The gene regulatory basis of genetic compensation during neural crest induction.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues

Dicer1 is required for pigment cell and craniofacial development in zebrafish.

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1sa9205, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1sa9205 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1sa9205 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1sa9205 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS mutant melanoma and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK-inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK-inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo.This project has received funding from the European Union’s Horizon 2020 432 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641458. The 433 work carried out at the University of Edinburgh was partly funded by EEP, MRC HGU Programme 434 (MC_UU_00007/9), European Research Council (ZF-MEL-CHEMBIO-648489), and L'Oreal-Melanoma 435 Research Alliance (401181)

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38–high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS mutant melanoma and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK-inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK-inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo.This project has received funding from the European Union’s Horizon 2020 432 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641458. The 433 work carried out at the University of Edinburgh was partly funded by EEP, MRC HGU Programme 434 (MC_UU_00007/9), European Research Council (ZF-MEL-CHEMBIO-648489), and L'Oreal-Melanoma 435 Research Alliance (401181)

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1.

Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing

Heritage Quay: What Will You Discover? Transforming the Archives of the University of Huddersfield, Yorkshire, UK

The Heritage Quay project is changing how archive services at the University of Huddersfield are delivered. This article examines how the Staff/Space/Collections dependency model and Customer Service Excellence framework have been used, and what lessons can be drawn for other archives

A high-resolution mRNA expression time course of embryonic development in zebrafish.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl

PRL3-DDX21 transcriptional control of endolysosomal genes restricts melanocyte stem cell differentiation

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer

Quantum state preparation and macroscopic entanglement in gravitational-wave detectors

Long-baseline laser-interferometer gravitational-wave detectors are operating at a factor of 10 (in amplitude) above the standard quantum limit (SQL) within a broad frequency band. Such a low classical noise budget has already allowed the creation of a controlled 2.7 kg macroscopic oscillator with an effective eigenfrequency of 150 Hz and an occupation number of 200. This result, along with the prospect for further improvements, heralds the new possibility of experimentally probing macroscopic quantum mechanics (MQM) - quantum mechanical behavior of objects in the realm of everyday experience - using gravitational-wave detectors. In this paper, we provide the mathematical foundation for the first step of a MQM experiment: the preparation of a macroscopic test mass into a nearly minimum-Heisenberg-limited Gaussian quantum state, which is possible if the interferometer's classical noise beats the SQL in a broad frequency band. Our formalism, based on Wiener filtering, allows a straightforward conversion from the classical noise budget of a laser interferometer, in terms of noise spectra, into the strategy for quantum state preparation, and the quality of the prepared state. Using this formalism, we consider how Gaussian entanglement can be built among two macroscopic test masses, and the performance of the planned Advanced LIGO interferometers in quantum-state preparation