Integrin Signaling Regulates Spindle Orientation in Drosophila to Preserve the Follicular-Epithelium Monolayer

SummaryEpithelia act as important physiological barriers and as structural components of tissues and organs. In the Drosophila ovary, follicle cells envelop the germline cysts to form a monolayer epithelium. During division, the orientation of the mitotic spindle in follicle cells is such that both daughter cells remain within the same plane, and the simple structure of the follicular epithelium is thus preserved. Here we show that integrins, heterodimeric transmembrane receptors that connect the extracellular matrix to the cell's cytoskeleton [1, 2], are required for maintaining the ovarian monolayer epithelium in Drosophila. Mosaic egg chambers containing integrin mutant follicle cells develop stratified epithelia at both poles. This stratification is due neither to abnormal cell proliferation nor to defects in the apical-basal polarity of the mutant cells. Instead, integrin function is required for the correct orientation of the mitotic apparatus both in mutant cells and in their immediately adjacent wild-type neighbors. We further demonstrate that integrin-mediated signaling, rather than adhesion, is sufficient for maintaining the integrity of the follicular epithelium. The above data show that integrins are necessary for preserving the simple organization of a specialized epithelium and link integrin-mediated signaling to the correct orientation of the mitotic spindle in this epithelial cell type

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. [Results]: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. [Conclusions]: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.This study was funded by the CNIC Translational Grant Programme (CNIC-08-2009 to MM and DF), the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 to MM, BFU2013-41322-P to JLGS, BFU2012-38111 to AA, and CSD2007-00008 to JLGS and MM), the Comunidad Autónoma de Madrid (grant CELLDD-CM to MM), and the Andalusian Government (grant BIO-396 to JLGS). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation.Peer Reviewe

Generation and characterization of mecp2 mutant.

Motivation: Mecp2 gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an important regulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances or silences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms in Rett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mental and physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in the MeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study is the generation of a knock-out (KO) mutant of the mecp2 gene in zebrafish by employing the CRISPR/Cas9 technique. Once we obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq), focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will give us an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a useful model system aimed at understanding RTT etiology in vivo.Methods: To create the mecp2 KO, we designed sgRNA guides targeting the mecp2 exon 2, using the CRISPRscan software. We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed to develop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCR followed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of the WT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interest were named as follows: (i) mecp2 26⚦, with a deletion of 181bp, (ii) mecp2 27, ⚦with deletion of 15bp and (iii) mecp2 13,⚦ with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspring was genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); it is expected that 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the mecp2 gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we will genotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the qiagen DNAeasy blood and tissue kit for DNA and Trizol for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.Results and conclusions: We have generated heterozygous mecp2 mutants, that we have to genotype, from the founders mentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not be expressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotype and perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be able to explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RTT

Generation and characterization of mecp2 mutant.

Motivation: Mecp2 gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an importantregulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances orsilences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms inRett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mentaland physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in theMeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study isthe generation of a knock-out (KO) mutant of the mecp2 gene in zebrafish by employing the CRISPR/Cas9 technique. Oncewe obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq),focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will giveus an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a usefulmodel system aimed at understanding RTT etiology in vivo.Methods: To create the mecp2 KO, we designed sgRNA guides targeting the mecp2 exon 2, using the CRISPRscan software.We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed todevelop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCRfollowed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of theWT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interestwere named as follows: (i) mecp2 26⚦, with a deletion of 181bp, (ii) mecp2 27 ,⚦ with deletion of 15bp and (iii) mecp2 13 ,⚦with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspringwas genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); it is expectedthat 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the mecp2gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we willgenotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the qiagen DNAeasy blood andtissue kit for DNA and Trizol for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.Results and conclusions: We have generated heterozygous mecp2 mutants, that we have to genotype, from the foundersmentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not beexpressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotypeand perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be ableto explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RT

Identification and Analysis of Conserved cis-Regulatory Regions of the MEIS1 Gene

Meis1, a conserved transcription factor of the TALE-homeodomain class, is expressed in a wide variety of tissues during development. Its complex expression pattern is likely to be controlled by an equally complex regulatory landscape. Here we have scanned the Meis1 locus for regulatory elements and found 13 non-coding regions, highly conserved between humans and teleost fishes, that have enhancer activity in stable transgenic zebrafish lines. All these regions are syntenic in most vertebrates. The composite expression of all these enhancer elements recapitulate most of Meis1 expression during early embryogenesis, indicating they comprise a basic set of regulatory elements of the Meis1 gene. Using bioinformatic tools, we identify a number of potential binding sites for transcription factors that are compatible with the regulation of these enhancers. Specifically, HHc2:066650, which is expressed in the developing retina and optic tectum, harbors several predicted Pax6 sites. Biochemical, functional and transgenic assays indicate that pax6 genes directly regulate HHc2:066650 activity.This work was funded through grants BFU2009-07044 (MICINN) and Proyecto de Excelencia CVI 2658 (Junta de Andalucía) to FC and BFU2010-14839 (MICINN), CSD2007-00008 and Proyecto de Excelencia CVI-3488 to JLGS. JLR is a recipient of a JAE-DOC contract from the Spanish National Research Council (CSIC)

Profiling of conserved non-coding elements upstream of SHOX and functional characterisation of the SHOX cis-regulatory landscape

Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox (SHOX) gene, regulated by seven CNEs located downstream and upstream of SHOX, with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis-regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS, and (iii) the chromatin architecture of the SHOX cis-regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells, and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis-regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis-regulatory domain spanning more than 1 Mb and harbouring putative new cis-regulatory elementsThis study is supported by FWO grant G079711N (CIS-CODE). Spanish and Andalusian government grants BFU2010-14839, BFU2013-41322-P, and BIO-396 to J.L.G.S. and SAF2012-30871 to K.E.H. and S.B.S. Andalusian government supports A.F.M. as the scientific manager of the CABD´s Aquatic Vertebrates Platform. CIBERER supports S.B.S

Comparative epigenomics in distantly related teleost species identifies conserved cis-regulatory nodes active during the vertebrate phylotypic period

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months. After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).The complex relationship between ontogeny and phylogeny has been the subject of attention and controversy since von Baer's formulations in the 19th century. The classic concept that embryogenesis progresses from clade general features to species-specific characters has often been revisited. It has become accepted that embryos from a clade show maximum morphological similarity at the so-called phylotypic period (i.e., during mid-embryogenesis). According to the hourglass model, body plan conservation would depend on constrained molecular mechanisms operating at this period. More recently, comparative transcriptomic analyses have provided conclusive evidence that such molecular constraints exist. Examining cis-regulatory architecture during the phylotypic period is essential to understand the evolutionary source of body plan stability. Here we compare transcriptomes and key epigenetic marks (H3K4me3 and H3K27ac) from medaka (Oryzias latipes) and zebrafish (Danio rerio), two distantly related teleosts separated by an evolutionary distance of 115-200 Myr. We show that comparison of transcriptome profiles correlates with anatomical similarities and heterochronies observed at the phylotypic stage. Through comparative epigenomics, we uncover a pool of conserved regulatory regions (≈700), which are active during the vertebrate phylotypic period in both species. Moreover, we show that their neighboring genes encode mainly transcription factors with fundamental roles in tissue specification. We postulate that these regulatory regions, active in both teleost genomes, represent key constrained nodes of the gene networks that sustain the vertebrate body plan.The Andalusian government (JA) supported A.F-.M. as scientific manager of the Aquatic Vertebrates Platform at CABD. J.W.C. was supported by a studentship from The Institute of Cancer Research. Spanish and Andalusian government grants BFU2010-14839, CSD2007-00008, and P08-CVI-3488 to J.L.G-.S.; and BFU2011-22916 and P11-CVI-7256 to J.R.M-.M. supported this work.Peer Reviewe

A role for insulator elements in the regulation of gene expression response to hypoxia

Hypoxia inducible factor (HIF) up-regulates the transcription of a few hundred genes required for the adaptation to hypoxia. This restricted set of targets is in sharp contrast with the widespread distribution of the HIF binding motif throughout the genome. Here, we investigated the transcriptional response of GYS1 and RUVBL2 genes to hypoxia to understand the mechanisms that restrict HIF activity toward specific genes. GYS1 and RUVBL2 genes are encoded by opposite DNA strands and separated by a short intergenic region (~1 kb) that contains a functional hypoxia response element equidistant to both genes. However, hypoxia induced the expression of GYS1 gene only. Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF. Enhancer blocking assays, performed in cell culture and transgenic zebrafish, confirmed the existence of an insulator element within this inhibitory region that could explain the differential regulation of GYS1 and RUVBL2 by hypoxia. Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements. This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1)1,3,4. Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113)