Temporal lack of DNA methylation-mediated repression is a universal feature of vertebrate development

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.-- et al.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 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

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

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

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.This work is supported by the following grants: (I) To J.-R.M.-M.: From the Spanish Ministry of Science, Innovation, and Universities (MICINN): BFU2017-86339P with FEDER funds, MDM-2016-0687 and PY20_00006/Junta de Andalucía. (II) To O.B. Australian Research Council (ARC) Discovery Project (DP190103852). (III) To F.-J.D.-C.: Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018). (IV) To P.B.: BFU2016-75412-R with FEDER funds; PCIN-2015-176-C02-01/ERA-Net Neuron ImprovVision, and a CBMSO Institutional grant from the Fundación Ramón Areces. (V) To both J.-R.M.-M. and P.B.: BFU2016-81887-REDT, as well as Fundación Ramón Areces-2016 (Supporting L.B.)

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5ʹ-3ʹ RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favourable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.This work was supported by the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008). P.A.C. and D.R.G. were supported by Grains Research and Development Council scholarships (GRS184 and GRS10683), and S.R.E. was supported by an Australian Research Council Discovery Early Career Researcher Award (DE150101206). R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowshi

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 emergence of the brain non-CpG methylation system in vertebrates

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.This work was supported by the Australian Research Council (ARC) Centre of Excellence programme in Plant Energy Biology (grant no. CE140100008). R.L. was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship (no. FT120100862) and Howard Hughes Medical Institute International Research Scholarship. A.d.M. was funded by an EMBO long-term fellowship (no. ALTF 144-2014). J.L.G.-S. was supported by the Spanish government (grant no. BFU2016- 74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (no. MDM-2016-0687). B.V. was supported by the Biomedical Research Council of the Agency for Science, Technology and Research of Singapore. F.G. was supported by an ARC Future Fellowship (no. FT160100267). C.W.R. was supported by an NSF grant (no. IOS-1354898). J.R.E. is an investigator of the Howard Hughes Medical Institute. Genomic data was generated at the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome

Background: Basenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness. Results: Here, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection. Conclusions: The growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies