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W4384209396 abstract "Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor FruitlessC (FruC) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fruC function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. FruC negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fruC loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans. Editor's evaluation This is an important study that defines the role of the FruC transcription factor in key developmental decisions during neurogenesis in Drosophila. The authors combine genetics and genomic profiling to provide convincing evidence that FruC-regulated gene expression is correlated with changes in repressive histone marks. This study will be of wide general interest to the developmental biology field. https://doi.org/10.7554/eLife.86127.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest From neurons to sperm, our bodies are formed of a range of cells tailored to perform a unique role. However, organisms also host small reservoirs of unspecialized ‘stem cells’ that retain the ability to become different kinds of cells. When these stem cells divide, one daughter cell remains a stem cell while the other undergoes a series of changes that allows it to mature into a specific cell type. This ‘differentiation’ process involves quickly switching off the stem cell programme, the set of genes that give a cell the ability to keep dividing while maintaining an unspecialized state. Failure to do so can result in the differentiating cell reverting towards its initial state and multiplying uncontrollably, which can lead to tumours and other health problems. While scientists have a good understanding of how the stem cell programme is turned off during differentiation, controlling these genes is a balancing act that starts even before division: if the program is over-active in the ‘mother’ stem cell, for instance, the systems that switch it off in its daughter can become overwhelmed. The mechanisms presiding over these steps are less well-understood. To address this knowledge gap, Rajan, Anhezini et al. set out to determine how stem cells present in the brains of fruit flies could control the level of activity of their own stem cell programme. RNA sequencing and other genetic analyses revealed that a protein unique to these cells, called Fruitless, was responsible for decreasing the activity of the programme. Biochemical experiments then showed that Fruitless performed this role by attaching a small amount of chemical modifications (called methyl groups) to the proteins that ‘package’ the DNA near genes involved in the stem cell programme. High levels of methyl groups present near a gene will switch off this sequence completely; however, the amount of methyl groups that Fruitless helped to deposit is multiple folds lower. Consequently, Fruitless ‘fine-tunes’ the activity of the stem cell programme instead, dampening it just enough to stop it from overpowering the ‘off’ mechanism that would take place later in the daughter cell. These results shed new light on how stem cells behave – and how our bodies stop them from proliferating uncontrollably. In the future, Rajan, Anhezini et al. hope that this work will help to understand and treat diseases caused by defective stem cell differentiation. Introduction Expression of genes that promote stemness or differentiation must be properly controlled in stem cells to allow their progeny to transition through various intermediate stages of cell fate specification in a timely fashion (Pollen et al., 2015; Bhaduri et al., 2021; Michki et al., 2021; Ruan et al., 2021; Dillon et al., 2022). Exceedingly high levels of stemness gene transcripts that promote an undifferentiated state in stem cells can overwhelm translational control that downregulates their activity in stem cell progeny and perturb timely onset of differentiation (San-Juán and Baonza, 2011; Xiao et al., 2012; Zacharioudaki et al., 2012; Zhu et al., 2012; Larson et al., 2021; Ohtsuka and Kageyama, 2021). Conversely, excessive transcription of differentiation genes that instill biases toward terminal cellular functions in stem cells can overcome the mechanisms that uncouple these transcripts from the translational machinery and prematurely deplete the stem cell pool (Lennox et al., 2018; Baser et al., 2019; de Rooij et al., 2019; Marques et al., 2023). Thus, fine-tuning stemness and differentiation gene transcription in stem cells minimizes inappropriate gene activity that could result in developmental anomalies. Coordinated regulation of stemness and differentiation gene activity in stem cells by transcriptional and translational control poise stem cell progeny for a timely cell-state transition during differentiation (Ables et al., 2011; Koch et al., 2013; Kobayashi and Kageyama, 2014; Bigas and Porcheri, 2018; Rajan et al., 2021). Mechanistic investigation of the fine-tuning of stemness and differentiation gene transcription in vivo is challenging due to the compensatory effect of translational control, a lack of sensitized functional readouts, and a lack of insight into relevant transcription factors. Neuroblast lineages of the fly larval brain provide an excellent in vivo paradigm for mechanistic investigation of gene regulation during developmental transitions because the cell-type hierarchy is well-characterized at functional and molecular levels (Janssens and Lee, 2014; Homem et al., 2015; Doe, 2017). A larval brain lobe contains approximately 100 neuroblasts, and each neuroblast asymmetrically divides every 60–90 min, regenerating itself and producing a sibling progeny that commits to generating differentiated cell types. Most of these neuroblasts are type I, which generate a ganglion mother cell (GMC) in every division. A GMC undergoes terminal division to produce two neurons. Eight neuroblasts are type II, which invariably generate an immature intermediate neural progenitor (immature INP) in every division (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008). An immature INP initiates INP commitment 60 min after asymmetric neuroblast division (Janssens et al., 2017). An immature INP initially lacks Asense (Ase) protein expression and upregulates Ase as it progresses through INP commitment. Once INP commitment is complete, an Ase+ immature INP transitions into an INP and asymmetrically divides 5–6 times to generate more than a dozen differentiated cells, including neurons and glia (Viktorin et al., 2011; Bayraktar and Doe, 2013). All type II neuroblast lineage cell types in larval brains can be unambiguously identified based on functional characteristics and protein marker expression. Single-cell RNA-sequencing (scRNA-seq) of sorted, fluorescently labeled INPs and their differentiating progeny from wild-type brain tissue has led to the discovery of many new genes that contribute to the generation of diverse differentiated cell types during neurogenesis (Michki et al., 2021). This wealth of information on the type II neuroblast lineage allows for mechanistic investigations of precise spatiotemporal regulation of gene expression during developmental transitions. A multilayered gene regulation system ensures timely onset of INP commitment in immature INPs by coordinately terminating Notch signaling activity (Komori et al., 2018). Activated Notch signaling drives the expression of downstream-effector genes deadpan (dpn) and Enhancer of (splitz)mγ (E(spl)mγ), which promote stemness in type II neuroblasts by poising activation of the master regulator of INP commitment earmuff (erm) (San-Juán and Baonza, 2011; Xiao et al., 2012; Zacharioudaki et al., 2012; Zhu et al., 2012; Zacharioudaki et al., 2016). During asymmetric neuroblast division, the basal protein Numb and Brain tumor (Brat) exclusively segregate into immature INPs, where they terminate Notch signaling activity and promote the timely onset of Erm expression (Bello et al., 2006; Betschinger et al., 2006; Lee et al., 2006a; Lee et al., 2006b; Wang et al., 2006). Numb is a conserved Notch inhibitor and prevents continual Notch activation in immature INPs (Frise et al., 1996; Zhong et al., 1996; Lee et al., 2006a; Wang et al., 2006; Wirtz-Peitz et al., 2008). Asymmetric segregation of the RNA-binding protein Brat is facilitated by its adapter protein Miranda, which releases Brat from the cortex of immature INPs, allowing Brat to promote decay of Notch downstream-effector gene transcripts and thus initiate differentiation (Loedige et al., 2014; Laver et al., 2015; Loedige et al., 2015; Komori et al., 2018; Reichardt et al., 2018). Complete loss of numb or brat function leads to unrestrained activation of Notch signaling in immature INPs driving them to revert into type II neuroblasts leading to a severe supernumerary neuroblast phenotype. Similarly, increased levels of activated Notch or Notch transcriptional target gene expression in immature INPs can drastically enhance the moderate supernumerary neuroblast phenotype in brat- or numb-hypomorphic brains (Xiao et al., 2012; Janssens et al., 2014; Komori et al., 2014b; Komori et al., 2018; Larson et al., 2021). Collectively, these findings suggest that precise transcriptional control of Notch and Notch target gene expression levels during asymmetric neuroblast division is essential, safeguarding the generation of neurons that are required for neuronal circuit formation in adult brains. We defined the fine-tuning of stemness gene transcription as a function that is mild enough to not effect INP commitment when lost alone but enough to enhance immature INP reversion to supernumerary neuroblasts induced by decreased post-transcriptional control of stemness gene expression. We established three key criteria to identify regulators which fine-tune stemness gene transcription in neuroblasts, (1) an established role in transcriptional regulation, for example a DNA-binding transcription factor, (2) clear expression in neuroblasts with no protein expression in immature INPs, and (3) acts as a negative regulator of its targets. From a type II neuroblast lineage-specific single-cell gene transcriptomic atlas, we found that fruitless (fru) mRNAs are detected in type I & II neuroblasts but not in their differentiating progeny. One specific Zn-finger containing isoform of Fru (FruC) is exclusively expressed in all neuroblasts. FruC binds cis-regulatory elements of most genes uniquely transcribed in type II neuroblasts, including Notch and Notch downstream-effector genes that promote stemness in neuroblasts. A modest increase in Notch or Notch downstream gene expression induced by loss of fruC function alone has no effect on INP commitment, but enhances immature INP reversion to type II neuroblasts in numb- and brat-hypomorphic brains. To establish how FruC might fine-tune gene transcription in neuroblasts, we examined the distribution of established histone modifications in the presence or absence of fruC. We surprisingly found FruC-dependent low-level enrichment of the repressive histone marker H3K27me3 in most FruC-bound peaks in genes uniquely transcribed in type II neuroblasts including Notch and its downstream-effector genes. The Polycomb Repressive Complex 2 (PRC2) subunits are enriched in FruC-bound peaks in genes uniquely transcribed in type II neuroblasts, and reduced PRC2 function enhances the supernumerary neuroblast phenotype in numb-hypomorphic brains, identical to fruC loss-of-function. We conclude that the FruC-PRC2-H3K27me3 molecular pathway fine-tunes stemness gene expression in neuroblasts by promoting low-level H3K27me3 enrichment in their cis-regulatory elements. The mechanism by which PRC2-H3K27me3 fine-tune stem cell gene expression will likely be relevant throughout metazoans. Results A gene expression atlas captures dynamic changes throughout type II neuroblast lineages To identify regulators of gene transcription during asymmetric neuroblast division, we constructed a single-cell gene transcription atlas that encompasses all cell types in the type II neuroblast lineage in larval brains. We fluorescently labeled all cell types in the lineage in wild-type third-instar larval brains, sorted positively labeled cells by flow cytometry, and performed single-cell RNA-sequencing (scRNA-seq) using a 10x genomic platform (Figure 1A; Figure 1—figure supplement 1A). This new dataset displays high levels of correlation to our previously published scRNA-seq dataset which were limited to INPs and their progeny. The harmonization of these two datasets results in a gene transcription atlas of the type II neuroblast lineage consisting of over 11,000 cells (Figure 1B). Based on the expression of known cell identity genes, we were able to observe clusters consisting of type II neuroblasts (dpn+,pnt+), INPs (dpn+,opa+), GMCs (dap+,hey-), immature neurons (dap+,hey+), mature neurons (hey-,nSyb+), and glia (repo+) (Figure 1C). The UMAP positions of these clusters match well with the results of pseudo-time analyses from a starter cell that was positive for dpn, pnt, and RFP transcripts (Figure 1D). Leiden clustering of the data was able to capture these major cell types (Figure 1E), and quality control metrics showed most clusters captured on average 1.5 k genes and showed low mitochondrial gene expression (Figure 1—figure supplement 1B). Thus, the harmonized scRNA-seq dataset captures molecularly and functionally defined stages of differentiation in the type II neuroblast lineage (Figure 1F). Figure 1 with 1 supplement see all Download asset Open asset A single-cell gene expression atlas of type II neuroblast lineages. (A) Summary illustration of gene and Gal4 driver expression patterns in the type II neuroblast lineage. The type II NB Gal4 driver: Wor-Gal4,Ase-Gal80. imm INP driver: R9D11-Gal4. (B) Harmonization of the scRNA-seq dataset from the entire type II neuroblast (NB) lineage generated in this study (blue) and our previously published scRNA-seq dataset which were limited to INPs and their progeny (orange). The genotype of larval brains used for scRNA-seq in this study: UAS-dcr2; Wor-Gal4, Ase-Gal80; UAS-RFP::stinger. (C) UMAPs of known cell-type-specific marker genes. Color intensity indicated scaled (log1p) gene expression value. (D) Pseudotime analysis starting from cells enriched for dpn, pnt, and RFP transcripts. (E) Left: Leiden clustering of the scRNA-seq atlas. Right: Representative UMAPs of dynamically expressed transcription factors from clusters 14 (NBs and immature INPs) and 1 (INPs). Color intensity indicated scaled (log1p) gene expression value. (F) Annotated gene expression atlas of a wild-type type II neuroblast lineage. To determine whether the new scRNA-seq dataset encompasses neuroblast progeny undergoing dynamic changes in cell identity during differentiation, we examined transcripts that were transiently expressed in neuroblast progeny undergoing INP commitment or asymmetric INP division. We found that cluster 14 contains type II neuroblasts (dpn+,erm-,ase-,ham-), Ase- immature INPs (dpn-,erm+,ase-,ham-) and Ase+ immature INPs (dpn-,erm-,ase+,ham+) (Figure 1E), which are well-defined rapidly changing transcriptional states during INP commitment (Xiao et al., 2012; Janssens et al., 2014; Rives-Quinto et al., 2020). Furthermore, cluster 1 contains proliferating INPs that express known differential temporal transcription factors (Bayraktar and Doe, 2013; Tang et al., 2022), including young INPs (D+,hbn-,ey-,scro-) and old INPs (D-,hbn+,ey+,scro+) (Figure 1E). These data led us to conclude that the type II neuroblast lineage gene transcription atlas captures neuroblast progeny undergoing dynamic changes in cell identity during differentiation (Figure 1F). FruC negatively regulates stemness gene expression in neuroblasts We hypothesized that regulators that fine-tune stemness gene expression in neuroblasts should (1) be transcription factors, (2) be exclusively expressed in type II neuroblasts, and (3) negatively regulate gene transcription. We searched for candidate genes that fulfill these criteria in the cluster 14 of the type II neuroblast lineage gene transcription atlas. dpn serves as a positive control because its transcripts are highly enriched in type II neuroblasts and rapidly degraded in Ase- immature INPs, allowing us to distinguish neuroblasts from immature INPs (Figure 2A–B). We found the expression of fru mirrors dpn expression, with transcript levels high in type II neuroblasts but lower in Ase- immature INPs (Figure 2A). fru is a pleiotropic gene with at least two major functions: one that controls male sexual behavior and another that is essential for viability in both sexes (Goodwin and Hobert, 2021). fru transcripts are alternatively spliced into multiple isoforms that encode putative transcription factors containing a common BTB (protein-protein interaction) N-terminal domain and one of four C-terminal zinc-finger DNA-binding domains (Dalton et al., 2013; Neville et al., 2014; von Philipsborn et al., 2014; Figure 2A). We used the Fru-common antibody that recognizes all isoforms to determine the spatial expression pattern of Fru protein in green fluorescent protein (GFP)-marked wild-type neuroblast clones. We detected Fru in neuroblasts but found that Fru is rapidly downregulated in their differentiating progeny in type I and II lineages (Figure 2B). To determine which Fru isoform is expressed in neuroblasts, we examined the expression of isoform-specific fru::Myc tagged allele where a Myc epitope is knocked into the C-terminus of the FruA, FruB, or FruC coding region (von Philipsborn et al., 2014). While FruA::Myc and FruB::Myc appear to be ubiquitously expressed at low levels, FruC::Myc is specifically expressed in both types of neuroblasts but not in their differentiating progeny, including immature INPs, INPs, and GMCs (Figure 2C, Figure 2—figure supplement 1A–B). These data indicate that FruC is the predominant Fru isoform expressed in neuroblasts. Figure 2 with 1 supplement see all Download asset Open asset Fruc functions through transcriptional repression to regulate stemness gene expression. (A) Top: fru and dpn mRNAs are highly enriched in neuroblasts in cluster 14 of the scRNA-seq dataset, but only dpn mRNAs are detected in INPs in cluster 1. Bottom: Domains in Fru protein isoforms. ZF: zinc-finger DNA-binding domain. (B) Fru protein is detected in the neuroblast but not in INPs in a GFP-marked type II neuroblast lineage clone. The genotype used in this experiment is Elav-Gal4,UAS-mCD8::GFP,hs-flp; FRT82B,Tub-Gal80/FRT82B. (C) Endogenously expressed FruC tagged by a Myc epitope (fruC::Myc) is detected in type I and II neuroblasts but not in their differentiating progeny. (D–E) Immature INPs in GFP-marked wild-type type II neuroblast clones never show detectable Dpn expression (100%). 1–2 Ase- immature INP per fru-null (frusat15) type II neuroblast clone show detectable Dpn expression (100%). The genotype in this experiment is Elav-Gal4,UAS-mCD8::GFP,hs-flp; FRT82B,Tub-Gal80/FRT82B,fruSat15. (F–I) The newborn immature INP marked by cortical Miranda staining show undetectable Dpn and E(spl)mγ::GFP expression in the majority of wild-type clones (85.7%). Reducing fruC function leads to ectopic Dpn and E(spl)mγ::GFP expression in the newborn immature INP in most type II neuroblast lineages (94.4%). NB-Gal4: Wor-Gal4. (J–L) Type II neuroblasts overexpressing Fruc display characteristics of differentiation including a reduced cell diameter and aberrant Ase expression. Quantification of the cell diameter is shown in L. (type II NB>: Wor-Gal4, Ase-Gal80). (M–P) Overexpressing full-length Fruc or a constitutive transcriptional repressor form of FruC (Fruc,zf::ERD) is sufficient to partially restore differentiation in brat-null type II neuroblast clones (brat11/Df(2 L)Exel8040,hs-flp; Act5C-Gal4>FRT > FRT>UAS-GFP/UAS-fruC or ERD::fruC,zf). The percentage of GMCs per clone is shown in P. Yellow dashed line encircles a type II neuroblast lineage. White dotted line separates optic lobe from brain. white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP; magenta arrow: type I neuroblast; magenta arrowhead: GMC. Scale bars: 10 μm. p-values: ***<0.0005, and ****<0.00005. To define the function of Fru in neuroblasts, we assessed the identity of cells in the GFP-marked mosaic clones derived from single type II neuroblasts. The wild-type neuroblast clone always contains a single neuroblast that can be uniquely identified by cell size (10–12 μm in diameter) and marker expression (Dpn+Ase-) as well as 6–8 smaller, Dpn- immature INPs (Figure 2D). The neuroblast clone carrying deletion of the fru locus (fru-/-) contains a single identifiable neuroblast but frequently contains multiple Ase- immature INPs with detectable Dpn expression (Figure 2E). Over 80% of newborn immature INPs (marked by intense cortical Mira expression) generated by fruC-mutant type II neuroblasts ectopically express Dpn and E(spl)mγ while less than 15% of newborn immature INPs generated by wild-type neuroblasts expressed these genes (Figure 2F–I). These results support a model in which loss of fruC function increases the expression of Notch downstream-effector genes that promote stemness in neuroblasts. Consistently, type II neuroblasts overexpressing FruC prematurely initiate INP commitment, as indicated by a reduced cell diameter and precocious Ase expression (Figure 2J–L). Thus, loss of fruC function increases stemness gene expression whereas gain of fruC decreases stemness gene expression during asymmetric neuroblast division. To determine whether FruC negatively regulates the transcription of stemness genes in type II neuroblasts, we overexpressed wild-type FruC in GFP-marked neuroblast lineage clones in brat-null brains. Ectopic translation of Notch downstream-effector gene transcripts that promote stemness in neuroblasts drives immature INP reversion to supernumerary type II neuroblasts at the expense of differentiating cell types in brat-null brains (Loedige et al., 2015; Komori et al., 2018; Reichardt et al., 2018). Control clones in brat-null brains contain mostly type II neuroblasts and few differentiating cells that include Ase+ immature INPs, INPs, and GMCs (Figure 2M and P). By contrast, overexpressing full-length FruC increases the number of INPs, GMCs, and differentiating neurons (Ase-Pros+) in brat-null neuroblast clones (Figure 2N and P). This result indicates that FruC overexpression is sufficient to partially restore differentiation in brat-null brains. To test if Fruc restores differentiation by promoting transcriptional repression, we generated fly lines carrying the UAS-fruC,zf::ERD transgene that encodes the zinc-finger DNA-binding motif of FruC fused in frame with the Engrail Repressor Domain. The ERD domain is well conserved in multiple classes of homeodomain proteins as well as many transcriptional repressors across the bilaterian divide and binds to the Groucho co-repressor protein to exert its repressor function (Smith and Jaynes, 1996; Jiménez et al., 1997; Bürglin and Affolter, 2016). Several previously published studies have used this strategy to demonstrate that neurogenetic transcription factors exert transcriptional repression function in neuroblasts (Xiao et al., 2012; Janssens et al., 2014; Bahrampour et al., 2017; Rives-Quinto et al., 2020). Similar to full-length FruC overexpression, overexpressing FruC,zf::ERD was also sufficient to partially restore differentiation in brat-null brains (Figure 2O–P). Thus, we conclude that FruC negatively regulates stemness gene expression in type II neuroblasts. Fruc binds cis-regulatory elements of the majority of genes uniquely transcribed in neuroblasts If FruC directly represses stemness gene expression, FruC should bind their cis-regulatory elements. To identify FruC-bound regions in neuroblasts, we applied a protocol of Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to brain extracts from dissected third-instar brat-null larvae homozygous for the fruC::Myc knock-in allele. brat-null brains accumulate thousands of supernumerary type II neuroblasts at the expense of INPs and provide a biologically relevant source of type II neuroblast-specific chromatin (Komori et al., 2014a; Janssens et al., 2017; Komori et al., 2018; Rives-Quinto et al., 2020; Larson et al., 2021). We used a specific antibody against the Myc epitope or the Fru-common antibody to confirm that FruC::Myc is detected in all supernumerary type II neuroblasts in brat-null brains homozygous for fruC::Myc (Figure 3—figure supplement 1A). We determined the genome-wide occupancy of FruC::Myc in type II neuroblasts using the Myc antibody and Fru-common antibody, and found that FruC::Myc binding patterns revealed by these two antibodies are highly correlated (Figure 3—figure supplement 1B; Pearson correlation = 0.94). FruC binds 9301 regions in type II neuroblasts (Figure 3A). Overall, 59% of FruC-bound regions are promoters whereas 29% are enhancers in the intergenic and intronic regions (Figure 3A). By contrast, 15% of randomized control regions are promoters and 55% are enhancers (Figure 3A). 50.1% of FruC-bound regions in promoters and enhancers overlap with regions of accessible chromatin (Larson et al., 2021; Figure 3B). Consistent with the finding that FruC negatively regulates stemness gene expression, FruC binds promoters and neuroblast-specific enhancers of Notch, dpn, E(spl)mγ, klumpfuss (klu) and tailless (tll) that were previously shown to maintain type II neuroblasts in an undifferentiated state (Figure 3C; Figure 3—figure supplement 1C). Based on our scRNA-seq data, we classified genes as NB genes, imm INP genes, or invariant genes expressed throughout the lineage based on differential expression within cluster 14 (Figure 1E; Supplementary file 2). Seventy-four percent of genes uniquely transcribed in type II neuroblasts (NB genes) are bound by FruC whereas 41% of these genes are in randomized control (Figure 3D–E). By contrast, the percentage of FruC-bound genes transcribed in immature INPs or throughout the type II neuroblast lineage is similar to random control (Figure 3D–E). Because stemness gene transcripts are highly enriched in type II neuroblasts, these results suggest that FruC preferentially binds cis-regulatory elements of stemness genes. Figure 3 with 1 supplement see all Download asset Open asset Fruc preferentially binds regulatory elements of genes uniquely expressed in neuroblasts. (A) Genomic binding distribution of FruC-bound peaks (total # of peaks shown in parentheses) from CUT&RUN or random (set of Fruc peaks shuffled to randomly determined places in the genome) in type II neuroblast-enriched chromatin from brat-null brains (brat11/Df(2L)Exel8040). Fruc preferentially binds promoters. (B) Heatmap is centered on promoters or regulatory regions showing accessible chromatin as defined by ATAC-seq with 500 bp flanking regions and ordered by signal intensity of Fruc::Myc binding. (C) Representative z score-normalized genome browser tracks showing regions with accessible chromatin (ATAC-seq) and bound by Fruc::Myc (detected by the Myc antibody or FruCOM antibody), Su(H), Trl, or IgG at Notch, dpn, and E(spl)mγ loci. (D) Genes in cluster 14 from the scRNA-seq dataset were separated into neuroblast-enriched genes (right), immature INP-enriched genes (left), and invariant genes. The middle circle is the set of genes bound by Fruc or Su(H) (shown in parentheses). UMAPs show gene enrichment score for Fruc-bound genes uniquely expressed in neuroblasts (right), uniquely expressed in immature INPs (left), and ubiquitously expressed (invariant genes) (middle). (E) Percentage of genes defined in D bound by either Fruc, Su(H), random Fruc peaks, or random Su(H) peaks (set of Su(H) peaks shuffled to randomly determined places in the genome). (F–G) Genomic binding distribution of identified Su(H)-bound peaks (total # of peaks shown in parentheses) from CUT&RUN in type II neuroblast-enriched chromatin. Heatmap is centered on promoters or regulatory regions and ordered by signal intensity of Su(H) binding. (H–I) Genomic binding distribution of identified Trl-bound peaks (total # of peaks shown in parentheses) from CUT&RUN in type II neuroblast-enriched chromatin. Heatmap is centered on promoters or regulatory regions and ordered by signal intensity of Trl binding. A mechanism by which FruC can negatively regulate stemness gene expression levels is to modulate the activity of the Notch transcriptional activator complex activity. Using Affymetrix GeneChip, a pr" @default.
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W4384209396 title "Editor's evaluation: Low-level repressive histone marks fine-tune gene transcription in neural stem cells" @default.
W4384209396 doi "https://doi.org/10.7554/elife.86127.sa0" @default.
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