the formation of the limb.

A pre-validation transferability study of the GreenScreen HC GADD45a-GFP assay with a metabolic activation system (S9).

A new protocol has recently been developed and validated for the GreenScreen HC GADD45a-GFP genotoxicity reporter assay, enabling the incorporation of an S9 metabolic activation system into the assay. The S9 protocol employs flow-cytometric methodology for the detection of both reporter GFP fluorescence and propidium iodide fluorescence for the estimation of cellular viability. In the spirit of assay validation by bodies such as the European Centre for the Validation of Alternative Methods (ECVAM), the adapted metabolic activation protocol for the GADD45a-GFP assay has been undergoing 'pre-validation'. Results of phases I and II of this pre-validation, namely protocol refinement and protocol transfer, respectively, are presented here. In phase I the protocol was transferred to a second laboratory for initial assessment of method portability and subsequent refinement of the protocol. In phase II, the protocol was then transferred to two further laboratories along with the elaborated standard operating-procedure (SOP) for further assessment of transferability. The three transfer sites then undertook an assessment of the method's reproducibility by testing eight compounds. The outcome of the study was a refined protocol that was found to be highly transferable. It yielded 100% agreement in results between all four laboratories.

Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.

Antisense oligonucleotide-mediated alternative splicing has great potential for treatment of Duchenne muscular dystrophy (DMD) caused by mutations within nonessential regions of the dystrophin gene. We have recently shown in the dystrophic mdx mouse that exon 23, bearing a nonsense mutation, can be skipped after intramuscular injection of a specific 2'-O-methyl phosphorothioate antisense oligoribonucleotide (2OMeAO). This skipping created a shortened, but in-frame, transcript that is translated to produce near-normal levels of dystrophin expression. This expression, in turn, led to improved muscle function. However, because DMD affects muscles body-wide, effective treatment requires dystrophin induction ideally in all muscles. Here, we show that systemic delivery of specific 2OMeAOs, together with the triblock copolymer F127, induced dystrophin expression in all skeletal muscles but not in cardiac muscle of the mdx dystrophic mice. The highest dystrophin expression was detected in diaphragm, gastrocnemius, and intercostal muscles. Large numbers of fibers with near-normal level of dystrophin were observed in focal areas. Three injections of 2OMeAOs at weekly intervals enhanced the levels of dystrophin. Dystrophin mRNA lacking the targeted exon 23 remained detectable 2 weeks after injection. No evidence of tissue damage was detected after 2OMeAO and F127 treatment either by serum analysis or histological examination of liver, kidney, lung, and muscles. The simplicity and safety of the antisense protocol provide a realistic prospect for treatment of the majority of DMD mutations. We conclude that a significant therapeutic effect may be achieved by further optimization in dose and regime of administration of antisense oligonucleotide.