Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.

CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.

Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish.

Due to its genetic amenability coupled with advances in genome editing, zebrafish is an excellent model to examine the function of (epi)genomic elements. Here, we repurposed the Ac/Ds maize transposition system to efficiently characterise zebrafish cis-regulated elements, also known as enhancers, in F0-microinjected embryos. We further used the system to stably express guide RNAs enabling CRISPR/dCas9-interference (CRISPRi) perturbation of enhancers without disrupting the underlying genetic sequence. In addition, we probed the phenomenon of antisense transcription at two neural crest gene loci. Our study highlights the utility of Ac/Ds transposition as a new tool for transient epigenome modulation in zebrafish.

miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms.

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-ß pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-ß pathway in this process.

Lim1, an embryonal transcription factor, is absent in multicystic renal dysplasia, but reactivated in nephroblastomas.

OBJECTIVE: Lim1 (Lim homeobox 1) plays an important role during rodent renal development; however, its rolein human kidney development and disease is still unclear. METHODS: We investigated LIM1 expression during human renal development, in dysplastic kidneys and in renal neoplasms using immunohistochemistry. RNA levels in renal carcinomas were determined by quantitative RT-PCR, and the potential roles of LIM1 in mesenchymal-epithelial transition and cell cycle were investigated in a cell culture model. RESULTS: LIM1 was detected in pretubular aggregates, S-shaped and comma-shaped bodies as well as immature glomeruli between 10 and 30 weeks of gestation. Eleven dysplastic kidneys showed no expression of LIM1. In contrast, 12 of 32 nephroblastomas showed nuclear positivity. One regressive nephroblastoma had diffuse expression of LIM1 in tubular structures, all others showed focal positivity in mesenchymal, blastemal and epithelial structures. Renal cell carcinomas revealed no expression of LIM1. Overexpression of LIM1 in a cell culture model led to an increase in KERATIN7 expression but no change in the cell cycle. CONCLUSION: Our study supports the concept of a causative role of LIM1 deficiency in the development of multicystic kidney. In a small subset of nephroblastomas with a more diffuse expression pattern LIM1 might also contribute to the pathogenesis of these lesions.

Tissue-Specific In Vivo Biotin Chromatin Immunoprecipitation with Sequencing in Zebrafish and Chicken.

Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019).

Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.

Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of the multipotent neural crest (NC) embryonic cell population. By coupling NC-specific epigenomic and transcriptional profiling at population and single-cell levels with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors, and cis-signatures allowing reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates early during NC ontogeny. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.

From Pioneer to Repressor: Bimodal foxd3 Activity Dynamically Remodels Neural Crest Regulatory Landscape In Vivo.

The neural crest (NC) is a transient embryonic stem cell-like population characterized by its multipotency and broad developmental potential. Here, we perform NC-specific transcriptional and epigenomic profiling of foxd3-mutant cells in vivo to define the gene regulatory circuits controlling NC specification. Together with global binding analysis obtained by foxd3 biotin-ChIP and single cell profiles of foxd3-expressing premigratory NC, our analysis shows that, during early steps of NC formation, foxd3 acts globally as a pioneer factor to prime the onset of genes regulating NC specification and migration by re-arranging the chromatin landscape, opening cis-regulatory elements and reshuffling nucleosomes. Strikingly, foxd3 then gradually switches from an activator to its well-described role as a transcriptional repressor and potentially uses differential partners for each role. Taken together, these results demonstrate that foxd3 acts bimodally in the neural crest as a switch from "permissive" to "repressive" nucleosome and chromatin organization to maintain multipotency and define cell fates.

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome.

Interrogation of gene regulatory circuits in complex organisms requires precise tools for the selection of individual cell types and robust methods for biochemical profiling of target proteins. We have developed a versatile, tissue-specific binary in vivo biotinylation system in zebrafish termed biotagging that uses genetically encoded components to biotinylate target proteins, enabling in-depth genome-wide analyses of their molecular interactions. Using tissue-specific drivers and cell-compartment-specific effector lines, we demonstrate the specificity of the biotagging toolkit at the biochemical, cellular, and transcriptional levels. We use biotagging to characterize the in vivo transcriptional landscape of migratory neural crest and myocardial cells in different cellular compartments (ribosomes and nucleus). These analyses reveal a comprehensive network of coding and non-coding RNAs and cis-regulatory modules, demonstrating that tissue-specific identity is embedded in the nuclear transcriptomes. By eliminating background inherent to complex embryonic environments, biotagging allows analyses of molecular interactions at high resolution.

Active DNA demethylation at enhancers during the vertebrate phylotypic period.

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

The pluripotent renal stem cell regulator SIX2 is activated in renal neoplasms and influences cellular proliferation and migration.

Embryonal renal mesenchyme contains pluripotent progenitor cells characterized by expression of SIX2, which suppresses cellular differentiation. Additionally hypomethylation of the promotor region in renal neoplasms indicates a role of SIX2 in tumorigenesis. This study focuses therefore on the investigation of SIX2 in different renal neoplasms and the mode and consequences of SIX2 activation. Expression of SIX2 was determined in renal cell carcinomas, nephroblastomas, and dysplastic kidneys using immunohistochemistry and quantitative real-time polymerase chain reaction. Its potential mode of activation was assessed by measuring upstream activators by quantitative real-time polymerase chain reaction and the level of methylation of the promoter region by quantitative DNA methylation analysis. Consequences of SIX2 activation were investigated by overexpressing SIX2 in a cell line. Forty-seven of 49 renal clear cell carcinomas showed nuclear staining of SIX2, whereas all papillary carcinomas were negative. In nephroblastomas of various subtypes blastema showed a significant up-regulation (P < .01) and a strong nuclear protein expression of SIX2 in contrast to negative epithelial and mesenchymal areas. 11 cases of dysplastic kidneys were entirely negative. Upstream activators of SIX2 indicated an activation of the signal transduction pathway in most samples. No difference of promoter methylation status was observed between blastema and epithelial structures. A significantly higher percentage of cells in the S-phase and an increased migration were detected in the cell-line overexpressing SIX2. Our study suggests that activation of SIX2 might contribute to the pathogenesis of renal clear cell carcinomas and nephroblastomas. SIX2 also appears to be a valuable marker for minimal residual blastema contributing to the prognosis of nephroblastomas.