eLife, Journal Year: 2018, Volume and Issue: 7

Published: Feb. 23, 2018

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials methods Data availability References Decision letter Author response author information Metrics During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible are used extensively to identify genomic regulatory elements. Whilst landscapes pluripotent stem cells well characterised, accessibility the somatic lineages not defined. Here we show that cell-specific can be produced via ectopic E. coli Dam methylase vivo, without requirement for cell-sorting (CATaDa). We have profiled individual cell-types Drosophila neural midgut lineages. Functional cell-type-specific enhancers were identified, as novel motifs enriched at different stages development. Finally, global between stem-cells their differentiated progeny. Our results demonstrate nature tissues during cell differentiation provide a approach understanding mechanisms underlying https://doi.org/10.7554/eLife.32341.001 For an embryo successfully develop into adult animal, specific genes must act types cells. Though all same encoded within DNA, looking way DNA packaged indicate which parts important particular type. If regions “open” one infer those actively involved regulation, whereas “closed” considered less important. It currently difficult determine open type complex organ, such brain. Existing require physically isolated from tissue, technically challenging. To overcome this issue, Aughey et al. now developed method does isolation The new technique involves using genetic engineering introduce enzyme called living fruit flies. This adds chemical label on then detected. tested various developing brain gut, able see differences openness corresponded action each also contain trends help understand role example, mature shown overall than divide generate them. hope will use other researchers working with either flies or mammalian tissues. knowledge scientists gain identifying how contributes both healthy diseased tissues, further our human biology diseases cancer. https://doi.org/10.7554/eLife.32341.002 multicellular organism, tightly regulated spatially temporally restricted signals. Changes accompanied concomitant structure composition. Therefore states vary widely across developmental types. genome, including promoters enhancers, identified relative lack nucleosomes. These ‘open chromatin’ assayed extrinsic factors. Consequently, profiling techniques commonly investigate (reviewed [Tsompana Buck, 2014]). Chromatin highly embryonic (ES) cells, but compacted following (Meshorer Misteli, 2006). has been suggested represents permissive state multiple programmes regulation may rapidly applied upon (Gaspar-Maia al., 2011). vivo understood. Imaging studies gross structure, example distribution heterochromatin observed post-mitotic (Francastel 2000; Le Gros 2016). Molecular investigating tended utilise heterogeneous due fact epigenome frequently requires physical nuclei, laborious prone error (McClure Southall, 2015). Therefore, there regarding models. recently ATAC-seq become popular address many limitations inherent earlier DNAse-seq (i.e. fewer increased assay speed), these still separation before (Buenrostro 2013). untethered adenine methyltransferase (Dam) methylation whilst nucleosome bound protected (Wines 1996; Bulanenkova 2007; Boivin Dura, 1998; Singh Klar, 1992). However, efficacy scale clear. Furthermore, manner, levels low enough avoid toxicity oversaturated signal, possible until now. Transgenic fusions DNA-binding proteins well-established assess transcription factor occupancy (DNA identification - DamID) (van Steensel Henikoff, 2000). Recently, it was demonstrated DamID could adapted profile DNA-protein interactions manner utilising ribosome re-initiation attenuate transgene (Marshall 2016; Southall referred Targeted (TaDa). Here, take advantage TaDa express produce profiles separation. Accessibility (CATaDa) yields comparable FAIRE methods, indicating reliable reproducible states. By assaying throughout central nervous system (CNS) enabled us elements dynamically change differentiation, functional enhancers. compared progeny, Dam-methylation signals more distributed greater level accessibility. CATaDa produces ATAC FAIRE-seq eye discs reasoned low-level transgenic Dam, tissue-specific GAL4 drivers Drosophila, would specifically methylate exclusively cell-type interest. Detection methylated sequences yield defined populations (Figure 1). if accurate reflection accessibility, acquired alternative techniques. A recent study generated imaginal (Davie Using CATaDa, expressed disc third instar larvae so compare previously collected data. Figure 1 Download asset Open Schematic illustrating technique. (A–B) interest (C) GATC areas condensed prevent access thereby precluding methylation. (D) Methylated detected mixed population https://doi.org/10.7554/eLife.32341.003 replicates (r2 = 0.947) 2—figure supplement showed good agreement FAIRE-seq. Visual inspection represented reliably inaccessible 2A,B). observe exhibited features consistent transcriptional start sites (TSS) 2C). 2 3 supplements Validation (A) chromosome three determined ATAC-seq, FAIRE-seq, CATaDa. Note reduced amount proximal centromere datasets. y-axes reads per million (rpm). (B) Example locus showing obtained FAIRE, ATAC, Peaks broadly Aggregation plot signal TSS kb up downstream. Aggregated shows expected enrichment Dam. peaks, loci. (E) Identification peaks 48.6% 55.9% respectively. overlap promoter (2 TSS), outside regions. https://doi.org/10.7554/eLife.32341.004 frequency increases dramatically towards centre 2D).The 49.4%, respectively (In comparison, – 2E). Monte Carlo simulation significant (p<1 × 10−5) peak heights shared correlation 10-16, r2 0.138) 2A). found increasing stringency calling notably decreased number coincided had relatively little impact unique discovery 2B). Given data, suggest majority false positives, genuinely ATAC-seq. Further examination indicates they significantly smaller present motifs, suggests cases negatives resolution achievable 3A–B). investigated detection features. (when ATAC), better non-promoter adjacent again explained depletion upstream 3C). high experimentally validated eye-antennal similarly 3A,B). 57.9% FlyLight corresponding (333 575 enhancers). 48% 68.7% respectively, loci Flylight coincide Corresponding pattern (i) (ii) (eye images database [http://flweb.janelia.org/cgi-bin/flew.cgi]). average (blue) (green) expression. Both Venn diagram profiling, most (305 total https://doi.org/10.7554/eLife.32341.008 In neurons derived asymmetrically dividing (NSCs). NSC divisions self-renewing daughter ganglion mother (GMC), divides once glia (Homem Knoblich, 2012). test local process cover four lineage. include NSCs (worniu- GAL4), GMCs newly born (R71C09-GAL4 [Figure 4—figure 1B, Li 2014]), larval (nSyb-GAL4), (nSyb-GAL4) 4A). 4 5 CNS. CNS lineage progression examined study. resulting Genomic region encompassing Wnt2 bruchpilot shown. Multiple Y-axes Clustering differentially two major groupings neurons. Motif analysis (e.g. ase E-box motif loci), motifs. Most cluster All E-values < 10−5. log2 scores selected GO terms Clear seen progresses. (NSC, GMC, L3 neuron, neuron left right). becoming progresses vice versa. https://doi.org/10.7554/eLife.32341.009 examining candidate development, relates manner. intronic (brp) locus, (L3) neurons, absent progenitor 4B). corresponds brp, transcribed synapse function (Wagh contrast, Wnt2, displays apparent intermediate Wnt signalling known control populations, therefore, (Ring 2014). Similar patterns At asense (ase) (a NSC-specific factor), intergenic considerably fully expressed. Interestingly, detectable GMCs/newly factors deadpan (dpn), CyclinE (CycE) prospero (pros) 2). GMC/newly loci, required immediately rendered 4B marked regulating given neurons). differential extent changed system. Hierarchical clustering reveals clusters versa 4C). Intriguingly, where maximal GMCs/early simple progressively closing neuronal gradually opening. There large NSCs, stage being terminally addition, demonstrates landscape although similar, distinct Regions thought belonging neurogenesis. binding enriched. CAGCNG proneural 4D) (Southall Brand, 2009; Jarman 1993). yielded sequence factor, Ci. groups, no partner 3). Analysis subdivision revealed yet examined, ase-like solely progeny 4). Gene ontology (GO) biological 5). ‘neuroblast fate determination’ ‘chromosome segregation’ ‘regulation behaviour’ ‘synaptic vesicle docking exocytosis’ 4E). Having CNS, asked whether similar contains pool cycling intestinal (ISCs) persists maintain mediate absorptive secretory functions organ (Jiang Edgar, 2011; Nászai contrast neurogenesis, single committed immature (enteroblast EB) divisions, differentiates epithelial (Ohlstein Spradling, 2007). examine midgut, ISCs EBs, enterocytes (ECs)(Figure 5A). midgut. escargot ECs. Upstream greatest ISCs, Similarly, 3’ 5’ distal (putative enhancer regions), nubbin gut Major ECs, down-regulated EBs. Principal component (mean replicates) (F) Correlation matrix (Spearman’s rank) means Individual denoted red outline. ISC (Asterisk R2 0.76), EC comparable. https://doi.org/10.7554/eLife.32341.016 As predictable variable (esg) self-renewal (Korzelius 2014), body surrounding ECs 5B). (encoding marker Pdm1), predominantly closed 5C). hierarchical groups (ECs) 5D). Smaller evident downregulated much pronounced that, lineages, specifying do occur after EB maturation. 5—figure fulfil roles respective organs production specialised exist short time fly long-lived persist animal’s lifetime, post-developmentally constantly replenish gut. comparing types, similarities growth division, tissue queried scale. >80% variance first principal components 5E). represent (CNS midgut) rather interesting Firstly, correlated closely direct 5F). recapitulate familial relationship progenitors cognate (R2 0.94/0.98 respectively). only weak (ISCs vs NSC, 0.51). maintenance multipotency, lineage-specific variation small. Enhancer prediction activity linked expression, orchestrate correct spatial temporal (Pennacchio 2015; Crawford cell-type- support this, covered 71C09-GAL4 line displayed higher clear reporter necessarily correspond active previous observations DNase hypersensitive often (Zhou 2017; Thurman least lineage, satisfied criteria designate them putative (see methods). available lines Vienna tiles (VT)(Kvon 2014) (Jenett 2012) collections contained predicted reporter, verified enhancer-GAL4 matched predictions activity. VT017417 GMR56E07 early part GFP 6A,B). readings prominent did not. detect line, VT004241, Delta positive 6C).

Language: Английский