Early alterations in the MCH system link aberrant neuronal activity and sleep disturbances in a mouse model of Alzheimer's disease.

Early Alzheimer's disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in AppNL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in AppNL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in AppNL-G-F mice. AppNL-G-F mice spend less time in rapid eye movement (REM) sleep. AppNL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions

Single cell profiling of an in vivo, RasV12 based tumour in the Drosophila eye primordium

The RasV12/scrib-/- tumour model has been widely used in Drosophila to study Ras-dependant oncogenesis and epithelial-mesenchymal transition (EMT). Recently, using a combination of ATAC-seq, FAIRE-seq and RNA-seq, we identified regulatory regions and two key transcription factors (AP-1 and Stat92E) involved in the development of these tumours. Using data from the whole tumour, it is impossible to determine whether these factors are part of the same gene regulatory networks (GRN) in a cell, or if they are controlling separate GRNs in different cells. In an attempt to answer this question, we used the C1 single cell auto-prep system (Fluidigm) and performed single-cell RNA-seq (SMART-seq2) and single-cell ATAC-seq on dissociated in vivo tumours. We envision that this approach will help answer this question and help enable us to start understand GRNs on the single cell level.status: publishe

Decoding epithelial enhancer logic using natural variation and single-cell ATAC-seq

Enhancers coordinate gene expression levels and the majority of disease related SNPs are located in these non-coding regions. Yet how genomic variation affects enhancer function is poorly understood. To investigate cis-regulatory variation in vivo, in an endogenous enhancer context, we profiled the genome-wide chromatin accessibility of epithelial tissues (imaginal discs), across 30 inbred Drosophila lines from the DGRP project. Statistical analysis identified 4289 chromatin accessibility QTLs (caQTL). We singled out the transcription factor Grainyhead as a key player, for which more than 50 caQTLs alter its binding site, thereby causing a concordant gain or loss in chromatin accesibility and in vivo enhancer activity. We further investigated the function and dynamics of Grainyhead bound enhancers using single-cell ATAC-seq on 322 cells, ranging from progenitors (high Grh), differentiated neurons (low Grh) and cells undergoing epithelial-to-mesenchymal transition (high to low Grh). We could construct developmental trajectories for these single-cells, driven by the changes in activity of the Grainyhead bound enhancers. Finally, to discriminate between functional and non-functional Grainyhead recognition motifs in the genome, we trained various machine learning algorithms and compared enhancers across different Drosophila species, allowing the identification of key enhancer features required for Grainyhead binding. In conclusion, we have directly linked non-coding SNPs to changes in enhancer activity and unveiled the basic regulatory logic of Grainyhead bound epithelial enhancers.status: publishe

Studying cellular differentiation in Drosophila using single cell ATAC-seq

Encoded within the genome is all of the information required to generate every cell type that exists in an organism, gene regulatory networks built from this information drive cellular differentiation from pluripotent cells to terminal differentiation. Although we are able to profile terminal cell states in many ways, it is currently unclear exactly how cells transition between states during differentiation. To unravel this transition, we aim to reverse engineer these gene regulatory networks and identify transcription factors and their targets which play a role in this process. To do this, we have taken advantage of single-cell ATAC-seq which allows us to profile the epigenome of individual cells. Particularly, we performed bulk and single-cell ATAC-seq across several hundreds of single cells, ranging from pluripotent progenitor cells to differentiating photoreceptor neurons in the Drosophila eye imaginal disc, as well as on RasV12/scrib-/- induced tumours, causing a block of differentiation in the same tissue. We compared the accessibility of various “cistromes” (sets of co-regulated enhancers) and identified meaningful cell clusters and possible developmental trajectories. The differentially accessible enhancers per cell cluster show motif enrichment for known transcription factors involved in the pluripotent state (e.g., Optix), the cancer-like cell state (e.g., AP-1, STAT92E), or the PR differentiation state (e.g., Tramtrack). In conclusion, scATAC-seq applied to in vivo tissue development allows us to identify changes in the chromatin landscape and infer regulatory programs of cellular differentiation at single cell resolution.status: accepte

A novel High-throughput Enhancer reporter assay reveals unsophisticated p53 enhancer logic

Deciphering the cis-regulatory logic encoded in enhancer sequences requires large-scale reporter assays to experimentally validate candidate enhancers predicted by genomic approaches such as chromatin accessibility and ChIP-seq. Here, we propose a novel high-throughput enhancer-reporter assay called CHEQ-Seq (Captured High-throughput Enhancer testing by Quantitative Sequencing). A set of candidate enhancers are pre-selected as regions of 0.5-1 kb and enriched from genomic, sheared DNA using custom-designed capturing baits. They are subsequently cloned into a reporter library and randomly combined with unique barcodes, before being tested under various conditions in cell culture. The relationship between each enhancer and its reporter-barcode is determined by PacBio long-read sequencing of the entire library; while the barcode expression level is determined by Illumina short-read cDNA sequencing. We have applied Cheq-seq to test the enhancer activity of 1526 p53 ChIP-seq peaks under p53 knock-down and p53 over-activating conditions. We obtained reproducible reporter expression for 1060 captured enhancers, of which 397 showed a significant p53-dependent activation. Strikingly, the large majority (99%) of p53 target enhancers can be characterized and distinguished from negative sequences by the occurrence of a single p53 binding site. Thus, the p53 enhancer logic represents a new ancestral class of enhancers, distinct from developmental enhancers that adhere to the billboard and enhanceosome models. The p53 enhancers do not contain obvious combinatorial complexity of binding sites for multiple transcription factors. This suggests that p53 acts alone on its target enhancers, and that context-dependent regulation of target genes is not encoded in the p53 enhancer sequences, but at different upstream or downstream layers of the cell’s gene regulatory network.status: accepte

Discovery of Transcription Factors and Regulatory Regions Driving In Vivo Tumor Development by ATAC-seq and FAIRE-seq Open Chromatin Profiling

Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.status: publishe

Mapping Gene Regulatory Networks in Drosophila Eye Development by Large-Scale Transcriptome Perturbations and Motif Inference

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, we performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, we derived direct TF-DNA interactions using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, we found network motifs, cis-regulatory codes, and regulators of eye development. We validate the predicted target regions of Grainyhead by ChIP-seq and identify this factor as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions

Natural Variation of Chromatin Accessibility reveals a new epithelial Enhancer model

Enhancers coordinate gene expression levels and the majority of disease related SNPs are located in these non-coding regions. Yet how genomic variation affects enhancer function is poorly understood. To investigate cis-regulatory variation in vivo, in an endogenous enhancer context, we profiled the genome-wide chromatin accessibility of epithelial tissues (imaginal discs), across 30 inbred Drosophila lines from the DGRP project. Statistical analysis identified 4289 chromatin accessibility QTLs (caQTL). We singled out the transcription factor Grainyhead as a key player, for which 70 caQTLs alter its binding site, thereby causing a concordant gain or loss in chromatin accessibility and in vivo enhancer activity. We show a clear uncoupling between enhancer accessibility and activity using an in vivo enhancer-reporter screen combined with cell-sorted ATAC-seq. The epithelial enhancers become accessible in all cells, due to the binding of the master regulatory Grainyhead, but activate gene expression only in a specific subpopulation of cells. Finally, to discriminate between functional and non-functional Grainyhead recognition motifs in the genome, we trained various machine learning algorithms and compared enhancers across different Drosophila species, allowing the identification of key enhancer features required for Grainyhead binding. In conclusion, we find a new epithelial enhancer model in which Grainyhead plays a similar role in epithelial tissues as Zelda in the embryo, uncoupling chromatin accessibility and enhancer activity for thousands of epithelial enhancers.status: publishe

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.status: publishe

Establishment of the mayfly Cloeon dipterum as a new model system to investigate insect evolution

Abstract The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects